The Na+-ionophore monensin enhances glucose uptake in mouse thymocytes

Inf. J. Biochem. Vol. 16, No. 7, pp. 837-840, Printed in Great Britain. All rights reserved

THE

1984 Copyright

Na+-IONOPHORE UPTAKE

MONENSIN ENHANCES IN MOUSE THYMOCYTES

JARDENA NORDENBERG,

KURT

The Rogoff-Wellcome

Medical Research University Sackler

0020-71 IX/84 S3.00 + 0.00 I(’ 1984 Pergamon Press Ltd

GLUCOSE

H. STENZEL* and ABRAHAM NOVOGRODSKY Institute, Beilinson Medical Center, School of Medicine, Tel-Aviv, Israel [Tel. 03-96-821 I]

Petah-Tikva,

Tel-Aviv

(Received 24 October 1983) Abstract--l. Monensin enhanced 2-deoxyglucose uptake and 3-O-methyl glucose transport in mouse thymocytes, but had no effect on L-glucose transport. 2. Cytochalasin B inhibited monensin induced as well as basal glucose uptake. 3. The enhanced 2-deoxyglucose uptake was time and dose-dependent. 4. The increase in the rate of 2-deoxyglucose uptake induced by monensin was more rapid than that of Na+ uptake. 5. Ouabain did not inhibit monensin-enhanced 2-deoxyglucose uptake. 6. Monensin failed to stimulate 2-deoxyglucose uptake at low concentrations of Na + (13 mM) or K+ (17 mM), higher concentrations of either cation were required for stimulation. Monensin enhanced glucose uptake also in Ca*+-free medium. 7. The data indicate that the stimulation of 2-deoxyglucose uptake by monensin results from activation of carrier-mediated transport.

INTRODUCTION Enhanced glucose uptake and alterations in cation flux are among the early cellular events induced in lymphocytes by a variety of mitogenic agents.These agents include Iectins (Yasmeen et al., 1977; Reeves, 1977; Peters and Hausen, 1971), tumor promoters (Blumberg, 1980; Nordenberg et al., 1983) and the Ca2+-ionophore A23187 (Reeves, 1976). Increased Na+ influx has recently been suggested to be an essential event in cell activation induced by various stimuli (Deutch et al., 1981; Deutch and Price, 1982; Koch and Leffert, 1979a,b). The potent tumor promoter tetradecanoyl-phorbol acetate (TPA) has been shown to induce in Swiss 3T3 cells an early increase in Na+ influx and activation of the Na+/K+ pump (Dicker and Rozengurt, 1981). Concanavalin A also caused a rapid increase in the intracellular Na+ content of thymocytes (Felber and Brand, 1983). These findings prompted us to explore the possibility that the early increase in glucose uptake in lymphocytes induced by different mitogens is related to Nat influx. In the present study we examined the effect of the carboxylic Na+ ionophore, monensin (Pressman and Fahim, 1982) on glucose uptake in murine thymocytes. The results show that monensin stimulates carrier-mediated 2-deoxyglucose uptake. However, monensin enhancement of the 2-deoxyglucose uptake does not appear to be directly related to Nat influx but rather to a direct effect on the cell membrane. MATERIALS AND METHODS Monensin was obtained from Calbiochem and dioctyl phthalate and dibutyl phthalate were obtained from Fluka AG. All other chemicals were obtained from Sigma, *Permanent address: Rogosin Kidney Center, Cornell versity Medical College, New York, U.S.A.

3-O-methyl-D-[ I -jH]glucose (4.5 Ci/mmol) and 2-deoxy -D-[ I-‘H]glucose (18.2Ci/mmol) were supplied by the Radiochemical Centre, Amersham. L-glucose [I -‘HI (10.4 Ci/mmol) and 22Na+ (carrier free) were obtained from New England Nuclear. Female Balb/c mice aged 4-6 weeks fed ad libitum were used. The thymus glands were rapidly removed and thymocytes isolated. For determination of uptake of glucose analogues, cells (1.2-2.5 x lO’/ml) were incubated at 37 ‘C in phosphate buffered saline containing sodium-b-hydroxy butyrate (1 mM) and sodium lactate (5 mM) supplemented with inactivated dialysed human AB serum (5?;) and bovine serum albumin (0X$. In all experiments cells were preincubated for 1&20min before additions were made. For the last 20 min of incubation 2-deoxy-[l-XH]glucose (2pCi/ml) or [ZH]-L-glucose (5 pCi/ml) were added. For 3-O-methyl glucose transport measurements 47 x IO’ cells/ml were incubated with 3-0-methyl-[1-‘HIglucose (7 pCi/ml) for 3 min at 23°C. Incorporation of [‘HIglucose analogues into cells was measured as described previously (Nordenberg et al., 1983). When the influence of ionic composition on monensin-induced 2-deoxyglucose uptake was examined NaCl was replaced by isotonic sucrose. The ionic composition is described in the figure legends. For measuring z2Naf uptake, cells (5-8 x lO’/ml) were incubated in modified phosphate buffered saline containing 50 mM NaCl and supplemented with 174 mM sucrose, at 37 ‘C. For the last 5 min of incubation ouabain (2 mM) and “Na+ (5 pCi/ml) were added, and “Na uptake into cells was measured as follows. Samples of 0.15 ml were transferred into 0.9 ml MgClz (0.1 M) layered over 0.2 ml dibutyl-dioctyl phthalate (1: 1) in microfuge tubes (embedded in ice). Tubes were centrifuged at 4 C for 30 set in a Beckman 152 microfuge. This procedure separates the cells from the isotope containing medium. After centrifugation the bottoms of the tubes were cut off and counted in a ~-Packard liquid scintillation counter. Viability of cells was examined using the Trypan Blue exclusion test. RESULTS Monensin

Uni-

induced

of 2-deoxyglucose 837

a marked

in murine

increase

thymocytes

in the uptake

(Table

1).

838

JARRENA

Table 1. The effect of monensin

NORDENEERG

on the uptake of D-glucose analoaues I-‘Hjglucose uptake (pmoli 10” cells)

Z-Deoxy-[

Treatment Cytochalasin

B

Control

6.8 * 0.3 {n = 30) 0.6+0.1 (n = 3) 17.5 * 1.4” (n = 30) 0.8 + 0.1

+ Monensin

+

ef al.

(f?

=

6)

bv mouse thvmocytes

3-D-Methyl-~l-3H]gluco~e

transport (pmolj 10’ cells) 3.7 [n 0.9 (n 6.9 (n 0.9 (n

* 0.2 = 5) * 0.1 =3) k 0.5” = 5) * 0.1 =

5)

Thymocytes were incubated with or without

monensin (7.2 PM) for SO min at 37°C. Cytochalasin 3 (4.~ig/mlf was added for the last 30 min of incubation. 2-deoxy-jl-3N]glucose uptake and 3-O-methyI-[l-3Hlglucose transport were measured as described in the text. Valuks are expressed as me& i %M. ‘Monensin vs control P < 0.001.

This glucose analogue is transported into the cell and is phosphorylated by hexokinase. The transport of 3-U-methyl glucose, a D-glucose analogue that is transported by the glucose carrier and is not further metabolized by the cell, was also enhanced by monensin. Cytochalasin B, which inhibits glucose uptake by its effect on the glucose carrier itself (Lin and Spudich, 1974; Shanahan, 1982; Schraw and Regen, 1983), markedly inhibited basal as well as monensininduced glucose uptake and transport (Table I). The transport of ~-glucose, a glucose analogue that is not recognized by the D-glucose carrier, was not enhanced by monensin (the basal level of [3H]-L-glucose transport was 0.8 & 0.1 pmoi/lO* c&Is and remained unchanged after treatment with monensin). The enhancement of Sdeoxyglucose and Na+ uptake by monensin was dose-dependent ‘over a wide concentration range (Fig. 1). The rates of 2-deoxy[ 1-3H]glucose and “Na uptake were measured

j

in cells incubated with mone~si~ (7.2 PM) (Fig. 2). The results indicate that the enhancement of 2ZNa uptake by monensin was rapid and preceded that of 2-deoxy-glucose uptake. After 15 min of incubation in the presence of monensin the rate of 22Na uptake was already maximally enhanced, whereas 2-deoxyglucose uptake was enhanced only by 37%. After 60 min of incubation with m~nensin~ the rate of 2deoxyglucose uptake was enhanced by approximately ISO%, The different time course for the enhancement of 2-deoxyglucose and 22Na uptake suggests that the glucose uptake induced by monensin is not coupled to Na+ uptake. To test whether the enhancement of Sdeoxyglucose uptake by monensin is related to the Na+jK+ pump activation, the effect of auabain on monensinenhanced 2-deoxyglacose uptake was determined. Millimolar concentrations of ouabain were used since high concentrations of this drug are required to

I 3ao-

-900

.

Fig. 1.The effects of monensin at various concentrations aa in mouse thy2-deoxy-[l-3H]glucose and 22Na uptake mocytes. (A) +-Thymocytes were incubated without and with monensin at various concentrations for 80min. 2-deoxy-[l-%]glucose was added for the last 20min of incubation. Uptake was measured as described in the text. (B) A-Thymocytes were incubated for 25 min. without and with monensin at various concentrations. “Na (5 pCi/ml) and ouabain (2 mM) were added for the last 5 min of incubation. 22Na uptake was measured as described in the text. The value at each concentration represents the mean % of control for 3-12 replicates done with various cell preparations.

Time

Iminl

Fig. 2. Time course of the effects of monensin on in mouse thy2-deoxy-[ 1--‘HIglucose and 22Na uptake mocytes. Thymocytes were incubated in the absence or presence of monensin (7.2 pM) for differe-t times. l-2-deoxy-[f-3Hfglucose uptake was measured over a 15 min period at each time point. A--**Na uptake was measured over a 5min period. Values are expressed as X enhancement. All measurements were made in 3-6 replicates with at least 2 different cell preparations.

Monensin

enhances

glucose

in thymocytes

uptake

839

Table 2. The effect of monensin on 2-deoxy-[l-1H]glucose uptake in the absence presence of ouabain and at various concentrations of Na+ and K+

or

2.Deoxy-[I-‘HIglucoseuptake (pmol/lo*

cells)

Monensin (7.2PM) _

Incubation medium Phosphate buffered saline, Na+ = 150mM Phosphate buffered saline Ouavain (2 mM) Sucrose, Na+ = 13 mM Sucrose, Nat = 63 mM Sucrose, K+ = 17 mM Sucrose. K+ = 67 mM

Enhancement (%)

+

7.0 * 0.5

15.4f 1.3”

+119

6.0 i. 0.4 8.8 * 0.3 7.1 * 0.4 9.8 +_0.4 6.7 + 0.3

12.2f 0.6” 10.9* 0.4 14.3f 0.5” 9.7 +_0.5 16.0+ 0.7”

+84 +24 flO1 -I +137

+

C&I, (0.9 mM), MgCI, (0.6 mM) and KH,PO, (1.47 mM) were included in all buffers. NaCI, KCI, Na,HPO, or K,HPO, were used at different concentrations to obtain the desired Nat or K+ concentration and sucrose was added to maintain isotonicity. Ouabain was added 20 minutes prior to monensin. Monensin was present for 70 min in all experiments. 2-Deoxy-[1-‘HIglucose uptake was measured as described in the text. Values are mean f SEM for 9-12 replicates of 3-4 different cell preparations. “With monensin YS without monensin P < 0.001.

the Na+ /K + pump activity in murine lymphocytes (Kaplan and Owens, 1982). Ouabain caused only a slight inhibition of monensin-enhanced 2-deoxyglucose uptake (Table 2). This small inhibition could be attributed to a decrease of about 15% in cell viability caused by ouabain. Thus it is unlikely that the Nat/K+ pump activity is involved in the enhancement of 2-deoxyglucose uptake induced by monensin. The effect of ionic strength on monensin-induced 2-deoxyglucose uptake was investigated (Table 2). At low ionic strength (approximately 15 mM), provided either by Na+ or K+, monensin failed to stimulate 2-deoxyglucose uptake. However, at higher ionic strength (approx 65 mM), monensin did enhance 2-deoxyglucose uptake (Table 2). Ca2+ has previously been implicated as a mediator in the activation of glucose transport by mitogens (Whitesell et al., 1977a,b). Monensin enhances Z-deoxyglucose uptake in Ca*+ free medium or in the presence of EGTA (Table 3). These results indicate that extracellular Ca2+ is not required for the stimulation of 2-deoxyglucose uptake by monensin. inhibit

DISCUSSION

A variety of agents including lectins, phorbol ester tumor promoters and the Ca*+ ionophore exert pleio-

tropic effects on lymphocytes and other cell types. Enhanced glucose uptake (Blumberg, 1980; Yasmeen et al., 1977; Driedger and Blumberg, 1977; Reeves, 1975; Whitesell et al., 1977a,b; Hume and Weidemann, 1978) and alterations in cation flux (Moroney et al., 1978; Smith and Rozengurt, 1978; Owens and Kaplan, 1980) were shown to be early events in cell activation by these agents. The present study indicates that monensin a polyether carboxylic acid monovalent cation ionophore mimicks the action of growth promoting agents on glucose uptake in lymphocytes. Polyene antibiotics were previously reported to enhance glucose uptake in adipose cells, similar to insulin (Kuo, 1968). Cytochalasin B, a compound known to inhibit glucose transport by direct binding to the glucose carrier (Lin and Spudich, 1974; Shanahan, 1982) inhibited the enhancement of glucose uptake by monensin. Furthermore, the transport of L-glucose, an analogue that is not recognized by the glucose carrier was not enhanced. These findings indicate that monesin-enhanced glucose uptake is mediated by a mechanism involving the glucose carrier rather than a result of non-specific alterations in the cell membrane leading to increased permeability. The observation that the transport of 3-O-methylglucose similar to that of 2-deoxyglucose is also enhanced by monensin provides evidence that the primary effect of

Table 3. The effect of monensin on 2-deoxy-[1-‘HIglucose uptake in mouse thvmocvtes incubated in Ca 2+ free medium or in the presence of EGTA 2-Deoxy-[l-3H]glucose uptake (pmol/lO” cells) Monensin Medium Phosphate buffered saline Phosphate buffered saline without Ca*+ + EGTA (2.5 mM) Phosphate buffered saline + Ca’+ + EGTA (5 mM)

_

+

6.3 + 0.3

13.7 i 1.0

6.5 f 0.4

15.4 + 1.0

5.9 * 0.1

Cells were incubated for 20 min with or without EGTA prior monensin (7.2 PM). Incubation without or with monensin an additional 70 min. 2-Deoxy-[1-‘HIglucose uptake was described in the text. Values are means f SEM of 3-12 replicates done with various

12.7 f 0.6 to the addition of was continued for then measured as cell preparations.

JARDENA NORDENBERG et al.

840

monensin on glucose uptake is on the carrier mechanism and not on hexokinase. Sodium-coupled glucose transport has been found in small intestinal epithelial cells (Schultz, 1977). The possibility that monensin enhanced the co-transport of Na+ and glucose in thymocytes seems unlikely since monensin enhanced 2-deoxyglucose uptake in Na+ free medium and ouabain did not decrease monensin-enhanced 2-deoxyglucose uptake. In addithe time course for enhancement of tion 2-deoxyglucose and Na+ uptake was different. The finding that monensin failed to stimulate glucose uptake in medium containing low concentrations of either Na+ or K+ appears to indicate a role for cations in mediating enhanced glucose uptake by monensin. It is unclear whether the requirement for either Na+ or K+ is related to their contribution to ionic strength or to a more specific effect. Ionophores, by virtue of their amphipatic properties, bind to membrane components and thus could directly or indirectly activate the transport system. The requirement of Na+ or K+ for this activation might be related to the effect of these cations on the structure of the ionophore itself. It is possible that monensin requires Na+ or K+ to maintain the proper conformation for interacting with the cell membrane. It has been reported that the Ca*+ ionophore A23187 is mitogenic for lymphocytes under conditions where Ca*+ influx was inhibited, suggesting that the ionophore activates lymphocytes by a direct mechanism (Resh et al., 1978). Taken together our data raise the possibility that increased glucose uptake induced by monensin might result from a direct interaction of the ionophore with the cell membrane and may not be directly related to alterations in ion flux. Acknowledgements-Dr

J. Nordenberg was supported by an Israel Cancer Research Fund fellowshin. We thank Dr Uri Pick, The Weizmann Institute, for helpful discussions. REFERENCES Blumberg P. M. (1980) In-vitro studies on the mode of action of the phorbol esters, potent tumor promoters: Part 1. CRC crit. Rev. Toxic. 8, 153-197.

Deutsch C. and Price M. (1982) Role of extracellular Na and K in lymphocyte activation. J. Cell Physiol. 113, 73-79. Deutsch C., Price M. A. and Johansson C. (1981) A sodium requirement for mitogen-induced proliferation in human peripheral blood lymphocytes. Exp/ Cell Res. 136, 359-369. Dicker P. and Rozengurt E. (1981) Phorbol ester stimulation of Na influx and Na-K pump activity in Swiss 3T3 cells. Biochem. biophys. Res. Commun. 100, 433-441. Driedger P. E. and Blumberg P. M. (1977) The effect of phorbol diesters on chicken embryo fibroblasts. Cancer Res. 37, 3257-3265. Felber S. M. and Brand M. D. (1983) Concanavalin A causes an increase in sodium permeability and intracellular sodium content of pig lymphocytes. Biochem. J. 210, 893-897. Hume D. A. and Weidemann M. J. (1978) On the stimulation of rat thymocyte 3-O-methyl-glucose transport by mitogenic stimuli. J. Cell. Physiol. 96, 303-308.

Kaplan J. G. and Owens T. (1982) The cation pump as a switch controlling mechanism proliferation and differentiation in lymphocytes. Biosci. Rep. 2, 577-58 I. Koch K. S. and Leffert H. L. (1979a) Increased sodium ion influx is necessary to initiate rat henatocvte _ __ nrohferation. Cell 18, 1533163: Kock K. S. and Leffert H. L. (1979b) Ionic landmarks along the mitogenic route. Nature 279, 104105. Kuo J. F. (1968) Stimulation of glucose utilization and inhibition of hpolysis by polyene antibiotics in isolated adipose cells. Archs Biochem. Biophys. 127, 406412. Lin, S. and Spudich J. A. (1974) Biochemical studies on mode of action of cvtochalasin B. Binding to red cell membrane in relation-to glucose transport. .?. biol. Chem. 249, 577885783. Moroney J., Smith A., Tomei L. D. and Wenner C. E. (1978) Stimulation of *‘Rb+ and 32P1movements in 3T3 cells by prostaglandins and phorbol esters. J. Cell. Physiol. 95, 287-294. Nordenberg J., Stenzel K. H. and Novogrodsky A. (1983) 12-O-tetradecanoylphorbol-13-acetate and concanavalin A enhance glucose uptake in thymocytes by different mechanisms. J. Cell. Physiol. 117, 183-188. Owens T. and Kaplan G. (1980) Increased cationic fluxes in stimulated lymphocytes of the mouse: response of enriched B- and T-cell subpopulations to B- and T-cell mitogens. Can. J. Biochem. %, 831-839. Peters J. H. and Hausen P. (1971) Effect of phvtohemagglutinin on lymphocyte membrane transport. Eur. J. Biochem. 19, 509-513. Pressman B. C. and Fahin M. (1982) Pharmacology and toxicology of the monovalent carboxylic ionophores. A. Rev. Pharmac. Toxic. 22, 465490. Reeves J. P. (1975) Calcium-dependent stimulation of 3-O-methylglucose uptake in rat thymocytes by the divalent cation ionophore A23187. J. biol. Chem. 250, 9428-9430. Reeves J. P. (1977) 3-0-methvlglucose transport by rat thymocyte subpopulations. J.- Cell. Physiol. 92, 309-3 18. Resch K.. Bouillon D. and Gemsa D. (1978) The activation of lymphocytes by the ionophore A23187. J. Immun. 120, 15141520. Schraw W. P. and Regen D. M. (1983) Reconstitution of the D-glucose transport of bovine lymphocyte plasma membranes. Partial purification of transport activity by chromatography on agarose lentil lectin and agarose ethanethiol. Archs Biochem. Biophys. 220, 214224. Schultz S. G. (1977) Sodium-coupled solute transport by small intestine: status report. Am. J. Physiol. 233, E249-E254. Shanahan M. F. (1982) Cytochaiasin B. a natural photoaffinity legand for labeling the human erythrocyte glucose transporter. J. biol. Chem. 250, 729&7293. Smith J. B. and Rozengurt E. (1978) Serum stimulates the Na+. K+ pumn in Quiscent, tibroblasts by increasing Na entry. Pro-c. natn. Acad. Sci. U.S.A. 75, 5560-5564.Whitesell R. R., Johanson R. A., Tarpley H. L. and Regen D. M. (1977a) Mitogen-stimulated glucose transport in thymocytes. Possible role of Ca2- and antagonism by adenosine 3’5-monophosphate. J. Cell Bioi. 72, 456469. Whitesell R. R.. Hoffman L. H. and Reaen D. M. (1977b) Dynamic aspects of glucose transport modulation in thymocytes. J. biol. Chem. 252, 3533-3537. Yasmeen D., Laird A. J., Hume D. A. and Weidemann M. J. (1977) Activation of 3-O-methyl-glucose transport in rat thymus lymphocytes by Concanavalin A. Temperature and calcium ion dependence and sensitivity to puromycin but not to cycloheximide. Biochim. biophys. Acta 500, 89-102.