Influence of Concanavalin A on 3-O-methylglucose uptake in cultured chick embryo fibroblasts

Differentiation

Differentiation (1984) 27: 192-195

0 Springer-Verlag 1984

Influence of Concanavalin A on 3-0-methylglucose uptake in cultured chick embryo fibroblasts Evidence for differences related to the age of embryos Chantal Berjonneau, Patrice Codogno, Joelle Botti, Mireme Giner, Bruno Bernard* and MicMle Aubery Institut National de la Sand et de la Recherche Mkdicale (INSERM) Unit6 180. Laboratoire de Biologie et Pathologie Molkulaires des Glycoprotkines UnitC d'Enseignement et de Recherche (UER) Biomklicale des Saints-PBres 45 rue des Saints Phes, F-75006 Paris, France

Abstract. Concanavalin A (Con A) was found to inhibit hexose uptake in cultured fibroblasts derived from 8day chick embryos and to stimulate this process in those derived from 16day embryos. (1) Con-A effects depended on the duration of contact with cells and lectin and were inhibited by a-methylmannopyrannoside. (2) Con A was shown to mask about 70% of the hexose carriers in both 8- and 16-day embryo fibroblasts. Lectin altered the hexose uptake very rapidly. (3) Con A only modified the V,, of the uptake system and did not alter the K,. This indicates that either the number or mobility of hexose carriers were modified by Con-A treatment. The differential effect of lectin could be due to a modification of the hexose-carrier mobility during the embryonic differentiation of fibroblasts. Secondary effects may affect cell growth.

Introduction Concanavalin A (Con A) differentially alters the proliferation of cultured chick-embryo fibroblasts according to the age of the embryo [1, 241. Although such alteration is known to occur after Con-A binding to cell-surface glycoproteins [7l, no direct correlation has yet been established between Con-A binding to embryo cells and its differential effects on cell growth [24]. Con A might also modify cellmembrane permeability to certain nutrients. The present report describes the effect of Con A on hexose uptake in cultured fibroblasts derived from 8- and 16day chick embryos. The analog 3-0-methylglucose (3-0-MeG) was chosen to explore the uptake system because it is accepted by the glucose carriers in the plasma membrane but not metabolized inside the cell [ 131.

Methods Cells and cultures. Fibroblasts were obtained from 8- and 16-day chick embryos (Centre National de Recherche Zoologique, CNRZ, France), as has been previously described 11, 21. Microscopic examination of the cultures revealed that only fibroblasts were present, and that the cultures were homogeneous and devoid of myoblasts. Fibroblasts derived from both 8- and 16day embryos mainly synthe-

*

Present address: Centre International de Recherches Dermatologiques (CIRD), Sophia Antipolis. 06565 Valbonne, France

tized type-I collagen and a little type-I11 collagen; this is normal for fibroblasts [6]. Cultures were grown in the absence or presence of Con A (Sigma grade 111) at a final concentration of 3 pg/ml, as has been previously described [l]. After various periods of culture, cells were harvested with trypsin as has been specified previously [28], and counted in a hemocytometer. As has been observed in an earlier study [l] under our conditions. Con A did not agglutinate embryo cells. Each measurement refers only to viable cells, which amounted to 95% or 100% of cell numbers, as checked in control and Con-A-treated cells by trypan blue exclusion and the lacticodehydrogenase test [31]. We have previously demonstrated that Con A has no cytotoxic effect on cultured fibroblasts from either 8- or 16day embryos [31]. Effect of Con A on 3-0-methylglucose uptake in 8- and 16-day-embryofibroblasts'. The total 3-0-MeG uptake was determined as has been previously described in subconfluent cultures of control cells and Con-A-treated cells. The K, and the ,V of 3-0-MeG specific uptake were determined as has been previously described [2, 31. Competition between Con A and cytochalasin B for binding to embryo cells. Because the dissociation constant of the transport system makes the direct binding of 3-0-MeG impossible [2], and it has been demonstrated that cytochalasin B (CB) binds to glucose carriers [9, 22, 251, the effect of Con A on 3-0-MeG binding to hexose carriers was tested with CB. Unlabelled CB was obtained from Sigma, and labelled CB (3H-CB) was purchased from the Radiochemical Centre, Amersham (sp. act., 11 Ci/mmol). Con A was labelled by the method of Miller and Great [16] using I4Cacetic anhydride, as has been described previously [5]. The specific radioactivity of the lectin was 1x lo6 dpm/mg. Con A and CB bindings were assayed as has been described earlier [4, 51. Briefly, for the binding of Con A, subconfluent monolayer cultures (1.0f0.1x lo6 cells/well) were washed three times with phosphate buffer, pH 7.4, and covered with a 0.5-ml layer of phosphate buffer containing 14C-labelledCon A or 3H-labelled CB. For the CB binding assay, the phosphate buffer contained 0.5% ethanol. 1 ,,8-day-embryo fibroblasts" means cultured fibroblasts derived

from 8day chick embryos. ,,16dayembryo fibroblasts" means cultured fibroblasts derived from 16-day chick embryos

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The procedure for the competition experiments was as follows: 3 . Cells were incubated at 21" C with unlabelled Con A at 3 or 40 pg/ml for 30 min, washed and assayed for binding to 1,OOO nM 'H-CB. 2. Conversely, other cells were first incubated with 1,000 nM unlabelled CB at 21" C for 15 min, and after washing, assayed for 30 min for binding to 40 p g / d 14CCon A [5]. Control experiments were run in the presence of 'H-CB or I4C-ConA only. The specific binding of Con A was determined by subtracting the amount bound in the presence of a-methylmannopyranoside (final concentration, 0.1 M) from the total amount bound. Specific binding of CB was calculated by subtracting nonspecific binding in the presence of 100 mM 3-0-MeG from total binding. After the binding assay, the cells were washed three times with phosphate buffer, pH 7.4, and dissolved in 0.5 ml 0.01 N NaOH before being added to 3 ml scintillation fluid (ACS; Amersham Searle). The radioactivity of each sample was counted in a liquid scintillation spectrometer (Intertechnique SL30). Results and discussion Con-A effects (3 pg/ml) were studied on 3-0-MeG uptake in both 8- and 16day-embryo fibroblasts (Fig. 1). In 16-dayembryo fibroblasts, Con A raised the V, of the uptake system by 70% (from 10nM/min per 108 cells to 17 nM/min per lo6 cells), while in 8-day-embryo fibroblasts lectin reduced the V,,, by 25% (from 2.8nMImin per lo6 cells to 2.1 nM/min per lo6 cells). Neither in 8- or Iddayembryo fibroblasts, in the presence or absence of Con A, were there significant differences in the K, (1 mM) of the 3-0-MeG uptake system. The effects induced by Con-A treatment could have resulted from a modification of either the accessibility and/or the mobility of the hexose carriers. The carrier number might be modified by masking or unmasking due to direct or indirect interaction(s) of Con A with carrier molecules [19, 251. The number of accessible carriers was measured by CB binding [4]. Table 1 shows that a final 3 p g / d concentration of Con A reduced specific CB binding by 56% in both 8- and 16day-embryo fibroblasts. Eightday-embryo fibroblasts bound 1.1 pmol/106 cells, of CB and Con A reduced this specific binding to 0.5 pmol:106 cells. In iddayembryo fibroblasts, 1.8 pmol/106 cells of CB were bound, and here. Con A also reduced specific binding by 56% (0.8 pmol/ lo6 cells). At saturation (40 pg/ml), Con A masked about 70% of the hexose carriers in both 8- and 16-day-embryo fibroblasts (Table 1). These results were not altered by higher (up to 180 pg/ml) lectin concentration. Therefore, the differential lectin effect on 3-0-MeG uptake was not due, at least directly, to the alteration of carrier accessibility. It might also have been possible that hexose-carrier mobility was modified by Con A, which is known to alter the distribution of cell surface components [q.So, in 16day-embryo fibroblasts. Con A might increase hexose-carrier mobility and thus raise hexose uptake. Conversely, in 8-day-embryo fibroblasts. Con A might gel the membrane architecture, thereby reducing hexose-carrier mobility and, consequently, hexose uptake. The change observed in the carrier mobility involved in hexose uptake and due to Con-A interaction with embryo cell surfaces might be attributed to the fact

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Fig. 1. Lineweaver-Burk plots of specific 3-0-MeG uptake in cultured fibroblasts derived from 8- ( 0 )and 16-day (A) chick embryos in the absence Vdl lines) or presence (dotted lines) of 3 pg/ml Con A. Cells were incubated with Con A for 30 min at 37" C and then processed as described in Methods. Labelled 3-0-MeG concentrations ranged from 30 to 5.000 pM.Each point is the average of three separate experiments, and SE does not exceed 10% Table 1. Assay of competition between Con A and CB for binding to cultured fibroblasts from 8- and 16-day chick embryos

Binding of labelled Con A Without CB incubation With CB incubation Binding of labelled CB Without Con A incubation With Con A incubation 3 pg 40 Fg

8-Dayembryo fibroblasts (pmol/ 106 cells)

1&Dayembryo fibroblasts (Pmol/ 106 cells)

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34 33

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Embryo cells were first incubated with unlabelled C o n A or CB and then with labelled CB or Con A. For the binding assay, cells were processed as described in Methods. Each value is the mean of the values from two separate experiments run in duplicate, and SE does not exceed 10%

that the fluid character of the membrane facilitates the redistribution of cell surface components [18,25]. The latter possibility is supported by our previous findings that the membrane became more rigid as the embryo ages [17], and the re-arrangement of cell surface components due to Con A is more important in 16-day than in 8-day-embryo fibroblasts [5]. Moreover, it has been observed that many glycoproteins, especially those of the embryonic plasma membrane, are capable of rapid topographical re-arrangement [ l l , 14, 15, 19, 27, 291, and such a re-arrangement is modified by lectins like Con A [19]. Figures 2 and 3 show that the effects of Con A increased with duration of contact with the cells and lectin concentration up to plateau levels which corresponded to the binding of the lectin (30 min, 40 pg/ml [5, 241). These effects on hexose uptake were inhibited by 95% in the presence of 0.1 M a-methylmannopyranoside. The change in 3-0-MeG uptake induced by Con A in 8- and 16-day-embryo fibroblasts thus appears to be a result of lectin attachment to the membrane.

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Fig. 2. EFFcct of Con A (3 pg/ml) IS a function of the time of contact between lectin and the cells. Cultured fibroblasts derived from 8- ( 0 ) and 16-day (0)chick embryos were incubated with Con A at 37" C for 10, 20, 30 or 60 min. After washing. 3-0-MeG uptake was measured at 21°C for 1 min. as described in Methods. Each value is the average of the values obtained from four separate experiments, and SE does not exceed 10%. Results are expressed as the percentage of inhibition (-) or stimulation (+) compared to the control value which is noted as 0

Fig. 3. Effect of Con A on 3-0-MeG uptake as function of the lectin concentration in cultured fibroblasts derived from 8- ( 0 ) and 16-day (0)chick embryos. Cells were incubated for 30 min at 37" C with various concentrations of Con A and washed as described in Methods. 3-0-MeG uptake was then measured at 21" C for 1 min. Each point is the average of values obtained from four separate experiments and SE does not excced 10%. Results are expressed as the percentage of inhibition (-) or stimulation (+) compared to the control value which is noted as 0

Time

[h]

Fig. 4. Effect of Con A on total 3-0-MeG uptake and cell growth in cultured fibroblasts derived from 8- and 16day chick embryos. Cells were incubated with 3 pg/ml Con A for various times and processed as described in Methods. Each value is the mean of the values from three separate experiments, and SE does not exceed 15%. Rcsults are expressed as the percentage of inhibition (-) o r stimulation (+) compared to the control value which is noted as 0.Black syuure, 16-day-embryo fibroblasts; white square. S-dayembryo fibroblasts

The effects brought about by C o n A depended on its interaction with the membrane [7], and the consequences of this interaction led either to a general stimulation of cell anabolism in 16-day-embryo fibroblasts or the inhibition of such anabolism in 8-day-embryo fibroblasts [l, 241, as has been reported for other cells (for a review, see [30]).

Here we demonstrated that, after 48 and 72 h culture in the presence of lectin. Con A modified hexose uptake. Thus, in 8-day-embryo fibroblasts. Con A inhibited hexose uptake by 39% after 24 h culture in the presence of lectin and by 29% after 48 h culture. Con A also inhibited the growth of 8-day-embryo fibroblasts (Fig. 4). In 16-day-embryo fibroblasts, it only slightly increased hexose uptake : 13% after 24 h culture. This rise was more marked with time since, after 48 h culture, Con A enhanced hexose uptake by 24% (Fig. 4). In 16-day-embryo fibroblasts, it stimulated cell growth as shown in Fig. 4. Therefore, the differential effects induced by Con A on both 3-0-MeG uptake and cell proliferation were parallel to each other according to duration. Our results for 16-day-embryo fibroblasts are comparable to those concerning the effects of phytohemagglutinin on lymphocytes [20, 211 and brown fat cells [lo]. All these findings show that the lectin-induced increase in hexose uptake that accompanies the transition from a resting or slow rate of proliferation to one of enhanced activity might be a general phenomenon, as has been observed under hormone-stimulated conditions in fat cells [8] and hepatocytes [12], and during albumin treatment of thymocytes [23]. In 8-day-embryo fibroblasts, the decline in hexose uptake observed here has not, to our knowledge, been described for other cell types. In conclusion, Con A might exert an effect on the cell membrane which leads to the modulation of hexose uptake and, consequently, the modification of cell growth. Acknowledgements. We are indebted to Dr. C. Rosenfeld for several useful discussions regarding the manuscript. We wish to thank Pr. Y. Goussault for his helpful advice and criticism, and we also thank A. de Cadoret for typing thc manuscript. This work was supported by grants from INSERM. Contrat de Recherche Libre 78-1-131-1, the UER Biomedicale des Saints-P6res and the Laboratoires Metabio (Egic. France). M.A. is Maitre de Recherches INSERM.

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Received December 1983 / Accepted in revised form April 1984