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Insulin-Like Activity of Concanavalin a and Wheat Germ Agglutinin-Direct

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Insulin-Like Activity of Concanavalin a and Wheat Germ Agglutinin-Direct Proc. Nat. Acad. Sci. USA Vol. 70, No. 2, pp. 485-489, February 1973 Insulin-Like Activity of Concanavalin A and Wheat Germ Agglutinin-Direct Interactions with Insulin Receptors (glucose transport/lipolysis/adenylate cyclase/affinity chromatography/lymphocyte transformation/growth factors) PEDRO CUATRECASAS AND GUY P. E. TELL Department of Medicine, and Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Communicated by Saul Roseman, December 7, 1972 ABSTRACT Concanavalin A and wheat germ agglu- mined by a modification (10) of the technique of Pohl et al. tinin are as effective as insulin in enhancing the rate of (11). Fat cell membranes (10) were freshly prepared for each glucose transport and in inhibiting epinephrine-stimu- lated lipolysis in isolated adipocytes. These lectins, also like experiment by homogenization (Polytron), centrifugation, and insulin, inhibit basal as well as epinephrine-stimulated suspension of the pellet in 50 mMI Tris - HCl (pH 7.6); aden- adenylate cyclase activity of membranes obtained from ylate cyclase assays were begun within 10 min. Concanavalin homogenates of fat cells. Low concentrations of wheat A (three-times crystallized) was from Miles. Wheat germ germ agglutinin enhance the specific binding of insulin to Dr. V. was by receptors of fat cells and liver membranes. Higher con- agglutinin, a gift from Marchesi, purified centrations of this plant lectin, as well as of concanavalin affinity chromatography and was homogeneous on Na A, competitively displace the binding of insulin to recep- dodecylsulfate disc gel electrophoresis (12). tors in these tissues. These effects are equally apparent Agarose derivatives of the lectins were prepared by the in insulin-binding proteins solubilized from membranes, CNBr procedure (13), or by reaction with activated N- indicating that the plant lectins interact directly with insulin receptors. All of the effects observed with the plant hydroxysuccinimide esters of diaminodipropylaminosuccinyl- lectins are reversed by simple sugars that bind specifically agarose (14). 40 ml of Sepharose 4B was activated with 6 g of to these plant proteins. Agarose derivatives of the plant CNBr and reacted with 60 ml of ice-cold 0.1 MI sodium phos- lectins effectively adsorb solubilized insulin-binding pro- phate buffer (pH 7.4) containing 500 mg of concanavalin A teins, and these can be eluted with buffers containing After 16 hr at 2 g specific simple sugars. The possible implications of these and 0.1 MI a-methyl-D-mannopyranoside. 40, findings to certain biological properties (mitogenicity) of of glycine was added and the incubation was continued for 8 these lectins and to the mechanism of action of other hr at 240. This adsorbent contained 5.5 mg of protein per ml growth-promoting substances are considered. of gel. Wheat germ agglutinin (1.4 mg//ml) was similarly coupled to activated agarose in the presence of 0.1 M N-acetyl- Concanavalin A and wheat germ agglutinin are plant proteins D-glucosamine; 1.1 mg of protein was coupled per ml of gel. that can bind to specific carbohydrate determinants on the '251-labeled plant lectins were used during the coupling surface of mammalian cells, and they can agglutinate various procedures. By phase-contrast microscopy, the lectin-beads normal and neoplastic animal cells. In addition concanavalin A became heavily coated with erythrocytes when these were can, by unknown mechanisms, stimulate mitosis and blasto- mixed at 240 for 1-2 hr. The cells could be rapidly desorbed genic transformation of lymphocytes (1), inhibit phagocytosis with 50 mM N-acetyl-D-glucosamine. by polymorphonuclear leukocytes (2), and prevent lympho- cyte cap and patch formation induced by anti-immunoglobulin RESULTS (3). In this report, we demonstrate that very low concen- Effects on glucose transport trations of these plant lectins have profound insulin-like effective effects on metabolic processes of isolated fat cells, and that Concanavalin A and wheat germ agglutinin are very in fat they can interact directly with the cell surface receptors for in enhancing the rate of [14C]glucose oxidation isolated to those that insulin in these cells. These observations may be of importance cells (Fig. 1). The maximal effects are similar in understanding the basis of the biological properties (e.g., can be achieved with insulin. The concentration required for A lymphocyte transformation) of these lectins, and they may the half-maximal effect is about 20 nM for concanavalin 4 wheat also provide a better understanding of the mechanism of action (molecular weight: 100,000) and about nMI for germ of insulin. agglutinin (molecular weight: 25,000). These effects result from interactions of the lectins with high-affinity binding sites METHODS that represent only a fraction of the total number of lectin- Isolated fat cells were prepared from 80-140 g Sprague- binding sites on the fat cells, since direct binding studies with Dawley rats (4). The procedures for the preparation of liver iodinated lectins demonstrate that saturation requires con- (5, 6) and fat cell membranes (7), and for measurement of the centrations greater than 0.1 mg/ml (manuscript in prepara- specific binding of [1251 linsulin to cell (8) and to membrane (7) tion). Fat cells do not agglutinate with either lectin under receptors have been described. Insulin receptor proteins were various conditions studied. solubilized from liver membranes with Triton X-100 (6). The increased rates of glucose oxidation induced by con- Glycerol in the incubation medium was determined by the canavalin A and by wheat germ agglutinin can be completely method of Ryley (9). Adenylate cyclase activity was deter- and selectively abolished by addition of the specific sugars 485 Downloaded by guest on September 29, 2021 486 Biochemistry: Cuatrecasas and Tell Proc. Nat. Acad. Sci. USA 70 (1973) TABLE 2. Combined effects of insulin, wheat germ agglutinin, and concanavalin A on glucose oxidation by isolated fat cells a. Production of 14CO2* + Insulin >13 W>=G (0.2 munit/ Addition No insulin ml) CONCANAVALIN A O None 4,100 ± 200 45,300 ± 600 0 Wheat germ agglutinin 0.15 Agg/ml 29,000 ± 400 45,300 ± 700 0 2.1 /ug/ml 39,100 ± 500 46,500 4± 700 10 ,sg/ml 44,700 i 700 45,900 i 600 Concanavalin A LECTIN CONCENTRATION, )Lg/mI 0.2 jutg/ml 9,100 ± 300 44,500 ± 700 FIG. 1. Effect of concanavalin A and of wheat germ ag- 2.4,Ag/ml 47,300 ±L 1,100 45,200 ± 900 glutinin (WGA) on the conversion of [14C]glucose to 14CO2 by 50 Mg/ml 49,100 ± 1,000 48,300 ±t 800 isolated fat cells. Fat cells were incubated for 2 hr at 370 in 1.25 ml of Krebs-Ringer bicarbonate buffer containing 1% *See Table 1. (w/v) albumin, and 0.2 mM [14C]glucose (6.4 Ci/mol). The maximal insulin (0.2 munit/ml) response is indicated by the insulin (Table 2). However, the combined effect of insulin arrow. and the plant lectins does not surpass the maximal effect that can be achieved by either agent alone. Furthermore, simul- a-methyl-D-mannopyranoside or N-acetyl-D-glucosamine taneous addition of both plant lectins in the absence or pres- (Table 1). Since, as will be described below, wheat germ ence of insulin does not result in greater effects than can be agglutinin at low concentrations increases the binding of achieved by any one of these three compounds. Tryptic iodoinsulin to fat cells, the possibility exists that the lectins digestion of fat cells, a process that decreases the apparent could be exerting their effect by facilitating effects of endog- affinity of insulin for its biological receptor (15), also results in enous insulin possibly retained on the surface of the fat cells. an apparent fall in the binding affinity of wheat germ agglu- The inability of a large excess of insulin antiserum to alter the tinin for the sites responsible for enhanced glucose transport metabolic effects of the cells in the presence or absence of the (unpublished results). The data suggest that the two plant lectins (Table 1) makes this possibility unlikely. lectins may be acting by a similar mechanism, and that this The effects on glucose oxidation of suboptimal concentra- mechanism may resemble that by which insulin normally tions of the lectins can be further increased by addition of exerts its effects. TABLE 1. Reversal by specific sugars of plant lectin Effects on lipolysis stimulation of glucose oxidation by fat cells Concanavalin A and wheat germ agglutinin are also as effective as insulin in reversing epinephrine-induced lipolysis in Production fat cells (Table 3). This antilipolytic property, which does not Additions of 14CO2* None 4,400 200 TABLE 3. Suppression by wheat germ agglutinin and Insulin (0.2 munits/ml) 34,200 I 1,900 concanavalin A of epinephrine-stimulated lipolysis in fat cells a-Methyl-D-mannopyranoside (25 mM) 4,500 400 N-Acetyl-D-glucosamine (25 mM) 4,800 300 Glycerol Insulin + a-methyl-D-mannopyrano- 32,200 1,100 Additions released* side + N-acetyl-D-glucosamine Concanavalin A (5 ,g/rml) 38,700 ±t 2,100 None 6.8 ± 0.5 Concanavalin A + a-methyl-D-manno- 4,600 500 Epinephrine, 0.16 jug/ml 24.0 ± 1.1 pyranoside Epinephrine, 0.16 ,ug/ml + Concanavalin A + N-acetyl-D-glucos- 36,800 800 Wheat germ agglutinin, 0. 1 ,ug/ml 20.5 ± 0.8 amine Wheat germ agglutinin, 0.33 ,sg/ml 14.0 ± 0.6 Wheat germ agglutinin (2 Mug/ml) 33,000 1,800 Wheat germ agglutinin, 1. 0 ,sg/ml 10.3 ±4 0.7 Wheat germ agglutinin + N-acetyl-D- 5,400 200 Wheat germ agglutinin, 5.0 gg/ml 6.4 ± 0.5 glucosamine Concanavalin A, 2,ug/ml 25.7 ± 1.4 Wheat germ agglutinin + a-methyl-D- 33,300 900 Concanavalin A, 6 ,ug/ml 20.1 ± 0.8 mannopyranoside Concanavalin A, 30,jg/ml 10.9 i 0.7 Insulin antiserum (1:250 dilution) 4,300 ±t 300 Concanavalin A, 100 ,g/ml 8.8 ± 0.9 Insulin + insulin antiserum 4,200 300 Insulin, 20 Munits/ml 6.3 ± 0.5 Concanavalin A + insulin antiserum 38,500 2,200 Insulin, 5 Muunits/ml 12.9 ± 0.6 Wheat germ agglutinin + insulin anti- 34,100 2,000 serum Fat cells (3 X 104 cells per ml) were incubated for 2 hr at 370 in Krebs-Ringer bicarbonate buffer containing 3% (w/v) The conditions are as described in Fig.
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