Carbohydrate determinants involved in both the binding and action of insulin in rat adipocytes

Molecular and Cellular Endocrinology, 28 (1982) 627-643 Elsevier Scientific Publishers Ireland, Ltd.

627

CARBOHYDRATE DETERMINANTS INVOLVED IN BOTH THE BINDING AND ACTION OF INSULIN IN BAT ADIPOCVTES

Gisele CHERQUI, Martine CARON, Jacqueline CAPEAU and Jacques PICARD * Luboratoire de Biochimie, INSERM U-181, Fact& 27 rue Chaligny, 75571 Paris Cidex I2 (France) Received

29 March

1982; revision received

de Mhdecine Saint-Antoine,

9 July 1982; accepted

6 August

1982

The insulin receptor apparent affinity was markedly decreased in fat cells treated with lectins specific either for D-@aCtOSe (Ricinus communis agglutinin I, RCA,), D-mannOSe (concanavalin A, Con A, Lens culinaris agglutinin, LCA) or N-acetyl-D-ghrcosamine (wheat germ agglutinin, WGA), as indicated by a rightward shift of the binding competition curves and almost lineared Scatchard plots. Limulus polyphemus agglutinin (LPA), specific for sialic acid, was ineffective. All lectins enhanced 2-deoxy-D-glucose uptake with relative bioactivities (maximal lectin effect/maximal insulin effect) of 68-868. Insulin and lectin stimulatory effects were antagonized by specific carbohydrates used as competitors and inhibited by cytochalasin B (70 pM). Maximal effects of insulin and lectins were not additive and were completely abolished in neuraminidase-treated fat cells. Lectins did not affect insulin degradation. These data show that sialylated glycosidic moieties containing D-galactose, D-mannose and N-acetyl-D-ghmosamine units are involved in both processes of insulin ‘high affinity’ binding and activation of glucose transport but are not implicated in hormone degradation. They suggest that N-linked carbohydrate chains of the complex type may be essential for functional insulin receptor and post-receptor systems. Keywords: insulin receptor affinity; glycoproteins; rat adipocytes.

glucose

transport;

insulin

degradation;

lectins;

There is considerable evidence to indicate that the insulin receptor located on the plasma membrane of target cells is an integral membrane glycoprotein: cells or membranes pretreated with glycosidases or lectins showed impairment of insulin binding (Cuatrecasas, 197 1, 1973; Caron et al., 1978; Capeau et al., 1978; Sandra et al., 1979; Capeau and Picard, 1980; Caron and Picard, 1981); Triton X- 100 soluble receptors were retained on Sepharose-immobilized lectin columns (Cuatrecasas and Tell,

* To whom all correspondence

should be addressed.

0303-7207/82/0000-OOOOooo/%O2.75

0 Elsevier Scientific

Publishers

Ireland,

Ltd.

628

G. Cherqui et al.

1973; Maturo and Hollenberg, 1978; Harrison and Itin, 1980); tunicamycin, an antibiotic that blocks glycosylation of N-linked glycoproteins, inhibited synthesis of functional insulin receptors (Rosen et al., 1979; Kohno et al., 1980; Reed et al., 1981; Ronnett and Lane, 1981); purified insulin receptor subunits showed altered electrophoretic mobilities after neuraminidase digestion (Jacobs et al., 1980a, b). Moverover, in a recent study designed to characterize further the glycosidic moiety of the insulin receptor in rat adipocytes, we identified D-galactose, D-mannose and N-acetyl-D-glucosamine units as carbohydrate determinants of the insulin receptor macromolecule; these findings suggested involvement of N-linked oligosaccharide side-chains of the complex type in the insulin-receptor interaction (Cherqui et al., 1981). Moreover, Hedo et al. (1981a) directly demonstrated that both major insulin receptor subunits were glycosylated and contained carbohydrate chains of the complex N-linked type. The present study was designed to gain further insight into the participation of the identified carbohydrates in the insulin-receptor interaction and to investigate their involvement in the action and degradation of insulin. With this aim, we studied in isolated rat adipocytes the effects of various lectins upon (i) insulin binding characteristics as estimated by inhibition competition curves and Scatchard plots, (ii) glucose transport as measured by 2-deoxy-D-glucose uptake and (iii) insulin degradation as determined by TCA precipitation. Our data indicate that sialylated glycosidic moieties containing Dgalactose, D-mannose and N-acetyl-D-glucosamine units are involved in both processes of insulin ‘high affinity’ binding and activation of glucose transport but are not implicated in the degradation of the hormone. These results stress the biological significance of cell surface carbohydrate units for functional insulin receptors and suggest that N-linked oligosaccharide side-chains of the complex type play a key role at both the insulin receptor and post-receptor levels in adipocytes.

MATERIALS

AND

METHODS

Materials Clostridium histolyticum collagenase (EC 3.4.24.3), type I (158 U/mg), was obtained from Worthington; Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18) (0.5 U/mg) from Boehringer Mannheim. Concanavalin A (Concanavalia ensiformis), Lens culinaris agglutinin (lentil seeds), Limulus polyphemus agglutinin (haemolymph), Ricinus communis agglutinin I (castor beans), wheat germ agglutinin (Triticum vulgare) and cw-methylD-mannopyranoside were purchased from Reactifs IBF, Pharmindustrie.

Carbohydrate

involvement

in insulin

binding

and action

629

D-Galactose, N-acetyl-D-glucosamine, gangliosides from bovine brain, type III, &deoxy-D-glucose, cytochalasin B and fatty-acid-free bovine serum albumin were from Sigma; dinonylphthalate from Merck Darminsulin (100 uCi/pg), 2-deoxy-D-[ l-‘4C]glustadt; ‘251-monoiodinated case (45-55 mCi/mmole) and [3H]inulin (265.9 mCi/g) from New England Nuclear; porcine insulin (25 U/mg) from Novo Research Institute. Lectin specificities (Goldstein and Hayes, 1978) Concanavalin A (Con A), Lens culinaris agglutinin (LCA): terminal or internal a-D-mannose, a-D-glucose. Limuluspolyphemus agglutinin (LPA): N-acetylneuraminic acid. Ricinus communis agglutinin I (RCA,): terminal or internal D-galactose. Soybean agglutinin (SBA): terminal cw-Nacetyl-D-galactosamine. Ufex europeus (UEA-F): terminal cY-r_-fucose. Wheat germ agglutinin (WGA): terminal or internal N-acetyl-D-glucosamine. Preparation of isolated adipocytes Adipocytes were prepared by a modification of the method of Rodbell (1964) as previously described (Cherqui et al., 1981). The cell number was estimated by counting an aliquot of the cell suspension on a haemocytometer. Fat-cell viability was assessed by the trypan blue exclusion test. ‘251-labelled insulin binding studies Fat cells (lo5 cells/ml) were suspended in Krebs-Ringer phosphate buffer, 1% bovine serum albumin (pH 7.6) and incubated in plastic test-tubes at 22°C with ‘25-labelled insulin (0.6 ng/ml) and O-l x lo5 ng/ml of native insulin. The incubations were terminated after 30 min (steady-state binding equilibrium) by removing aliquots (300 ~1) from the cell suspension and rapidly centrifuging the cells in plastic microtubes through 100 ul dinonylphthalate (Gammeltoft and Gliemann, 1973). Data are reported as specific binding obtained by subtracting the amount of radioactivity that remained bound to the cells in the presence of 1 X lo5 rig/n’’ 1:” dnlabelled insulin. The non-specific binding in all experiments v 9s about 15% of the total binding. All binding studies were done in quadruplicate. ‘251-labelled insulin degradation studies The amount of ‘251-labelled insulin (0.6 ng/ml) degraded in the medium in the presence of O-l X lo5 ng/ml of unlabelled insulin was measured by transferring aliquots (100 ~1) of separated cell-free incuba-

630

G. Cherqui et a/.

tion medium in 650 l.~l of chilled Krebs-Ringer phosphate buffer, 1% bovine serum albumin (pH 7.6). 750 ~1 of 10% trichloroacetic acid (TCA) were immediately added and the tubes centrifuged for 5 min at 3500 ‘pm. The radioactivity was then counted in the precipitate and supernatant. The percentage of insulin which is intact according to this method is compared to that of ‘251-labelled insulin which had been incubated in the absence of adipocytes. The percentage of ‘251-labelled insulin which remains intact was then determined by the following formula: ‘%intact of incubated CR,intact of unexposed In all experiments hormone.

insulin insulin

there

x loo

was less than

4% degradation

of the control

Effect of lectins on ‘2sI-labelIed insulin binding and degradation Fat cells (lo5 cells/ml) were incubated with the indicated concentrations of lectins in Krebs-Ringer phosphate buffer, 1% bovine serum albumin (pH 7.6), for 45 min at 22°C in the absence or presence of 50 mM of the specific saccharide competitors. ‘251-Labelled insulin binding and degradation were then determined as indicated above. Estimation of the agglutination of fat cells by lectins was monitored as previously described (Cherqui et al., 1981). 2-Deoxy-D-[I-‘4CJglucose transport studies Transport studies were performed with the same cell centrifugation technique as described for binding studies. Isolated adipocytes (lo5 cells/ml) were incubated at 37°C with 0.25 mM %deoxy-D-1[ 1-‘4C]glucase (0.2 uCi/tube) in Krebs-Ringer phosphate buffer, pH 7.4, containing fatty-acid-free bovine serum albumin (40 mg/ml). The assay is terminated at the end of 2 min incubation by transferring 300 ul aliquots from the assay mixture to plastic microtubes containing 100 ~1 aliquots of dinonylphthalate. The tubes are centrifuged for 30 set in a Beckman microfuge and the assay is considered terminated at the beginning of centrifugation. The microfuge was cooled, sliced at the viscous oil layer and the upper portion containing packed cells analysed for radioactivity in a vial containing 10 ml Biofluor (New England Nuclear). At least 60 min elapsed between the time the packed cells were added to vials and the time the vials were vortexed and their radioactive content (dpm) determined in a liquid scintillation counter (Intertechnque SL 4000). In experiments in which the effects of insulin, lectins, saccharide competitors and cytochalasin B were studied, the cells were preincubated with

Carbohydrate

involvement

in insulin binding and action

631

these agents for 30 min at 37°C either separately or simultaneously. The amount of sugar trapped in the extracellular fluid was measured using [ 3Hlinulin according to the method of Gliemann et al. (1972); this ranged from 2 to 10% of the total sugar uptake. All results were corrected for this factor. Neuraminidase treatment of fat cells As previously described (Cherqui et al., 1981), neuraminidase activity (from A. ureafaciens) was estimated by measuring the sialic acid released in the medium, using the procedure of Warren (1959). Preliminary assays monitoring the enzymatic digestion of fat cells in the presence or absence of 0.2 mM PMSF indicated that the enzyme preparation used in this study was devoid of proteinases. For current assays, fat cells were incubated with neuraminidase (50 mu/ml) for 20 min at 37°C; at the end of this period, an additional 30 min incubation at 37°C was performed in the absence or presence of insulin or lectins. 2-Deoxy-Dglucose uptake was then measured as previously described. Cell viability was verified in each experiment by the trypan blue exclusion test. Statistical analysis The statistical comparison of 2 individual group means was accomplished using Student’s t-test for non-paired data. Differences between groups resulting in P values < 0.05 were considered significant.

RESULTS Effect of lectin preincubation on insulin binding characteristics Binding data derived from steady-state equilibrium studies with control and lectin-treated fat cells were presented as inhibition competition curves (Fig. 1, upper panel) and Scatchard plots (Fig. 1, lower panel). The concentration of unlabelled insulin that produced half-maximal inhibition of ‘251-labelled insulin binding in steady-state competition experiments (INS,,,) was considered as an operational index of the receptor affinity (Clark et al., 1980). In control fat cells, the concentration of unlabelled insulin causing half-maximal inhibition of ‘251-labelled insulin binding (100 PM) was 3.3 k 0.2 nM (Table 1). Scatchard analysis of the binding data yielded a typical curvilinear plot (Fig. 1, lower panel). Fat cell preincubation with LPA (3 pg/ml) did not significantly modify either the apparent affinity (Fig. IA, upper panel, and Table 1) or the Scatchard plot, as compared to the control (Fig. lA, lower panel).

G. Cherqui et al.

632

INSULINCONCENTRATION hIEm)

INSULIN

BOUND

(f~mtomol~./t05e~ll.)

Fig. 1. Upper panel: Effect of lectin preincubation on competition inhibition curves of ‘251-labelled insulin binding to rat adipocytes. Fat cells (10’ cells/ml) were incubated for 45 min at 22°C in the absence or presence of LPA (3 ug/ml), Con A, LCA, RCA, or WGA (30 &ml). Binding assays with ‘251-labelled insulin (100 PM) in the presence of 0- 1 X 10s ng/ml unlabelled insulin were then conducted as described in Materials and Methods. Specifically bound t2SI-labelled insulin is expressed as a percentage of the initial maximal amount bound which was 0.744 f 0.083, 0.891 + 0.014, 0.297 + 0.030, 0.318 f 0.018, 0.209 f 0.021 and 0.338 f 0.040 fmoles/105 cells for the control, LPA-, Con A-, LCA-, WGAand RCA,-treated fat cells, respectively. Results were corrected for non-specific binding which was estimated as that tracer amount of ‘251-labelled insulin bound in the presence of 1 x 10’ ng/ml unlabelled insulin. Data represent the mean f SE of 16 (control), 8 (Con A, WGA), 5 (RCA,) or 3 (LPA, LCA) separate experiments performed in quadruplicate. Lower panel: Scatchard analyses of the results shown in the upper panel. For clarity, mean standard errors were not reported.

In contrast, preincubation of fat cells with Con A, LCA, RCA, or WGA (30 pg/ml) resulted in consistent rightward shifts of the binding competition curves (Fig. lB, C, upper panel) and increased the concentration of unlabelled insulin causing half-maximal inhibition of ‘251-labelled insulin binding by 1l-, 16 and 25-fold, respectively (Table 1). Corresponding Scatchard plots were almost linearized, indicating an important decrease

Carbohydrate

Table

involvemenr in insulin binding and action

633

1

Effect of lectin preincubation Treatment

None WA (3 pg/ml) Con A (30 gg/ml) LCA (30 ug/mQ RCA, (30 pg/ml) WCA (30 rg/d)

on the insulin receptor Receptor apparent

affinity

(INS,,,,

nW

3.3 4.1 31.3 37.3 52.1 83.0

f f f + f f

0.2 0.2 2.5 1.8 4.2 1.2

’

apparent

affinity

Number of experiments 16 3 8 3 5 8

in rat adipocytes P

< < c e

0.001 0.001 0.001 0.001

Values for the apparent affinity were determined from the data illustrated in Fig. 1 (upper panel). Differences between the mean values were evaluated by Student’s r-test. P values > 0.05 were considered non-significant. ’ Unlabelled insulin concentration required for half-maximal inhibition of ‘25i-labelled insulin binding in steady-state competition experiments.

of the insulin receptor binding affinity at low occupancy (Fig. lB, C, lower panel). Insulin-like biological actioities of Iectins on fat cells Table 2 shows the effects of insulin and lectins on Zdeoxy-D-[ l“C]glucose uptake. Insulin increased 2-deoxy-D-glucose uptake in a dose-dependent manner; maximal stimulation (268.0 f 23.4% of the control, P < 0.001) was observed at a concentration of 2 ng/ml and no further significant increase could be detected using 7.5 ng/ml. o+MethylD-mannopyranoside (100 mM) and N-acetyl-D-glucosamine (50 mM) decreased the insulin maximal effect by 20 and 43% respectively (Table 2). LPA, Con A, LCA, WGA and RCA, exhibited marked insulin-like activities. Maximal stimulatory effects were observed either at 20, 75 or 100 pg/rnl for LPA, WGA or Con A, LCA, RCA, respectively. At higher lectin concentrations (40 pg/ml for LPA or 250 ug/ml for the other lectins) no further significant increases in the stimulation of 2-deoxy-D-glucose uptake could be detected (not shown). Table 2 indicates that, as compared to insulin, LPA, Con A, LCA, WGA and RCA, showed relative bioactivities (maximal lectin effect/maximal insulin effect) of 81, 86, 68, 73 and 798, respectively. In the presence of their respective specific saccharide competitors, LPA, Con A, LCA and WGA did not significantly modify Zdeoxy-Dglucose uptake, indicating the specificity of the lectin insulin-like effects.

+ bovine brain gangliosides Con A ( ug/ml) 10.00 50.00 100.00

( u g/ml) 2.50 10.00 20.00 LPA (20 ug/ml)

( 10 mg/ml)

f 16.9 k 25.2

214.0 152.7

IPA

f + f f +

159.2 174.7 226.3 268.0 277.1

+ 3.3 + 10.5 k 20.2 f 15.0 f 6.4 + 11.4 f 9.5

141.2 179.8 224.0 75.0 153.0 197.5 237.0

3.9 9.7 18.4 23.4 28.0

+ 9.8

100.0

4 4 3

2

4

6 4

4 3

4 5 3 3 6

27

Number of experiments

in rat adipocytes

None Insulin (ng/ml) 0.50 1.00 1.50 2.00 7.50 Insulin (2 ng/ml) + cr-methyl-Dmannopyranoside (100 mM) + N-acetyl-D-glucosamine (50 mM)

uptake

2-Deoxy-D-[ 1-‘4C]glucose uptake (% of control + SE)

effects of lectins and insulin on 2-deoxy-D-[ l-‘4C]glucose

Treatment

Comparative

Table 2

c 0.05 c 0.001 < 0.001

-= 0.05 -= 0.01 < 0.001

< 0.05 -=z0.01 < 0.001 c 0.001 < 0.001

P

86

81

100

Relative bioactivity (% of insulin activity) a

E

(100 mM)

48.58+

110.5 171.5 219.7

86.0

123.7 153.9 201.9

8.0

f 9.5 f 8.5 +28.8

f 12.5

f 13.3 f 16.0 k26.9

4

2 3 6

2

3 4 4

2

3 4 3

2

< 0.05

< 0.05 < 0.001

< 0.05 < 0.001

< 0.05 < 0.01

79

73

68

Fat cells (IO5 cells/ml) were incubated for 30 min at 37°C with or without the indicated concentrations of insulin or lectins in the absence or presence of specific carbohydrates used as competitors. 2-Deoxy-D-[ I-t4Cjglucose (0.25 mM) was then added and the uptake was measured at the end of a 2 min incubation as described in Materials and Methods. All data were corrected for sugar trapped in the extracellular water space determined with [3H]inulin. The amount of 2-deoxy-D-glucose transported for the control was 67.1+-6.6 pmoles/min/105 cells. Each experiment was performed in quadruplicate. Differences between the mean values were evaluated by Student’s f-test. P values > 0.05 were considered non-significant. ’ Values are recorded relative to the effect of insulin at 7.5 ng/ml.

RCA, (100 rg/mU + n-galactose (100 mM)

RCA, (gg/nQ 10.00 20.00 100.00

WGA (75 ug/mU + N-acetyl-n-glucosamine

(100 mM)

119.31 f 15.0

WGA ( ug/mU 10.00 25.00 75.00

f 17.7 f 15.9 f 19.5

+ 12.5

LCA (100 cg/rN + a-methyl-n-mannopyranoside

101.3 148.6 159.5 188.0

(100 mM)

LCA (ug/mU 10.00 25.00 100.00

Con A (100 pg/ml) + a-methyl-D-marmopyranoside

636

G. Cherqui et al.

Exceptionally, in the simultaneous presence of RCA, (100 FL/ml) and D-galactose (100 mM), 2-deoxy-D-glucose transport was markedly decreased (Table 2): the competitive inhibition of D-glucose uptake by D-galactose probably accounted for this finding (Katzen, 1979). To determine the combined effects of lectins and insulin on 2-deoxyD-glucose uptake, mixing experiments were conducted in the simultaneous presence of lectins and insulin at maximally effective concentrations. In neither case were insulin and lectin effects additive: LPA (20 l.tg/ml), Con A, RCA, (100 pg/ml) and WGA (75 Pg/ml) could not enhance 2-deoxy-D-glucose uptake beyond the maximal effect of insulin (not shown). Effect of cytochalasin B on insulin- and lectin-stimulated 2-deoxy-D-glucose uptake In the presence of a 70 FM concentration of cytochalasin B, which completely inhibits glucose transport in isolated fat cells (Foley et al.,

LPI ww

(27)

(3)

(4)

Con A

LCA

1Wd

loqo/d

(31

(3)

(6)

Fig. 2. Effect of cytochalasin B (70 PM) on the basal and insulin- or &tin-stimulated 2-deoxy-D-[1-t4C]ghrcose uptake in rat adipocytes. Fat cells (10’ cells/ml) were incubated for 30 mm at 37T with or without the indicated concentrations of insulin or lectins either in the absence or presence of cytochalasin B (70 PM). 2-Deoxy-D-[l-‘4C]glucose uptake (0.25 mM) was then measured after a 2 min incubation at 37°C as described in Materials and Methods. Alf data were correctedfor sugar trapped in the extracellular water space using [3H]inulin. Numbers in parentheses indicate the number of separate experiments. Each experiment was performed in quadruplicate.

(50 mu/ml)

treatment

42.3 f 12.8 57.5* 7.5

Control

178.8 f 20.0 57.0* 9.1 P
Insulin (7.5 ng/ml)

uptake

130.5 f 16.7 51.7f 10.2 P < 0.02 143.3 If: 13.0 55.7* 4.5 P <: 0.01

Con A (100 ug/ml)

by insulin

LPA

uptake

(20 ug/ml)

of 2-deoxy-D-[ I-‘4C]glucose

Z-DCCOXY-D-[ l- 14C]glucose (pmoles/min/lOs cells)

on the stimulation

118.9& 13.4 50.1 f 6.0 P < 0.01

(75 Irg/ml)

WGA

and lectins in isolated

(

132.1 f 17.1 51.31f: 8.2 P <. 0.02

(100 I*g/ml)

RCA

rat adipocytes

Isolated fat cells were incubated at 37°C with or without neuraminidase from Arrhrobucter ureafuciens (50 mu/ml). After 20 min, fat cells were incubated at 37OC for an additional 30 min in the absence or presence of insulin or leetins at the indicated concentrations. At this point, 2-deoxy-o-[ l-‘4C]glucose was added at a final concentration of 0.25 mM and the uptake was measured at the end of a 2 mm incubation as described in Materials and Methods. All data were corrected for sugar trapped in the extracellular water space as determined with [ ‘Hlinulin. Data represent the mean *SE of 3 separate experiments performed in quadruphcate. Differences between the mean values were evaluated by Student’s r-test. P values > 0.05 were considered non-significant.

None Neuraminidase

Treatment

Effect of neuraminidase

Table 3

ft: 3 El L f? P 9

0” 5

3’ z -2

<.

638

G. Cherqui et al.

1978) lectins, like insulin, did not significantly modify Zdeoxy-D-glucose uptake as compared to the control, indicating that increased diffusion did not account for their effects (Fig. 2). Effect of neuraminidase on insulin- and lectin-stimulated 2-deoxy-D-glucose uptake In an attempt to gain further insight into the insulin-like activities of lectins, the effect of neuraminidase treatment of fat cells was comparatively studied on basal or insulin- and lectin-stimulated 2-deoxy-D-glucase uptake. As shown in Table 3, basal Zdeoxy-D-glucose uptake was similar in control and neuraminidase-treated fat cells, indicating that neuraminidase digestion did not damage the glucose transport system. In contrast, neuraminidase treatment of fat cells dramatically affected stimulation of glucose transport by insulin or lectins. Insulin, LPA, Con A, WGA and RCA,, at maximally effective concentrations, were no longer able to enhance 2-deoxy-D-glucose uptake over the basal level in neuraminidase-treated fat cells. Effect of lectin preincubation on insulin degradation To examine whether carbohydrate residues were involved in the degradation of insulin, we comparatively studied in control and lectintreated fat cells the degradation of a 100 pM ‘251-labelled insulin concentration in the presence of O-l X lo5 ng/ml unlabelled insulin. These studies, which were conducted under the same conditions as insulin binding, indicated that none of the lectins tested (LPA, Con A, WGA and RCA,) appreciably modified the insulin degradation in fat cells (not shown).

DISCUSSION Specific lectins were used (i) to gain further insight into the participation of sialic acid, D-galactose, D-mannose and N-acetyl-D-glucosamine units in the insulin-receptor interaction and (ii) to investigate the involvement of these carbohydrate components in the action and degradation of insulin. Methodological problems concerning the choice of appropriate conditions for fat-cell isolation, enzymatic digestion or lectin treatment have been examined in detail elsewhere (Cherqui et al., 198 1). Measurement of labelled 2-deoxy-D-glucose uptake was used to assess glucose transport. Two arguments indicate that, under our conditions, true unidirectional flux was measured: (i) preliminary assays showed that uptake of 0.25

Carbohydrate

involvement

in insulin binding and action

639

mM of labelled 2-deoxy-D-glucose was linear for at least 5 min, and this largely exceeded the 2 min incubation used in the present study; (ii) it was previously reported (Olefsky and Saekow, 1978) that even at a 10 mM extracellular concentration of Zdeoxy-D-glucose in the presence of 25 ng/ml insulin, hexokinase activity was not rate-limiting, thus implying that all the intracellular sugar was irreversibly trapped in the phosphorylated form. The insulin receptor apparent affinity of control fat cells (3.3 nM), which was reproducibly evaluated from inhibition competition curves, fell within the range of values reported by others (Gliemann et al., 1975; Clark et al., 1980; Cushman et al., 1981). Scatchard analysis of these data yielded a typical curvilinear plot which is consistent with various hypotheses (Kahn et al., 1974; De Meyts et al., 1976; Jacobs and Cuatrecasas, 1977; Pollet et al., 1977; Olefsky and Chang, 1978; Donner, 1980). Preincubation of fat cells with Con A, LCA, RCA, and WGA markedly affected the initial part of the Scatchard curvilinear plot without substantially modifying the second part (Fig. lB, C, lower panel). This finding was consistent with previous results reported for Con A (Caron et al., 1978; Herzberg et al., 1980; Sorge and Hilf, 198 1) and showed that lectin binding to cell surface glycosylated components chiefly altered the insulin ‘high affinity’ binding process while not affecting the ‘low affinity’ one nor presumably the total receptor-binding capacity. However, the mechanism involved remains unclear: from the above data it is not possible to ascertain whether the effects of lectins resulted from direct binding to carbohydrate determinants of the insulin-binding structures themselves or whether they reflected the lectin interaction with different glycosylated portions of the insulin receptor or even with extrareceptor structures. Such interaction might involve a modification of insulin binding through steric factors or conformational changes. In any case, the selective lectin-induced inhibition of insulin binding at low receptor occupancy pointed to the importance of N-linked glycosidic moieties in the ability of the insulin receptor to bind insulin with a high affinity. In support of this view, it was previously demonstrated that both major subunits of the insulin receptor contained N-linked glycoproteins of the complex type (Hedo et al., 1981a) and that a glycosylated non-recognition moiety of the receptor oligomer induced the appearance of a ‘high affinity’ component in the binding of insulin (Maturo and Hollenberg, 1978). The failure of LPA to modify steady-state equilibrium characteristics of insulin binding (Fig. 1A) could not be attributed to the low lectin concentration used (3 pg/ml) as compared with other lectins (30 pg/ml): indeed, the lectin concentration used in insulin-binding assays was usu-

640

G. Cherqui et al.

ally chosen in relation to the concentration range of their insulin-like activities (Table 2). Thus, this finding, in addition to the failure of neuraminidase to modify insulin binding (Cuatrecasas and Illiano, 197 l), indicated that terminal sialic acid residues are not directly involved in the insulin-receptor interaction although present in the insulin receptor glycosidic moiety (Jacobs et al., 1980a, b; Hedo et al., 1981a). In an attempt to investigate involvement of carbohydrate residues in the insulin activation of glucose transport, the lectin effects on 2-deoxyD-glucose uptake were evaluated in comparison with insulin. LPA, Con A, LCA, RCA, and WGA exhibited various degrees of insulin-like activities (Table 2). The fact that lectins mimicked the effect of insulin on glucose transport does not necessarily imply that they are interacting at the same site or in the same way as insulin. Insulin effects may be mimicked by antibodies to the insulin receptor whether or not these inhibit insulin binding (Kahn et al., 1977; Jacobs et al., 1978, 1980b) and also by anti-membrane antibodies which do not interact with the insulin receptor at all (Pillion et al., 1980). However, the present study emphasized several aspects of lectin insulin-like activities which, in the final analysis, provided support for the concept of carbohydrate involvement in insulin action: (i) A suggestive correlation was observed between the abilities of lectins (Con A, LCA, RCA,, WGA) to inhibit the binding of insulin and to mimic its effect on glucose transport. Soybean and UIex agglutinins (5-75 p-g/ml), which did not decrease insulin binding (Cherqui et al., 1981), did not enhance glucose transport (not shown). Similar observations were reported elsewhere (Katzen et al., 1981). (ii) Lectin effects were inhibited by a maximally effective concentration of cytochalasin B, showing that, like insulin, lectins act by stimulating a specific carrier-mediated glucose transport. (iii) Lectin and insulin maximal stimulatory effects were not additive, indicating a common pathway for both lectin and insulin biological activities. (iv) Lectin and insulin effects were both abolished by neuraminidase treatment of fat cells; this finding indicated that sialylated glycosidic moieties are implicated in lectin and insulin bioactivities and added further support to the concept of a common glycosylated effector system. (v) Simple sugar residues were able to antagonize both lectin and insulin effects, although to different extents. The fact that carbohydrates exhibited antagonistic effects on the insulin biological activity agreed with previous findings (Katzen, 1979) and implied that the interaction of insulin with cell surface glycosylated components is important for its action on glucose transport. Moreover, the involvement of N-linked

Carbohydrate

involvement

in insulin binding and a&ion

641

glycoproteins in post-receptor events was previously indicated by the findings of Rosen et al. (1979), who reported a profound loss of insulin responsiveness in tunicamycin-treated 3T3-Ll adipocytes and a decreased sensitivity of these cells to the insulin-like properties of an antireceptor antibody which did not compete for insulin binding. Thus, taking into account all these data, it can be envisaged that N-linked sialylated glycosidic moieties of the insulin receptor-effector system participate in the insulin activation of glucose transport in fat cells. The failure of LPA, Con A, RCA, and WGA to modify the insulin degradation indicates that sialic acid, D-galactose, D-mannose and Nacetyl-D-glucosamine residues were not implicated in the insulin degradative system. This finding, obtained by using a different experimental approach, provided further support to the hypothesis of a lack of direct correlation between the binding or action of insulin on the one hand and insulin degradation on the other (Freychet et al., 1972; Hammons and Jarett, 1980). In conclusion, the methodological approach developed in the present study, using specific lectins as probes of glycoprotein involvement in insulin receptor and post-receptor events, provided insight into the physiological importance of cell surface carbohydrate determinants in the insulin receptor function. Our data indicate that sialylated glycosidic moieties, containing D-galactose, D-mannose and N-acetyl-D-glucosamine units, are involved in both processes of insulin ‘high affinity’ binding and activation of glucose transport but are not implicated in hormone degradation. These results are consistent with the hypothesis that N-linked oligosaccharide side-chains of the complex type mediate essential functions at insulin receptor and post-receptor levels. Further experiments are required to indicate whether similar glycosylated structures might also be involved in insulin stimulation of different glucose metabolic pathways in fat cells.

ACKNOWLEDGEMENTS This work was supported by grants from the Institut National de la Sante et de la Recherche Medicale (CRAT 49.77.81 and CRL 77.1.191.7), and the Centre National de la Recherche Scientifique (ERA 691). We thank Olivier Lascols for helpful discussions, Christiane Horn (Melbourne, Australia) for revising the manuscript and Jocelyne Berton for secretarial assistance.

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