Clinical Endocrinology (1 984) 20, 23-30

HUMAN ERYTHROCYTE INSULIN RECEPTORS IN NORMAL MALE AND FEMALE SUBJECTS

B. L. WAJCHENBERG, A. C. LERARIO, W. EL-ANDERE, L. Y. OHNUMA AND I . T. TOLEDO E SOUZA

The Diabetes and Adrenal Unit, Hospital das Clinicas and Institute of Energetic and Nuclear Research, SCo Paulo, Brazil

(Received I March 1983; reuised 2 June 1983; uccepied 6 June 1983)

SUMMARY

Human red blood cells (RBC) have been shown to have highly specific insulin receptors. We have studied the binding characteristics of insulin to these receptors in erythrocytes from normal male and female subjects on their usual diet and physical activity. There were no significant differences in insulin binding in erythrocytes from females between the two phases of the menstrual cycle. However, the receptor concentration was higher in the 2nd half of the cycle accompanied by a reduction in affinity.

Binding curves of 125-I-insulin to RBC from males were higher than females in either phases of the menstrual cycle, primarily due to an increase in receptor concentration when compared to females in the follicular phase and mediated by an increased affinity at low receptor occupancy when compared to females in the luteal phase.

We speculated that the differences in the binding characteristics of 'Z51-insulin to RBC insulin receptors are mediated by differences in the levels of sex hormones.

Human erythrocytes (RBC) have recently been shown to have highly specific insulin receptors (Gambhir et al., 1977, 1978). At least three studies in different clinical situations have demonstrated a close correlation between changes in 1251-insulin-binding to RBC and monocytes (Bertoli et al., 1980; Pedersen et a1.,1980; Dons et al., 1981). Thus, it is clear that the RBC as well as monocyte (Bar et al., 1978) can be used to detect changes in insulin binding in body tissues relevant to glucose homeostasis. However, this does not imply that changes in metabolism, readily detectable in monocytes, are also reflected in RBC (Dons et al., 1981).

In this report we present data on the interaction of insulin with its specific receptors on RBC from normal males and from females studied in both phases of menstrual cycle since reports of changes in binding during the menstrual cycle in females are controversial.

Correspondence: Bernard0 Leo Wajchenberg, Diabetes and Adrenal Unit, Hospital das Clinicas, SBo Paulo, Brazil.

23

24 B. L. Wajchenberg et al.

MATERIAL A N D METHODS

We have studied 12 normal adult females (age 29.4k5.5 years; height 159.4k6.5 cm; weight 54.3 f 6.7 kg: mean f SD), Table 1, and 12 normal males (age 27.6 f 3.8 years; height 173 5.7 cm; weight 67.6f 8.5 kg). The composition of the diets was similar for all subjects (30% fat, 50% carbohydrate and 207; protein) with an approximate total caloric intake of 2800 kcal/d for the males and 2200 kcal/d for females. Body weights were within the normal range (within 10% of ‘ideal’ body weight) according to the Metropolitan Life Insurance Company tables. All subjects appeared to undertake equivalent degrees of physical activity.

Blood samples were collected early in the morning after a 12 h fast. All the females had normal menstrual cycles, confirmed by appropriate hormonal changes and cycle lengths varying from 27 to 30 d (Table I) . Blood was collected in both the first and second halves of the cycles, corresponding to the mid follicular and mid-luteal phases respectively.

Ten ml of venous blood was drawn into heparinized tubes. After centrifugation and separation of plasma (used to measure immunoreactive insulin; Higa ef a/ . , 1974) the remaining cells were diluted 1 : 1 with an aqueous solution of sodium chloride (0.1 54 molil). For the insulin measurements, intra-assay variation was 7.4% and interassay variation was 7.6%. Sensitivity was found to be 1.25 pU/ml (Higa et al., 1974). Separation of RBC from the circulating cells was done according to the method of Boyum (1968).

The label for binding studies was prepared by iodinating monocomponent porcine insulin with lZ51 using HzOz/lactoperoxidase at neutral pH followed by anion-exchange chromatography (J~rrgensen & Larsen, 1980), and was provided by NOVO Research Institute. The RBC were re-suspended to a final concentration of 4.5 x lo9 erythrocy- tes/ml in HEPES-Tris buffer (pH 8.0) incubated with 100 pg/ml of the ~ O ~ O - ~ ~ ~ I O - do-(Tyrosine A 14)-insulin and unlabelled porcine insulin over a range of concentration from approximately 0.1 to lo5 ng/ml for 90 min at 15°C in a final volume of 0.5 ml. After incubation, 200 p1 of the suspension was aliquoted into prechilled microcentrifuge tubes containing 200 pl of assay buffer and 200 p1 of dibutyl phthalate, which were then centrifuged in a Beckman microcentrifuge B for 2.5 min. After centrifugation, the supernate was aspirated and discarded (free radioactivity). The cell pellet in the tip of microfuge tube was excised and counted in a gamma spectrometer (bound radioactivity). “on-specific binding’ was defined by the presence of a constant radioactivity in the cell pellet, despite further increases of unlabelled insulin. To obtain specific insulin bound levels, non-specific binding was subtracted from the total insulin bound a t each concentration of unlabelled insulin.

The data were presented in the following ways: (a) competition-inhibition curves: In this presentation, the percentage of total radioactivity specifically bound to the cell pellet is graphically expressed as a function of the log of the total insulin concentration. (b)

Scatchard plot: the ratio of bound to free ‘251-insulin IS plotted as a function of bound

insulin (B) for each individual. The intercept of the prolongation of the curve with the abcissa determines the maximal capacity for insulin binding, from which the total number of receptors per cell (Ro) can be calculated by the formula:

(;).

Insulin bound (M) x 6.02 x 10” Cell concentration/l

Ro =

Insulin receptors on human erythrocytes 25

Although the explanation of the curvilinear shape of this plot remains debatable, we decided arbitrarily to describe it in terms of a negative cooperative model (De Meyts & Michiels-Place, 1978; Gambhir et al., 1978). In this way, we constructed the 'average affinity profile' as proposed by De Meyts and Roth (1975). The high affinity state at low levels of receptor occupancy is designated K,. The low affinity state at high levels of occupancy is designated Kf. The level of receptor occupancy at which the affinities start declining is called Ye whereas the level of occupancy at which affinity constants attain Kfis designated Yf.

Student's t-test was used to evaluated differences between means (Snedecor, 1965).

RESULTS

Table 1 indicate the laboratory data from the 12 women studied. Figure 1 shows the mean (_+ 1 SD) inhibition-competition curves separately for male and female subjects. Inhibition-competition curves of females did not differ statistically between the two phases of the menstrual cycle. However, males showed significantly higher specific bound '251-insulin at low insulin concentrations than females in either phase of the menstrual cycle.

Figure 1 (inset) shows the comparison of the specific and non specific binding, both expressed as percentage of total counts. The mean non-specific binding was indistinguish- able among the groups varying in the range of 1.2 to 2.8% of the total counts added. Figure 2 shows the Scatchard plot of the mean values obtained for the males and female subjects in the first and second halves of the menstrual cycle. The mean intercept of the terminal slope with the abcissa was higher for females in the second half (Ro = 3.4 ng/ml), as compared to that in the first half of the menstrual cycle (Ro= 1.9 ngiml), being similar to that observed in males (Ro= 3.3 nglml).

The affinity profiles obtained from the mean values for male and female subjects are shown in Fig. 3. Affinity constants at low occupancy levels were significantly higher for

Table 1. Clinical and hormonal data from the 12 women studied

Phase of the menstrual cycle

Mid-follicular Mid-luteal

Age (years) 29.4+ 5.5 Height (cm) 159.4 + 6.5 Weight (kg) 54.3 + 6.7 Blood glucose (mg/dl) 88 2 7 90+ 10 Insulin 6.3k3.2 6.7+ 1.6

Oestradiol 526k15.2 159.3f30.1

Progesterone 0.6+.0.2 11.425.7

(pU/ml)

(pg/ml)

(ng/mU

Results are expressed as mean* 1 SD.

26

0.10.

B. L. Wujchenberg et ul.

A I I

l -

0. I I 2 5 1020 5010020050010' lo4 I

Females ( I u teal I

Bound

! I I 4- Non-specific

I Females (f0lllC.)

Insulin (ng/rnl)

Fig. I . Competition-inhibition curves: the percentage of 'Z51-insulin specifically bound to RBC is plotted as function of insulin concentration. The inset shows the comparison between '251-insulin specifically bound at 0.1 ng/ml unlabelled insulin concentration and non-specific binding, both expressed as percentage of the total counts added. (0) Females (follicular), (0) females (luteal), (A) males. Mean & SD. b, c, see Table 2.

I n s u l i n bound (ng/ml)

Fig. 2. Scatchard analysis of insulin binding on RBC from normal male and female subjects Values shown are means. Symbols as Fig. 1.

females in the first half of the menstrual cycle as compared to either males or female subjects in the 2nd half of the menstrual cycle. At high occupancy levels, the affinities tend to converge to comparable values but they were still significantly higher in females in the follicular phase than in females in the luteal phase or males, in both of which the Kfvalues were similar.

No significant correlation was found between receptor concentration and correspond- ing fasting plasma insulin concentration in either male or female subjects.

Insulin receptors on human erythrocytes 27

Occupancy 'L

Fig. 3. Affinity profiles. The average affinities are plotted against theoccupancy. Valuesshown are means. Symbols as Fig. 1 .

DISCUSSION

In the present paper, we present data indicating that the characteristics of binding of the RBC insulin receptor are variable in normal human subjects. While female subjects in the first and second halves of the menstrual cycle exhibited inhibition-competition curves which appeared not to be statistically different, males showed significantly increased binding of '251-insulin in relation to the females at low unlabelled insulin concentrations.

According to current concepts, changes in binding may be due to changes in either receptor affinity (K) or binding capacity (Ro).

At any given level of binding, if the affinity has not changed, an increase in Ro is

followed by a proportionate decrease in Ke and Kf without changes in the ratio-. If,

however the affinity changes, the ratio -will also change independently of RO values.

Thus in situations of uncertain Ro, inherent in derivation by extrapolation, variations in Kr -will actually indicate changes of affinity.

B

Kr Kr K,

Ke

Ke Thus, in males, there must be an increase in either K, or Ro (or both), because at tracer

insulin concentration -tends to equal Ke x Ro. Our analysis suggests that this increase in

-is due to an increase in the number of receptors (Table 2) when compared to females in F the mid-follicular phase. In these the increase in affinity did not compensate for decreased receptor concentration, so that total binding decreased. Since the ratio -was not

significantly different, primary changes in affinity did not occur. On the other hand, the same comparison between males and females in the mid-luteal phase demonstrates that the increase in binding of the males was due to an increase in K, without a significant change in the index, - , probably related to inaccuracies of the derivation of Kr, as primary changes in receptor affinity should be expected.

B F

K r Ke

Kr Ke

4

Qi

Insulin receptors on human erythrocytes 29

Despite significant alterations in receptor concentration, when comparing the two phases of the menstrual cycle, the lack of differences in binding between them seems to be due to the proportional alterations in K, and Kf without any change in their ratio, compensating for the differences in receptor concentration. Therefore, no primary affinity changes are occurring (Muggeo et al., 1977).

Potential factors such as increased physical activity among the males or greater caloric intake among the females which might have contributed to sex-related differences in insulin binding were not apparently present, so our data suggest that sex hormones should be considered when interpretating studies of insulin binding to its receptor, and also indicate that binding to RBC may be affected by the phase of the menstrual cycle. However, the results obtained are somewhat different from those presented by Bertoli et al. (1980).

Furthermore, sex-related differences in insulin binding on RBC have been uniformly observed (Gambhir et al., 1978; Wachslicht-Rodbard et al., 1979).

The changes of RBC insulin receptor concentration from one phase of the menstrual cycle to the other, in a cell without the capacity for new protein synthesis, is probably related to the movement of the receptors from the cytoplasmic storage pools to the membrane, from which they could be either degraded and/or internalized by endocytosis (Kahn et al., 1977; Marshall & Olefsky, 1981).

We did not detect a significant correlation between receptor concentration and ambient fasting plasma insulin concentrations in either male and female subjects separately, or as a group. This lack of correlation was also found in previous studies using monocytes (Beck-Nielsen & Pedersen, 1980) and RBC (Kappy & Plotnick, 1979) from healthy persons, and may be the result of the small range of variation in basal insulin concentrations in normal subjects.

We did not find significant differences of Ye between the groups, but Yf values were higher for males in relation to females in the luteal phase. Since the differences occur at unphysiologically high levels of ambient insulin, their significance is difficult to evaluate and must await further study.

ACKNOWLEDGEMENTS

This study was supported by a grant from NOVO Research Institute, Copenhagen, Denmark and by ‘Fundaqiio de Amparo a Pesquisa do Estado de Sgo Paulo’, FAPESP, NO. 8010543-9.

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