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INSULIN RECEPTOR CHARACTERISTICS OF ERYTHROCYTES FROM HUMAN NEWBORNS Kanwal K. Gambhir, PhD, Shriniwas G. Nerurkar, PhD, Teresa Allen, MD, Ruth M. Hill, MS, H. S. Sekhon, PhD, and Lennox S. Westney, MD Washington, DC

Erythrocytes from human newborns were observed to have specific insulin receptors. The characteristics of these receptors were similar to those of the normal adult subjects. An observed slight increase in R, and a decrease in Ke of insulin receptors in erythrocytes may be speculated to facilitate the transfer of insulin from the fetal erythrocytes to other rapidly growing fetal tissues at a rate faster than that present in the circulation of the adult subjects.

An insulin radioreceptor assay for human erythrocytes was presented by Gambhir, Archer, and Carter' that established a means of measuring specific binding of insulin to this circulating cell.

Requests for reprints should be addressed to Dr. K.K. Gambhir, Molecular Endocrinology Laboratory, Department of Medicine, Howard University Hospital, Washington, DC 20060.

This assay was prompted by a competitive binding radioimmunoassay of Yalow and Berson2 that previously established a basis for clinical evaluation of circulating peptide hormones. Conventionally, as shown by Gavin et al,3 monocytes were used to study insulin action at the cell receptor level. A removal of 150 to 500 ml of blood is required from a subject in order to isolate enough monocytes for receptor studies. A leukophoresis is performed, and the plasma and erythrocytes are returned to the subject. In comparing this time consuming procedure to isolate monocytes, erythrocytes proved an ideal cell type due to their abundance and accessibility. Insulin cell receptors have been studied in various altered physiological states. A number of investigations-by Goldstein et a14 in diabetes, Bar et a15 in obesity, Muggeo et a16 in acromegaly, and Kahn et a17 in acanthosis nigricans-have shown alteration in insulin binding to monocytes. Soman and Felig8 demonstrated that increased insulin binding to monocytes from untreated anorexia nervosa patients returned to normal following treatment. In addition to these, a number of other studies-Olefsky,9 Harrison et al,10 Freychet et al,1' and Forgue and Freychet12-have established

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INSULIN RECEPTORS IN NEWBORNS

that the characteristics of insulin receptors in monocytes are similar to body tissues, including human adipocytes. Thus, monocytes were regarded as a representative cell type in man to study insulin receptors. However, with the introduction of the human erythrocyte insulin receptor assay by Gambhir et al,I erythrocytes were shown to possess insulin binding characteristics similar to monocytes and other human and animal tissues.13 Using erythrocytes, Robinson et al'4 showed decreased insulin binding in diabetics, WaschlischtRodbard et al'5 demonstrated increased insulin binding in untreated anorexia nervosa patients, and Gambhir et al'6 indicated a defect in insulin binding in uremic patients. Thus, erythrocytes can be considered as a representative cell type to study insulin receptor and altered insulin sensitivity in man.'7 The purpose of the present investigation is to study insulin receptor status in human newbornis by defining the insulin receptor parameters of the cord blood erythrocytes.

nated insulin was prepared as previously described by Gambhir et al.1

Buffer G The following composition of buffer G for radioreceptor assay was determined after a series of experiments by Gambhir et all: Hepes, 50 mM; TRIS, 50 mM; MgCl2*6H20, 10 mM; CaCl2, 10 mM; EDTA, 2 mM; dextrose, 10 mM; NaCl, 50; KCl, 5 mM; and 0.1 percent bovine serum albumin. The pH of this buffer was adjusted to 8.0 at room temperature (23 to 25 C). The osmolarity of the buffer was determined to be 300 mOsm/liter.

Preparation of Erythrocytes for Binding Studies Upon delivery, cord blood sample was imme-

Purified porcine insulinA was used both as the unlabeled ligand and for iodination. Radioiodi-

diately collected in a 15 cc sterile syringe and transferred to heparinized (green top) Vacutainer tubesB containing 143 USP units of sodium heparin per tube. Following centrifugation (15 minutes, 400g, 23 C), the plasma was aspirated. An erythrocyte pellet was mixed with two parts of physiological saline solution and layered in 3 ml of a mixture of 33.9 percent diatriozate (Hypaque) and 9 percent Ficoll (1:2.4 by volume) in a glass tube as described by Boyum.'8 Following centrifugation (400g) for 20 minutes and 23 C, the saline, monocytes, Hypaque-Ficoll, granulocyte phases, and the upper layer of the erythrocyte phase were aspirated. The cell pellet was then suspended in two parts of saline solution, and the above procedure was repeated. The resulting erythrocyte pellet was then resuspended at 4 C in two parts of buffer G to equilibrate the cells. After centrifugation of the cell suspension (15 minutes, 400g, 4 C), the supernate buffer was aspirated, and the cell pellet was resuspended with a volume of buffer G that re-

ALot No. 8GN814, Elanco Laboratories, Indianapolis, Indiana

BKimble-Terumo, Inc, Elkton, Maryland

METHODS AND MATERIALS

Subjects Following proper consent, cord blood erythrocyte samples for study were obtained from full term deliveries from nine black female volunteers. Their ages ranged from 16 to 38 years. All were nonobese and had no personal and family history of diabetes mellitus.

Hormones

332

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sulted in a suspension containing 4.4 x 109 cells per milliliter. The viability of the erythrocytes was assessed based on their degree of hemolysis. Most of the cell suspensions produced little or no detectable hemoglobin in the supernatants.

measured using C-peptide kit supplied by CalBiochem.c

Calculations Binding Studies A 400-,ll suspension (1.75 x 109 cells in buffer G) was incubated with 50 pg of 125I-labeled insulin in 25 ,u1 of buffer and various amounts of unlabeled insulin (0 to 0.5 x 105 ng) in a total volume of 0.5 (Table 1). After incubation at 15 C for 3.5 hours, 200 ,u of the incubation suspension was aliquoted into prechilled microfuge B tubes containing 200 ,u1 buffer G and 200 ,ul of dibutyl phthalate. Following centrifugation in Beckman Microfuge B at 4 C for 2.5 minutes, the buffer and dibutyl phthalate layers were aspirated, leaving about 10 percent of the dibutyl phthalate on the pellet. The tip of the microfuge tube containing the pellet was then cut with a hot scalpel and was counted in a gamma counter (Searle Model 1185). The following expression was used to determine the percent radioactivity bound:

The average number of insulin binding sites per erythrocyte was obtained by Scatchard analyses20 of the 1251-insulin binding data as modified by DeMeyts and Roth.2' The Scatchard curve was obtained by plotting ratio of bound insulin (B) to free insulin (F) on the Y axis against bound insulin (expressed as molar concentration) on the X axis. Since this plot was curvilinear, a constant slope was not possible, and, thus, estimation of the exact affinity constant could not be obtained. The average number of estimated sites per cell was calculated using the following expression: Sites per cell moles of insulin bound per liter cell concentration per liter (3.52 x 1012) x 6.03 x 1023 (Avogadro's number)

RBC pellet radioactivity x 100 Total radioactivity in 200 , Xl of the incubation suspension

RESULTS The percent radioactivity observed in erythrocyte pellet from incubation mixture containing 105 ng/ml of unlabeled insulin (tube 14, Table 1) was defined as nonspecific insulin binding. Subtraction of percent nonspecific insulin binding from the total percent insulin binding for each assay tube (tubes 1, 2, and 3, Table 1) gives percent specific binding.

The maximum specific insulin binding to 3.52 x 109 cord blood erythrocytes obtained at deliveries of nine black female volunteers after exposure to 100 pg of 125I-insulin was 11.1 + 2.9 (mean + SD) percent. As the amount of unlabeled insulin was gradually increased, there was a concomitant decrease observed in 1251-insulin binding. The specific 1251-insulin binding observed in each point was plotted on the Y axis against corresponding amounts of total insulin concentration (radioactive and nonradioactive) in logarithmic scale on the X axis. The average binding curve thus obtained is shown in

Measurement of Insulin and C-Peptide Insulin from cord blood serum was determined by a modification of double antibody radioimmunoassay of Morgan and Luzarow.19 C-peptide was

CCal-Biochem,

JOURNAL OF THE NATIONAL MEDICAL ASSOCIATION, VOL. 74, NO. 4,1982

PO Box 12087, San Diego, California

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INSULIN RECEPTORS IN NEWBORNS

0-12

X12

Scatchard Curve o

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10°

30

ro220

10

Total Insulin Bound (10(10 M)

0

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Figure 1. The shape of this binding curve displayed similarity with the shape of the average insulin binding curve obtained for erythrocytes of 30 normal adult subjects.'6 The amount of insulin required to inhibit 50 percent of maximum specific binding (ID5,,) was obtained from this binding curve (Figure 1). This value, which is also one of the important characteristics of insulin receptors, was 6.2 ng/ml for cord blood erythrocytes, as compared to 4.8 ng/ml for normal adult erythrocytes.'6 Data from Figure 1 were further used for Scatchard analyses. In these analyses the ratio of bound insulin to free insulin (B/F) and the amount of total insulin bound for every point (Table 1 and Figure 1) were calculated. The ratio B/F was plotted on the Y axis with corresponding total bound 334

idigi

0

n/i

erthocte

insulin values for each point on the X axis. The Scatchard analyses for average binding curve (Figure 1) have been shown in the inset of Figure 1. The Scatchard curve (Figure 1, inset) showed curvilinearity because of negative cooperative interaction between receptor sites. The negative cooperativity is a phenomenon wherein the affinity of the receptor for its ligand (in this case insulin) decreases as receptor occupancy increases. In other words, the receptors bind insulin molecules at a faster rate when most of them are unoccupied by insulin. This interpretation has been put forward by DeMeyts and Roth,21 based upon the assumption that there is only one type of insulin receptor on cell surfaces. The curvilinearity of the Scatchard plot can also be attributed to the pres-

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TABLE 1. PROTOCOL FOR RADIORECEPTOR ASSAY

Tube No.

Buffer G

1 2 3 4 5 6 7 8 9 10 11 12 13 14

75 65 50 25 65 50 25 50 25 50 25 50 25 25

Concentration Stock Insulin Standard

(ng/lI)

Volume of Insulin Standard (ng/ll)

0 0.01 0.01 0.01 0.1 0.1 0.1 1 1 10 10 100 100 1000

0 10 25 50 10 25 50 25 50 25 50 25 50 50

Final Unlabeled Insulin Concentration

1251-Labeled Insulin

(,Al)

Cell Suspension (A1)

0 0.2 0.5 1 2 5 10 50 100 500 1,000 5,000 10,000 100,000

25 25 25 25 25 25 25 25 25 25 25 25 25 25

400 400 400 400 400 400 400 400 400 400 400 400 400 400

(ng/ml)

Final volume = 500 /Il per tube

ence of two types of insulin receptors, but for simplicity the latter interpretation has not been considered in this investigation. The average Scatchard plot (Figure 1, inset) has been utilized to find out the three major parameters for erythrocyte insulin receptors viz the total number of receptor sites per erythrocyte (R), the average affinity at empty sites (K,), and the average affinity at filled sites (Kf). In this investigation, for the average Scatchard plot, B/F and B values for points 1 through 11 (Table 1) were utilized. The curve thus obtained was extrapolated to get the intercept on the X axis. The value of total insulin bound at this intercept was then applied to calculate R0, as shown in the calculations. The average affinity at empty sites (K,) was obtained by measuring the slope of the line joining the point of maximum B/F value along the Y axis and the point of intercept of the curve on the X axis. The average affinity at filled sites (Kf) was obtained by determining the tangent of the angle formed between the lower portion of the extrapolated Scatchard curve and X axis. The Scatchard analyses were carried out for all the in-

dividual insulin binding curves from nine subjects. These important parameters for cord blood erythrocytes are shown_in Table 2. The results show slight increase in Ke for cord blood erythrocyte insulin receptors.

DISCUSSION These studies demonstrate that erythrocytes from human newborns bind insulin specifically. The maximum mean specific insulin binding to cord blood erythrocytes (11.1 + 2.9 percent), obtained after full-term deliveries was not significantly higher (P > 0.1) as compared to normal adult erythrocytes'6 (9.9 + 1.4 percent). These results compared favorably with the mean maximum specific insulin binding reported under almost similar conditions by Kappy and Plotnick22 and Herzberg et a123 for the erythrocytes of full-term

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TABLE 2. PARAMETERS OF CORD BLOOD ERYTHROCYTES INSULIN RECEPTORS Percent Maximum

Specific

Subject

Insulin Binding (% ± SD)

Cord erythrocytes 11.1 ± 2.9* (n=9) Adult**erythrocytes 9.9 ± 1.4 (n = 30)

ID50

(ng/mI)

Ro

Ke

Kf

6.2

570*

0.38 x 108 M-1

0.8 x 107 M-1

4.8

420

0.45 x 108 M-1

0.62 x 107 M-1

*Statistically not significant, P>0.05 **Gambhir et all6

newborns. However, we do not find any significant difference in insulin binding to cord blood erythrocytes as compared to normal adult erythrocytes, which is contrary to the reports of Kappy and Plotnick22 and Herzberg et al.23 Generally the insulin receptors (R.) on cells decrease in the presence of higher concentrations of insulin. This fact is vividly manifested in cases of hyperinsulinemia ie, diabetes, insulinoma, and the cell cultures grown in the presence of excessive insulin. Therefore, determination of serum insulin and C-peptide helps in one of the ways to rationalize the increase or decrease in insulin receptors in body tissues. The determination of insulin and C-peptide values from cord blood sera was carried out and compared with the same from normal adult subjects. Insulin and C-peptide (0.9 + 0.3 and 2.9 + 1.7 ng/ml) values for cord blood samples were comparable to those from normal adult subjects (0.9 + 0.2 and 3.7 + 1.2 ng/ml, respectively). Thus, the insulin and C-peptide values cannot be

the controlling factors for the slight increase in insulin binding observed in cord blood erythrocytes. Increase in reticulocytes in the erythrocyte preparation can also be a cause of increased insulin binding.2426 However, the reticulocyte population in erythrocyte suspensions used in this investigation was below 0.1 percent, which was the same as in adult erythrocyte preparation. The chromotographic and electrographic analyses of cord blood 336

hemolysate were carried out to determine the presence of hemoglobin A. Since the presence of hemoglobin A was undetectable in these cell suspensions, the presence of mothers' erythrocytes in cord blood samples was found to be negligible. The parameters of insulin receptors, namely, R0, Ke Kf, and ID501 for cord blood erythrocytes and adult erythrocytes are comparable (Table 2). ID51) values for cord blood erythrocytes reported by Kappy and Plotnick22 and Herzberg et a123 were 6.0 and 4.8 ng/ml, respectively. As shown in Table 2, though there was 35 percent increase in cord blood erythrocyte receptor sites (R,) as compared to normal adult erythrocytes, it was not statistically significant (P > 0.1). Moreover, since the fetal erythrocytes have 25 percent more surface area as compared to adult erythrocytes,23 the R, per unit surface area may be almost similar for both of these erythrocytes. The comparison of R. in our investigation with those reported by Kappy and Plotnick22 and Herzberg23 has not been carried out since different criteria for the Scatchard analyses were utilized. However, it should be pointed out that the R, values for cord blood and adult erythrocytes calculated by them under a similar set of conditions are not similar. Also their Ro values for adult erythrocytes differ from each other and from those reported by Wachslicht-Rodbard et al.'5 The slight decrease in Ke of cord blood erythrocytes (Table 2) as compared to that of adult

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erythrocytes may make insulin bound to the erythrocyte more accessible to body tissues growing at a faster rate in newborns than in adults. Thus, the present investigation shows the presence of specific insulin receptors on erythrocytes in newborns which have characteristics similar to those of insulin receptors in erythrocytes from adult subjects. Slightly increased R0 in cord blood erythrocytes might be essential for storing and transporting insulin to rapidly growing fetal tissues. Similarly, decreased Ke might facilitate the transfer of insulin from cord blood erythrocytes to other fetal tissues at a faster rate than that present in the circulation of adult subjects.

Acknowledgments This work was supported by Biomedical Support Grant No. 5 S07 RR 05361 from the General Research Branch, Division of Research Resources, NIH, Bethesda, Maryland, and by the March of Dimes Birth Defects Foundation, 1275 Mamaroneck Avenue, White Plains, NY 10605, Grant No. 6-262. A portion of the work was carried out by Teresa Allen as a summer medical student research trainee.

Literature Cited 1. Gambhir KK, ArcherJA, Carter L. Insulin radioreceptor assay for human erythrocytes. Clin Chem 1977; 23: 1590-1595. 2. Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin in man. J Clin Invest 1960; 39:1157-1175. 3. Gavin IlIl JR, Roth J, Jen P, et al. Insulin receptors in human circulating cells and fibroblasts. Proc Natl Acad Sci USA 1972; 69:747-751. 4. Goldstein B, Blecher M, Binder R, et al. Hormone receptors binding of glucagon and insulin to human circulating mononuclear cells in diabetes mellitus. Endocr Res Commun 1975; 2:637-679. 5. Bar RS, Gordon P, Roth J, et al. Fluctuation in the affinity of insulin receptors on circulating monocytes of

obese patients. J Clin Invest 1976; 58:1123-1135. 6. Muggeo M, Bar RS, Roth J, et al. The insulin resistance of acromegaly: Evidence for two alterations in insulin receptors on circulating monocytes. J Clin Endocrinol Metab 1979; 48:17-25. 7. Kahn CR, Flier JS, Bar RS, et al. The syndrome of insulin resistance and acanthosis nigricans insulin receptors disorders in man. N Engl J Med 1977; 297:739-745. 8. Soman V, Felig P. Anorexia nervosa: Fluctuations in insulin binding and insulin sensitivity. Clin Res 1979; 27: 260A. 9. Olefsky JM. The insulin receptors: Its role in insulin resistance of obesity and diabetes. Diabetes 1976; 25: 1154-1165. 10. Harrison LC, Billington T, East IJ, et al. The effect of solubilization on properties of the insulin receptor of human placental membranes. Endocrinology 1978; 102: 1485-1495. 1 1. Freychet P, Kahn CR, Roth J, et al. Insulin interaction with liver plasmas membranes: Independence of binding of hormone and its degradation. J Biol Chem 1972; 247:3953-3961. 12. Forgue ME, Freychet P. Insulin receptors in the heart muscle. Demonstration of specific binding sites and impairment of insulin binding in plasma membranes of obese hyperglycemic mouse. Diabetes 1975; 24:715-723. 13. Gambhir KK, Archer JA, Bradley C. Characteristics of human erythrocyte insulin receptors. Diabetes 1978; 27: 701-708. 14. Robinson TJ, Archer JA, Gambhir KK, et al. Erythrocytes: A new cell type for the evaluation of insulin receptor defects in diabetic humans. Science 1979; 205:200-202. 15. Wachslicht-Rodbard H, Gross HA, Rodbard D, et al. Increased insulin binding to erythrocytes in anorexia nervosa: Restoration to normal with refeeding. N Engl J Med 1979; 300:882-887. 16. Gambhir KK, Archer JA, Nerurkar SG, et al. Erythrocyte insulin receptors in chronic renal failure. Nephron 1981; 28:4-10. 17. Nerurkar SG, Gambhir KK. Insulin receptor assay in human erythrocytes as an index to insulin sensitivity of body tissues. Clin Chem 1979; 25:1672-1673. 18. Boyum A. Separation of leukocyte from blood and bone marrow. Scand J Clin Invest 1968; 21(Suppl 97):77-89. 19. Morgan CR, Luzarow A. Immunoassay of insulin two antibody system: Plasma insulin levels of normal, subdiabetic and diabetic rats. Diabetes 1963; 12:115-126. 20. Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci 1949; 51:660-672. 21. DeMeyts P, Roth J. Cooperativity in lingand binding: A new graphic analysis. Biochem Biophys Res Commun 1975; 66:1118-1126. 22. Kappy MS, Plotnick L. Studies in insulin binding in children using human erythrocytes in small amounts of blood. Diabetes 1979; 28:1001-1005. 23. Herzberg VL, Boughter IM, Carlisle SK, et al. 1251Insulin receptor binding to cord blood erythrocytes of varying gestational age and comparison with adult values. Pediatr Res 1980; 14:4-7. 24. Thompoulos P, Berthelien M, Laudet M. Loss of insulin receptor on maturation of reticulocytes. Biochem Biophys Res Commun 1978; 85:1460-1465. 25. Eng J, Lee L, Yalow RS. Influence of the age of erythrocytes on their insulin receptors. Diabetes 1980; 29 164-166. 26. Kosmakos FC, Nagulespanan M, Bennet PH. Insulin binding to erythrocytes: A negative correlation with red cell age. J Clin Endocrinol Metab 1980; 51:46-50.

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