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Insulin receptor defect in diabetic man with chronic renal failure: A comparison of erythrocyte insulin binding in diabetic and nondiabetic patients on maintenance hemodialysis
BIOCHEMICAL
25, 62-73
MEDICINE
(1981)
insulin Receptor Defect in Diabetic Man with Chronic Renal Failure: A Comparison of Erythrocyte Insulin Binding in Diabetic and Nondiabetic Patients on Maintenance Hemodialysis KANWAL
K. GAMBHIR,SHRINIWASG.NERURKAR,ILUMINADO CRUZ, AND ADRIAN
Endocrine
and Renal
0.
Sections, Department of Medicine, University. Washington, D.C. Received
July
A.
HOSTEN College 20060
of
Medicine,
Howard
17, t980
Abnormal glucose metabolism in chronic renal failure (CRF) has been recognized for a long time (1). Unlike the glucose intolerance of diabetes mellitus, this abnormality in CRF has a relatively small impact on the management of the patients (2). Unlike diabetics, patients with CRF generally do not develop insulin-dependent hyperglycemia or ketoacidosis. However, since diabetic CRF treated by maintenance dialysis and/or renal transplantation have significantly higher morbidity and mortality than do nondiabetic patients (3), it is of more than academic interest to evaluate the insulin receptor status in nondiabetic and diabetic patients with CRF. The altered insulin action has been studied by determining insulin binding to insulin receptors in a number of pathological states. The determination of insulin binding to body tissues is a much simpler procedure than the perfusion or the clamp techniques previously utilized by a, number of investigators (4-6). The insulin binding and therefore, insulin sensitivity, has been shown to be decreased in diabetes (7-9), obesity, (IO), acromegaly (1 l), and acanthosis nigricans (12). Both the insulin binding and insulin sensitivity are increased in untreated anorexia nervosa and return to normal after treatment (13,14). Insulin receptors on human erythrocytes have been well characterized (15) and their characteristics have been shown to be reflective of insulin action in body tissues during various physiological conditions (9,13,16).. The present investigation is an attempt to establish a simpler parameter for the evaluation of the altered insulin receptor status in chronic renal failure. 62 ooo6-2944181/010062-12$02.00/0 Copyright All rights
@ 1981 by Academic Press. Inc. of repreduction in any form reserved.
INSULIN
BINDING
MATERIALS
TO RBC IN CRF
63
AND METHODS
Supplies Purified porcine, insulin (lot lJM!$SAF), was obtained from Elanco Laboratories, a division of Eli Lilly Research Laboratories, and was used for iodination as well as for unlabeled insulin in the binding studies. Bovine serum albumin, carrier-free Nalz51, dibutylphthalate, Microfuge B, and its accessories were purchased from Pentex, New England Nuclear, Aldrich Chemical Company, and Beckman Instrument Incorporated, respectively. Insulin and C-peptide assay kits were purchased from Sorin (Italy) and Calbiochem-Behring Corporation, respectively. The other chemicals were of reagent grade. Patient
Selection
Sixteen, nonobese (body weight: 99 + 14% of ideal weight), nondiabetic patients with CRF ranging in age from 36 to 70 years and who had been undergoing hemodialysis for a period of more than 1 year were studied. Except for the patient ((X-1 1 years) (Table 1) all the other patients had a history of 0.5 to 4 years of CRF prior to initial hemodialysis. Seven, nonobese (body weight: 101 f 4% of ideal body weight), diabetic (not receiving insulin or hypoglycemic agents during the period of dialysis treatment) patients with CRF were selected for the study. They ranged in age from 43 to 68 years, had a history of 6 to 30 years of diabetes prior to renal failure and had been undergoing hemodialysis for a period of more than 1 year. Many diabetic patients with CRF as well as nondiabetic patients with CRF undergoing dialysis were obese (body weight: >150% of ideal body weight). Since obesity has been shown to lower insulin binding to the cell receptors (lo), these obese patients were not included in the present study. Due to this limitation only seven diabetic patients with CRF could be selected for this investigation (Table 1). None of the CRF patients selected received blood transfusions at least 3 months prior to the study. Thirty, nonobese (body weight: 102 + 8% of ideal body weight), nondiabetic normal subjects of both sexes and with no personal or family history of diabetes or CRF were studied as controls. They ranged in age from 29 to 55 years. Proper informed written consent was obtained. Blood was drawn from all subjects before breakfast and after an overnight fast. Four to ten milliliters of blood was sufficient from normal subjects while 15 to 20 ml of blood was obtained from uremic patients just prior to dialysis. Zsolation Erythrocytes, Insulin Binding to Cells, and Data Analyses The erythrocytes were isolated by Hypaque-FicoIl gradient centrifugation and were equilibrated with buffer G as described by Gambhir et al.
JW Mean k SD
GW
43 67 62 45
CH IL MM EP BT
53 I
68 44 44
Age
Patient
F F M F M
F
M
Sex
4
101
107 97 102 101
103 100 97
Percentage IBW”
74 74 86 73 27
57 55 51 124
BUN
1
10.3
10.5
12
10
9
10 11 10
Insulin r&/ml
132 66
100
83 120 105 120 125 275 38 32
-
30 22 28 102 28 18
7.5 1.5
-
10 6.5 5.9 6.7 7.9 7.7
C-peptide rig/ml
Diabetics with renal failure
Glu’
TABLE 1 PATIENT PROFILE
6.7 12.8 7.2 9.2 2.4
11.7
7.3 8.7 10
glycol. hemogl.
6.7 1.9 4.9 3.1 7.0 4.6 4.7 4.7 1.8
Percentage lBd
Glomerulosclerosis Glomerulosclerosis Glomerulosclerosis Glomerulosclerosis Glomerulosclerosis Glomerulosclerosis Glomerulosclerosis
Diagnosis
2 b
44 52 60 41 36 70 67 58 65 47 63 45 70 53 59 37 54 12
M M F F M F F M M F M M F F M M
101 107 102 100 102 loo % 89 75 105 94 86 78 133 110 99 14
93 102 82 90 90 83 NDe 86 ND 152 ND 64 ND 116 ND 80 94 23
-
21 II 12 IO 29 IO ND” 19 ND 14 ND 18 ND 19 ND 12 16 6
Nondiabetics 80 104 100 100 110 85 ND 115 ND 115 ND 85 ND 115 ND 100 101 13
with renal failure 25 9.5 38 11.5 15 8.5 IO 4.3 28 8.4 22 6.2 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 23 8.1 9.9 2.5 9.3 12.8 8.0 9.0 7.2 ND ND ND ND ND ND ND ND ND ND 9.3 1.9
11.4 14.0 11.1 10.0 16.9 12.8 12.5 17.4 11.4 15.3 14.2 15.5 12.0 9.3 11.6 14.3 13.1 2.4
o Ideal body weight. b Creatinine. c Glucose. d Maximum percentage specific L451-insulin binding. p Not determined on the day of the receptor assay. However, patient charts indicated established uremic values.
RJ WM EP BT cw cs PE AE SN SM FW MR SM HS SJ JD Mean + SD
Chr. giomerulonephritis Chr. glomerulonephritis Nephrosclerosis Nephrosclerosis Nephrosclerosis Nephrosclerosis Nephroscierosis Nephrosclerosis Obstructive nephropathy Interstitial nephritis Nephrosclerosis Nephrosclerosis Nephrosclerosis Polycystic kidney Nephrosclerosis Nephrosclerosis
66
GAMBHIR ETAL,
(17). Isolated and purified erythrocyte suspension did not contain more than 0.1% reticulocytes as determined by methylene blue staining. The insulin receptor binding data and the Scatchard analyses of these data were obtained as described earlier ( 1.5). Determination of Insulin, C-peptide, Glycosylated Hemoglobins. and Glucose
From the fasting serum samples of all the subjects insulin determinations were carried out by the modification of double antibody radioimmunoassay of Morgan and Lazarow (18). Insulin values based on a similar RIA procedure were also determined by Ms. Carla Hendricks, and Ms. Janice Ryan in the Laboratory of Phillip Gorden, M.D., Diabetic Branch, NIAMDD, Bethesda, Maryland. The C-peptide values were estimated utilizing the RIA kit from Calbiochem-Behring Corporation. Glycosylated hemoglobin level was determined according to the method of Trivelli et al. (19) by Dr. G. Giridhar. Metpath Laboratories, Hackensack, New Jersey. The blood samples for these determinations were collected in NaF. Serum glucose determinations were carried out according to the glucose oxidase method utilizing Beckman GlucoseBUN analyzer II. RESULTS
As shown in Fig. 1, the maximum specific binding to 3.52 x 1tY erythrocytes when exposed to 100 pg of iz51-insulin was 4.7 2 1.8% (mean + SD) in the diabetic patients with CRF. This maximum specific i251insulin binding was significantly lower (P < 0.01) than that for the normal subjects (mean t SD, 9.9 ? I .4%) as well as for the nondiabetic patients with CRF (mean ? SD, 13.1 * 2.4%). Further, 1251-insulin binding to erythrocytes in the presence of a range of physiological insulin concentration up to 10 @ml was significantly lowered (P < 0.05) in the diabetic patients with CRF than in the normal subjects and in the nondiabetic patients with CRF. However, the amount of insulin required to inhibit 50% of the maximum specific lz51-insulin binding (ID,,), in the diabetic patients with CRF was similar to that in the normal subjects. The ID50 values for the diabetic patients with CRF, the nondiabetic patients with CRF and normal subjects were 4.8, 3.2, and 4.8 &ml, respectively (Table 2). The Scatchard analyses of binding data (Fig. 1) yielded curvilinear plots (Fig. 2). The Scatchard plots were interpreted by the procedure described by DeMeyts and Roth (20). The number of insulin receptors per cell (R,) and average affinity constants (R, and R,) depend upon the intercept of extrapolated Scatchard plot on X-axis. The extrapolation of the Scatchard plot in turn depends upon the selection of final insulin concentration used as a last point in DeMeyts’ analyses. The characteristic features of the erythrocytes’ insulin receptors are shown in Table 2. Since the specific
INSULIN
BINDING
0 NON-DIABETIC . NORMAL 4 DIABETIC CRF
ir
v)
10“
1CP
TOTAL
67
TO RBC IN CRF
10’
1W
INSULIN
CONCENTRATION
CRF
1P
1P
hg/mll
FIG. I. ‘051-insulin binding to erythrocytes. Erythrocytes from normal, nondiabetic, and diabetic patients on maintenance dialysis were isolated, washed, and resuspended in a Hepes-Tris buffer, pH 8.0, to constitute a suspension with 4.4 x 1fP erythrocytes per milliliter. Then 400 ~1 (I .76 x IP erythrocytes) of each cell suspension was incubated with 100 ~1 of the buffer, containing unlabeled insulin (0 to 0.5 x 106ng) and 50 pg of 1*51-insulin for 3.5 hr at 15°C. At the end of the incubation period, duplicate ahquots of each cell suspension were centrifuged through chilled buffer (4°C) and dibutylphthatate gradient. 1*51-insuiin bound was determined by counting cell pellets. The data are expressed as mean k SD for 30 normal subjects, I6 nondiabetic, and 7 diabetic patients with chronic renal failure on maintenance hemodialysis.
percentage 1251-insulin binding to erythrocytes when exposed to 500 and 1000 nglml of unlabeled insulin was significantly different (P c 0.05) from that exposed to 100 rig/ml of unlabeled insulin in the patients studied, the insulin binding data up to 1000 rig/ml of insulin concentration was used for the Scatchard plots. For comparison purposes only (13), R. and average affinity constants were also determined using 100 and 500 rig/ml of insulin concentration as the last point of the Scatchard plots (Table 2). To be consistent with our previous report (16), we have used total insulin binding up to 1000 rig/ml of insulin concentration for the Scatchard plots. The zxtrapolation from Scatchard plots resulted in 190 receptor sites per zrythrocyte in the diabetic patients with CRF compared to 410 sites per :ell in the nondiabetic patients with CRF and the normal subjects. In zontrast to R,, the receptor affinities for the diabetic patients with CRF
9.9 * 1.4 4.7 k 1.8 13.1 -c 2.4
Subject
Cutoff point (@nl)* Normal (30) Diabetic CRF (7) Nondiabetic CRF (16)
4.8 4.8 3.2
IDSO 100 80 40 80
500 230 loo 200
looo 410 190 410
No. of sites/cell (R,)
TABLE 2 INSULIN RECE~OR
100 2.3 2.0 3.1
500 0.83 0.86 1.3
K, x 108 M-’
CHARACTERISTICS
loo0 , 0.43 0.44 0.63
loo 5.4 6.6 6.3
Affinity constant
500 1.3 1.7 1.7
R, x IO’ tv-’ loo0 0.62 0.58 0.46
a Concentration @g/ml) of insulin required 10 inhibit 50% of maximum specific 1251-insulin binding to 3.52 x IO@erythrocytes in I ml of the incubation suspe‘nsion (Fig. I). b The cutoff point (100,500. and 1000 &ml) refers to that selected insulin concentration from the range of insulin concentrations in the receptor assay (Fig. 1) used as the final point for plotting the Scatchard curve. Extrapolation of these curves would result different X-axis intercepts and thus, will give different amounts of insulin bound to calculate the number of receptor sites per erythrocyte. However, to be consistent with our previous report (16). receptor number and affinity constants using 1000 rig/ml of insulin concentration as a cut off point (Fig. 2) were calculated for discussion in the text. R, refers to the average affinity constant at the unoccupied sites and K, refers to the average affinity constant at the filled receptor sites.
Percentage Max. sp. binding
ERYTHROCYTE
F
2
F 2 E j;;
69
INSULIN BINDING TO RBC IN CRF
0 NOM-DIASEIX l
. MASEW
TOTAL
INSULIN
BOUND
CRF
NORMAL
CRF
t1O-‘o MI
FIG. 2. Scatchard plots with affinity profiles. Scatchard analyses of the insulin-binding data (Fig. I) were performed. On the X-axis is the amount of insulin bound, calculated by multiplying the percentage 1*51-insulinbound by total insulin concentration in the incubation medium. The ratio of the bound to the free ins&n is plotted on the Y-axis. The inset depicts the relationship of the log of the percentage of occupancy of receptor sites 6 x MO), calculated by B/R, x 100andthe averageaftinity constant(K). R, equalsB/F /R,,-B. B represents the amountof msulinbound,F is the amountof free insulin,RI depictstotal receptorconcentration,andj is the fraction of receptoroccupancy.In order to plot the rangeof receptorsiteoccupancy,a numberof pointsontheScatchardcurveswerederived.
and the normal subjects were almost similar. The average affinity constants for unoccupied sites (I&.) were 0.46 x 108 and 0.44 x 108 M-’ for the diabetic patients with CRF and normal subjects respectively. By comparison, nondiabetic patients with CRF showed a marked increase in R, (0.63 x 108 M-l). In the normal subjects and the diabetic patients with CRF, the average affinities for filled sites, Et, were 0.62 x lo7 and 0.58 x 10’ M-I, respectively, compared with 0.46 x lo7 M+ in the nondiabetic patients with CRF. Serum insulin concentrations were 38 & 32 PUlml (mean rfr SD) for the diabetic patients with CRF, 22.8 f 5.7 @/ml for the normal subjects, and 23 ? 9.9 pU/ml for the nondiabetic patients with CRF. Since these diabetic CRF patients were not receiving insulin they did not show any presence of insulin antibodies in their plasma as determined by lack of insulin binding properties to their plasma. The levels of plasma insulin in these subjects were not significantly different (P > 0.05) when compared to each other. The C-peptide values were significantly higher (P < 0.05) in the patients than in normal subjects. These values were 7.5 2 1.5 rig/ml
70
GAMBHIR
ET AL.
(mean fr SD) for the diabetic patients with CRF, 3.7 F 0.6 rig/ml for the normal subjects, and 8.1 ? 2.5 rig/ml for the nondiabetic patients with CRF. C-Peptide clearance is lowered in CRF (21). Thus, the higher C-peptide values in CRF are feasible without noticeable hyperinsulinemia. It should be mentioned at this point that insulin binding to erythrocytes was not the lowest in that subject (EP-Table 1) whose serum insulin concentration was the highest among all the subjects studied. We did not see any relationship of insulin binding to endogenous insulin concentration (r = -0.32). when all the subjects were considered together or even when they were considered as three separate groups. The glycosylated hemoglobin levels were also not statistically different (P < 0.05) in these three groups. The diabetic patients with CRF had 9.2 ? 2.4 (mean t SD, percentage of total hemoglobins), the nondiabetic patients with CRF had 9.3 IT 1.9 (mean + SD, percentage of total hemoglobins) and the normal subjects had 7.3 + 1.3 (mean I~I SD, percentage of total hemoglobins). It should be noted here that one of the nondiabetic patients (WM-Table 1) with CRF had a higher glycosylated hemoglobin level in spite of the fact that this glucose level was not elevated (104 mg/ml). Although the exact reason for increased glycosylated hemoglobins level is not known, a possibility exists that isocyanate, which is known to be present in in vitro preparations of urea, may cause carbamylation of hemoglobin molecules (22) in the patients with CRF. These carbamylated hemoglobin molecules are eluted along with glycosylated hemoglobins in the procedure for the isolation of glycosylated hemoglobins. To investigate the possibility of a factor responsible for lowering insulin binding in the diabetic patients with CRF, the isolated and purified erythrocytes from a normal subject were preincubated in an equal volume of plasma from a diabetic patient with CRF at 37°C for I hr before the insulin radioreceptor assay. The ‘251-insulin binding to those erythrocytes remained unchanged. Similarly, the ‘251-insulin binding to erythrocytes from diabetic patients with CRF preexposed to normal plasma, did not change. However, preincubation of the erythrocytes from a diabetic CRF patient with serum containing anti-insulin receptor antibody lowered more than 90% of the insulin binding to these erythrocytes. DISCUSSION These studies demonstrate that there is a marked reduction in insulin binding to erythrocytes in the diabetic patients (4.7%) with CRF as compared to both the normal subjects (9.9%) and the nondiabetic patients with CRF (13.1%). A similar decrease in insulin binding to erythrocytes has been demonstrated in diabetic patients without renal failure (9) as well as in nondiabetic patients with renal failure who were not on dialysis (23). The diabetic patients without CRF (9) and nondiabetic patients with CRF
INSULIN
BINDING
TO RBC
IN CRF
71
but not on dialysis (23) had 5.9 and 7.9% specific insulin binding, respectively. However, diabetic patients with CRF in the study, showed a further decrease in insulin binding compared to the diabetic without CRF (9) and the uremic patients (23). A decrease in insulin binding has also been shown in monocytes (7), adipocytes (8), and placental membranes (24) of diabetic patients without CRF. Increased insulin binding to erythrocytes in nondiabetic patients with CRF who were undergoing hemodialysis cannot be explained at this time. There are reports showing increased insulin binding to younger erythrocytes than to older cells (25,26). One expects more younger cells in CRF patients. If increase in younger cells were to be the cause of increased insulin binding it should have also been evident in the nondiabetic patients with CRF who were not on dialysis. The Scatchard analyses indicated that the marked reduction in insulin binding observed in the diabetic patients with CRF was accompanied by a decrease in R,, while the average affinity constants, k, and &, remained almost the same as those of the normal subjects. Robinson et al. (9) also observed reduction in R, with little or no change in the average receptor affinities in erythrocytes from the diabetic patient without CRF. On the other hand, the nondiabetic patients with CRF had R, similar to the normal subjects but the average affinity constant, k, was increased (40%). In the acutely starved obese human subjects, Bar et al. (10) have reported variation in receptor affinities without any changes in R, compared to the unstarved obese subjects. Many investigators have reported the presence of reduced insulin sensitivity and down regulation of insulin receptors accompanied by hyperinsulinemia in diabetes (8,9), obesity (lo), acromegaly (1 I), and insulinoma (27). In the present investigations, such a relationship was not evident as shown by analyses of variance of endogenous insulin levels and insulin binding to erythrocytes (r = -0.32). A number of other studies also have been reported which do not support down regulation of insulin receptors (28,29). According to this study, the patient groups do not show any significant variations in levels of fasting glucose, insulin, and glycosylated hemoglobins as compared to those of normals. The C-peptide values for the patients with CRF were higher than normal as similarly shown by Jaspan et al. (21). The higher C-peptide levels in patients with CRF were due to the prolonged half life of endogenous C-peptide compared to that of nsulin and these increased C-peptide values did not constitute a hyperiniulinemia in patients with CRF (21). The only differentiating entity among ;he patients with CRF were higher than the normals as similarly shown by zrythrocytes. The metabolic studies by DeFronzo (6) have shown reduced glucose utilization in undialyzed patients with renal failure and return to almost normal following dialysis. We previously have demonstrated (23) that
72
GAMBHIR
ET AL
reduced insulin binding in the nondialyzed uremic patients was improved following chronic dialysis. In this investigation, we are reporting decreased insulin binding in the dialyzed diabetic patients with CRF and confirming the improved insulin binding after dialysis in the nondiabetic patients with CRF. The abnormal carbohydrate metabolism in the renal failure which has been related to reduced insulin sensitivity to peripheral tissues by very elaborate techniques and experiments (4-6) can also be evaluated as an insulin receptor defect by studying insulin binding to erythrocytes using the much simpler erythrocyte insulin receptor assay. Thus, if the receptor modulation acquires a status of treatment for diabetes, evaluation of insulin receptor status may become evident in the treatment of diabetes. In such a case, availability of representative cell type and a suitable receptor assay as well as measurement of receptor characteristics will be a necessity. The present investigation can be viewed as an attempt to satisfy these requirements. SUMMARY
1251-Insulin binding to most accessible and easily obtained circulating erythrocytes in 7 diabetic and 16 nondiabetic, nonobese patients with chronic renal failure (CRF) on maintenance hemodialysis was studied and compared with that of the 30 normal, nonobese volunteers. The percentage of 1251-insulin binding was 4.7 + 1.8 (mean + SD) in the diabetic and 13.1 + 2.4 in the nondiabetic patients with CRF while in the normal subjects it was 9.9 k 1.4. There was no statistical difference in the fasting levels of glucose, insulin, and glycosylated hemoglobins in all the subjects studied. No correlation (r = -0.32) between insulin binding and endogenous insulin was observed in these subjects. The diabetic patients with CRF had no circulating factor(s) that would reduce insulin binding to nornal erythrocytes. In comparison to the normal subjects, the reduction in insulin binding in the diabetic patients with CRF was accompanied by 47% reduction in insulin receptor sites (R,) but with no change in insulin receptor affinity, (R,, 0.46 x 108 M-I), while in the nondiabetic patients with CRF no change in insulin receptor sites (R,) of increased affinity (k,,, 0.63 X 108 M-l) was observed. These findings suggest that insulin bindin utilizing the much simpler erythrocyte insulin receptor assay can be use 1 to evaluate alteration in insulin receptor status in CRF. These investigai tions also provide a very useful assay and characteristic studies for insulin rei ceptors, if receptor modulation acquires a status of treatment for diabetes. REFERENCES 1. Linder, G. C., Hiller, A., and VanSlyke, D. D., J. Cfin. Invest. 1, 247 (1925). 2. DeFronzo, R. A., Andres, R., Edgar, P., and Walker, W. G., Medicine 52,469 (1973). 3. Simmons, R. G., and &hilling, K. .I., in “Rehabilitation: Diabetic vs. Non-Diabetiq Transplants” (M. Schesinger,R. E. Billingham, F. T. Rapaport, Eds.), Transplantal tion Today Vol. 3, p. 719. Grune & Stratton, New York, (1975).
INSULIN
BINDING
TO RBC IN CRF
73
4. Westervelt, F. B., J. Lab. Clin. Med. 74, 79 (1969). 5. Mondon, C. E., Dolkas, C. B., and Reaven, G. M., Diabetes 27, 571 (1978). 6. DeFronzo, R. A., Metabolism 27, 1866 (1978). 7. Goldstein, S., Blecher, M., Binder, R., Perrino, P. V., and Recant, L., Endocr. Res. Commun. 2, 367 (1975). 8. Olefsky, J. M., Diabetes 25, 1154 (1976). 9. Robinson, T. J., Archer, J. A., Gambhir, K. K., Hollis, V. W., Jr., Carter, L., and Bradley, C., Science 205, 200 (1979). IO. Bar, R. S., Gorden, P., Roth, J., Kahn, C. R., and DeMeyts, P., J. Clin. Invest. 58, 1123 (1976). Il. Muggeo, M., Bar, R. S., Roth, J., Kahn, C. R., and Gorden, P., J. Clin. Endocrinol. Med. 48, 17 (1979). 12. Kahn, C. R., Flier, J. S., Bar, R. S., Archer, J. A., Gorden, P., Martin, M. M., and Roth, J., N. Engl. J. Med. 294, 740 (1976). 13. Wachslicht-Rodbard, H., Gross, H. A., Rodbard, D., Ebert, M. H., and Roth, J., N. Engl.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
J. Med.
300, 882 (1979).
Soman, V., and Felig, P., Clin. Res. 27, 260A (1979). Gambhir, K. K., Archer, J. A., and Bradely, C., Diabetes 27, 701 (1978). Nerurkar, S. G., and Gambhir, K. K., Clin. Chem 25, 1672 (1979). Gambhir, K. K., Archer, J. A., and Carter, L., Clin. Chem 23, 1590 (1977). Morgan, C. R., and Lazarow, A., Diabetes 12, 1 I5 (1%3). Trivelli, L. A., Ranney, H. M., and Lai, H. T., N. Engf. J. Med. 284, 353 (1971). DeMeyts, P., and Roth, J., Rio&em. Biophys. Res. Commun. 66, 1118 (1975). Jaspan, J. B., Mako, M. E., Hideshi, K., Blix, P. M., Horowitz, D. L., and Rubenstein, A. H., J. Clin. Endocrinol. Metub. 45, 441 (1977). Lee, C. K., and Manning, J. M., J. Biol. Chem. 248, 5861 (1973). Gambhir, K. K., Archer, 1. A., Nerurkar, S. G., Cruz, I., and Sanders, M., Nephron, in press. Harrison, L. C., Billington, T., Clark, S.. Nichols, R., East, I., and Martin F. I. R., J. Clin. Endocrinol. Metab. 44, 206 (1977). Kosmakos, F. C., Nagulesparan, M., and Bennett, P. H., J. Clin. Endocrinof. Metab. 51, 46 (1980). Eng. J., Lee, L., and Yalow, R. S., Diabetes 29, 164 (1980). Bar, R. S., Gorden, P., Roth, J., and Siebert, C. W., J. Clin. Endocrinol. Metab. 44, 1210 (1977). Thorsson, A. V., and Hintz, R. L., N. Engl. J. Med. 297, 908 (1977). Misbin, R. I., O’Leary, J. P., and Pulkkinen, A., Science 205, 1003 (1979).
25, 62-73
MEDICINE
(1981)
insulin Receptor Defect in Diabetic Man with Chronic Renal Failure: A Comparison of Erythrocyte Insulin Binding in Diabetic and Nondiabetic Patients on Maintenance Hemodialysis KANWAL
K. GAMBHIR,SHRINIWASG.NERURKAR,ILUMINADO CRUZ, AND ADRIAN
Endocrine
and Renal
0.
Sections, Department of Medicine, University. Washington, D.C. Received
July
A.
HOSTEN College 20060
of
Medicine,
Howard
17, t980
Abnormal glucose metabolism in chronic renal failure (CRF) has been recognized for a long time (1). Unlike the glucose intolerance of diabetes mellitus, this abnormality in CRF has a relatively small impact on the management of the patients (2). Unlike diabetics, patients with CRF generally do not develop insulin-dependent hyperglycemia or ketoacidosis. However, since diabetic CRF treated by maintenance dialysis and/or renal transplantation have significantly higher morbidity and mortality than do nondiabetic patients (3), it is of more than academic interest to evaluate the insulin receptor status in nondiabetic and diabetic patients with CRF. The altered insulin action has been studied by determining insulin binding to insulin receptors in a number of pathological states. The determination of insulin binding to body tissues is a much simpler procedure than the perfusion or the clamp techniques previously utilized by a, number of investigators (4-6). The insulin binding and therefore, insulin sensitivity, has been shown to be decreased in diabetes (7-9), obesity, (IO), acromegaly (1 l), and acanthosis nigricans (12). Both the insulin binding and insulin sensitivity are increased in untreated anorexia nervosa and return to normal after treatment (13,14). Insulin receptors on human erythrocytes have been well characterized (15) and their characteristics have been shown to be reflective of insulin action in body tissues during various physiological conditions (9,13,16).. The present investigation is an attempt to establish a simpler parameter for the evaluation of the altered insulin receptor status in chronic renal failure. 62 ooo6-2944181/010062-12$02.00/0 Copyright All rights
@ 1981 by Academic Press. Inc. of repreduction in any form reserved.
INSULIN
BINDING
MATERIALS
TO RBC IN CRF
63
AND METHODS
Supplies Purified porcine, insulin (lot lJM!$SAF), was obtained from Elanco Laboratories, a division of Eli Lilly Research Laboratories, and was used for iodination as well as for unlabeled insulin in the binding studies. Bovine serum albumin, carrier-free Nalz51, dibutylphthalate, Microfuge B, and its accessories were purchased from Pentex, New England Nuclear, Aldrich Chemical Company, and Beckman Instrument Incorporated, respectively. Insulin and C-peptide assay kits were purchased from Sorin (Italy) and Calbiochem-Behring Corporation, respectively. The other chemicals were of reagent grade. Patient
Selection
Sixteen, nonobese (body weight: 99 + 14% of ideal weight), nondiabetic patients with CRF ranging in age from 36 to 70 years and who had been undergoing hemodialysis for a period of more than 1 year were studied. Except for the patient ((X-1 1 years) (Table 1) all the other patients had a history of 0.5 to 4 years of CRF prior to initial hemodialysis. Seven, nonobese (body weight: 101 f 4% of ideal body weight), diabetic (not receiving insulin or hypoglycemic agents during the period of dialysis treatment) patients with CRF were selected for the study. They ranged in age from 43 to 68 years, had a history of 6 to 30 years of diabetes prior to renal failure and had been undergoing hemodialysis for a period of more than 1 year. Many diabetic patients with CRF as well as nondiabetic patients with CRF undergoing dialysis were obese (body weight: >150% of ideal body weight). Since obesity has been shown to lower insulin binding to the cell receptors (lo), these obese patients were not included in the present study. Due to this limitation only seven diabetic patients with CRF could be selected for this investigation (Table 1). None of the CRF patients selected received blood transfusions at least 3 months prior to the study. Thirty, nonobese (body weight: 102 + 8% of ideal body weight), nondiabetic normal subjects of both sexes and with no personal or family history of diabetes or CRF were studied as controls. They ranged in age from 29 to 55 years. Proper informed written consent was obtained. Blood was drawn from all subjects before breakfast and after an overnight fast. Four to ten milliliters of blood was sufficient from normal subjects while 15 to 20 ml of blood was obtained from uremic patients just prior to dialysis. Zsolation Erythrocytes, Insulin Binding to Cells, and Data Analyses The erythrocytes were isolated by Hypaque-FicoIl gradient centrifugation and were equilibrated with buffer G as described by Gambhir et al.
JW Mean k SD
GW
43 67 62 45
CH IL MM EP BT
53 I
68 44 44
Age
Patient
F F M F M
F
M
Sex
4
101
107 97 102 101
103 100 97
Percentage IBW”
74 74 86 73 27
57 55 51 124
BUN
1
10.3
10.5
12
10
9
10 11 10
Insulin r&/ml
132 66
100
83 120 105 120 125 275 38 32
-
30 22 28 102 28 18
7.5 1.5
-
10 6.5 5.9 6.7 7.9 7.7
C-peptide rig/ml
Diabetics with renal failure
Glu’
TABLE 1 PATIENT PROFILE
6.7 12.8 7.2 9.2 2.4
11.7
7.3 8.7 10
glycol. hemogl.
6.7 1.9 4.9 3.1 7.0 4.6 4.7 4.7 1.8
Percentage lBd
Glomerulosclerosis Glomerulosclerosis Glomerulosclerosis Glomerulosclerosis Glomerulosclerosis Glomerulosclerosis Glomerulosclerosis
Diagnosis
2 b
44 52 60 41 36 70 67 58 65 47 63 45 70 53 59 37 54 12
M M F F M F F M M F M M F F M M
101 107 102 100 102 loo % 89 75 105 94 86 78 133 110 99 14
93 102 82 90 90 83 NDe 86 ND 152 ND 64 ND 116 ND 80 94 23
-
21 II 12 IO 29 IO ND” 19 ND 14 ND 18 ND 19 ND 12 16 6
Nondiabetics 80 104 100 100 110 85 ND 115 ND 115 ND 85 ND 115 ND 100 101 13
with renal failure 25 9.5 38 11.5 15 8.5 IO 4.3 28 8.4 22 6.2 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 23 8.1 9.9 2.5 9.3 12.8 8.0 9.0 7.2 ND ND ND ND ND ND ND ND ND ND 9.3 1.9
11.4 14.0 11.1 10.0 16.9 12.8 12.5 17.4 11.4 15.3 14.2 15.5 12.0 9.3 11.6 14.3 13.1 2.4
o Ideal body weight. b Creatinine. c Glucose. d Maximum percentage specific L451-insulin binding. p Not determined on the day of the receptor assay. However, patient charts indicated established uremic values.
RJ WM EP BT cw cs PE AE SN SM FW MR SM HS SJ JD Mean + SD
Chr. giomerulonephritis Chr. glomerulonephritis Nephrosclerosis Nephrosclerosis Nephrosclerosis Nephrosclerosis Nephroscierosis Nephrosclerosis Obstructive nephropathy Interstitial nephritis Nephrosclerosis Nephrosclerosis Nephrosclerosis Polycystic kidney Nephrosclerosis Nephrosclerosis
66
GAMBHIR ETAL,
(17). Isolated and purified erythrocyte suspension did not contain more than 0.1% reticulocytes as determined by methylene blue staining. The insulin receptor binding data and the Scatchard analyses of these data were obtained as described earlier ( 1.5). Determination of Insulin, C-peptide, Glycosylated Hemoglobins. and Glucose
From the fasting serum samples of all the subjects insulin determinations were carried out by the modification of double antibody radioimmunoassay of Morgan and Lazarow (18). Insulin values based on a similar RIA procedure were also determined by Ms. Carla Hendricks, and Ms. Janice Ryan in the Laboratory of Phillip Gorden, M.D., Diabetic Branch, NIAMDD, Bethesda, Maryland. The C-peptide values were estimated utilizing the RIA kit from Calbiochem-Behring Corporation. Glycosylated hemoglobin level was determined according to the method of Trivelli et al. (19) by Dr. G. Giridhar. Metpath Laboratories, Hackensack, New Jersey. The blood samples for these determinations were collected in NaF. Serum glucose determinations were carried out according to the glucose oxidase method utilizing Beckman GlucoseBUN analyzer II. RESULTS
As shown in Fig. 1, the maximum specific binding to 3.52 x 1tY erythrocytes when exposed to 100 pg of iz51-insulin was 4.7 2 1.8% (mean + SD) in the diabetic patients with CRF. This maximum specific i251insulin binding was significantly lower (P < 0.01) than that for the normal subjects (mean t SD, 9.9 ? I .4%) as well as for the nondiabetic patients with CRF (mean ? SD, 13.1 * 2.4%). Further, 1251-insulin binding to erythrocytes in the presence of a range of physiological insulin concentration up to 10 @ml was significantly lowered (P < 0.05) in the diabetic patients with CRF than in the normal subjects and in the nondiabetic patients with CRF. However, the amount of insulin required to inhibit 50% of the maximum specific lz51-insulin binding (ID,,), in the diabetic patients with CRF was similar to that in the normal subjects. The ID50 values for the diabetic patients with CRF, the nondiabetic patients with CRF and normal subjects were 4.8, 3.2, and 4.8 &ml, respectively (Table 2). The Scatchard analyses of binding data (Fig. 1) yielded curvilinear plots (Fig. 2). The Scatchard plots were interpreted by the procedure described by DeMeyts and Roth (20). The number of insulin receptors per cell (R,) and average affinity constants (R, and R,) depend upon the intercept of extrapolated Scatchard plot on X-axis. The extrapolation of the Scatchard plot in turn depends upon the selection of final insulin concentration used as a last point in DeMeyts’ analyses. The characteristic features of the erythrocytes’ insulin receptors are shown in Table 2. Since the specific
INSULIN
BINDING
0 NON-DIABETIC . NORMAL 4 DIABETIC CRF
ir
v)
10“
1CP
TOTAL
67
TO RBC IN CRF
10’
1W
INSULIN
CONCENTRATION
CRF
1P
1P
hg/mll
FIG. I. ‘051-insulin binding to erythrocytes. Erythrocytes from normal, nondiabetic, and diabetic patients on maintenance dialysis were isolated, washed, and resuspended in a Hepes-Tris buffer, pH 8.0, to constitute a suspension with 4.4 x 1fP erythrocytes per milliliter. Then 400 ~1 (I .76 x IP erythrocytes) of each cell suspension was incubated with 100 ~1 of the buffer, containing unlabeled insulin (0 to 0.5 x 106ng) and 50 pg of 1*51-insulin for 3.5 hr at 15°C. At the end of the incubation period, duplicate ahquots of each cell suspension were centrifuged through chilled buffer (4°C) and dibutylphthatate gradient. 1*51-insuiin bound was determined by counting cell pellets. The data are expressed as mean k SD for 30 normal subjects, I6 nondiabetic, and 7 diabetic patients with chronic renal failure on maintenance hemodialysis.
percentage 1251-insulin binding to erythrocytes when exposed to 500 and 1000 nglml of unlabeled insulin was significantly different (P c 0.05) from that exposed to 100 rig/ml of unlabeled insulin in the patients studied, the insulin binding data up to 1000 rig/ml of insulin concentration was used for the Scatchard plots. For comparison purposes only (13), R. and average affinity constants were also determined using 100 and 500 rig/ml of insulin concentration as the last point of the Scatchard plots (Table 2). To be consistent with our previous report (16), we have used total insulin binding up to 1000 rig/ml of insulin concentration for the Scatchard plots. The zxtrapolation from Scatchard plots resulted in 190 receptor sites per zrythrocyte in the diabetic patients with CRF compared to 410 sites per :ell in the nondiabetic patients with CRF and the normal subjects. In zontrast to R,, the receptor affinities for the diabetic patients with CRF
9.9 * 1.4 4.7 k 1.8 13.1 -c 2.4
Subject
Cutoff point (@nl)* Normal (30) Diabetic CRF (7) Nondiabetic CRF (16)
4.8 4.8 3.2
IDSO 100 80 40 80
500 230 loo 200
looo 410 190 410
No. of sites/cell (R,)
TABLE 2 INSULIN RECE~OR
100 2.3 2.0 3.1
500 0.83 0.86 1.3
K, x 108 M-’
CHARACTERISTICS
loo0 , 0.43 0.44 0.63
loo 5.4 6.6 6.3
Affinity constant
500 1.3 1.7 1.7
R, x IO’ tv-’ loo0 0.62 0.58 0.46
a Concentration @g/ml) of insulin required 10 inhibit 50% of maximum specific 1251-insulin binding to 3.52 x IO@erythrocytes in I ml of the incubation suspe‘nsion (Fig. I). b The cutoff point (100,500. and 1000 &ml) refers to that selected insulin concentration from the range of insulin concentrations in the receptor assay (Fig. 1) used as the final point for plotting the Scatchard curve. Extrapolation of these curves would result different X-axis intercepts and thus, will give different amounts of insulin bound to calculate the number of receptor sites per erythrocyte. However, to be consistent with our previous report (16). receptor number and affinity constants using 1000 rig/ml of insulin concentration as a cut off point (Fig. 2) were calculated for discussion in the text. R, refers to the average affinity constant at the unoccupied sites and K, refers to the average affinity constant at the filled receptor sites.
Percentage Max. sp. binding
ERYTHROCYTE
F
2
F 2 E j;;
69
INSULIN BINDING TO RBC IN CRF
0 NOM-DIASEIX l
. MASEW
TOTAL
INSULIN
BOUND
CRF
NORMAL
CRF
t1O-‘o MI
FIG. 2. Scatchard plots with affinity profiles. Scatchard analyses of the insulin-binding data (Fig. I) were performed. On the X-axis is the amount of insulin bound, calculated by multiplying the percentage 1*51-insulinbound by total insulin concentration in the incubation medium. The ratio of the bound to the free ins&n is plotted on the Y-axis. The inset depicts the relationship of the log of the percentage of occupancy of receptor sites 6 x MO), calculated by B/R, x 100andthe averageaftinity constant(K). R, equalsB/F /R,,-B. B represents the amountof msulinbound,F is the amountof free insulin,RI depictstotal receptorconcentration,andj is the fraction of receptoroccupancy.In order to plot the rangeof receptorsiteoccupancy,a numberof pointsontheScatchardcurveswerederived.
and the normal subjects were almost similar. The average affinity constants for unoccupied sites (I&.) were 0.46 x 108 and 0.44 x 108 M-’ for the diabetic patients with CRF and normal subjects respectively. By comparison, nondiabetic patients with CRF showed a marked increase in R, (0.63 x 108 M-l). In the normal subjects and the diabetic patients with CRF, the average affinities for filled sites, Et, were 0.62 x lo7 and 0.58 x 10’ M-I, respectively, compared with 0.46 x lo7 M+ in the nondiabetic patients with CRF. Serum insulin concentrations were 38 & 32 PUlml (mean rfr SD) for the diabetic patients with CRF, 22.8 f 5.7 @/ml for the normal subjects, and 23 ? 9.9 pU/ml for the nondiabetic patients with CRF. Since these diabetic CRF patients were not receiving insulin they did not show any presence of insulin antibodies in their plasma as determined by lack of insulin binding properties to their plasma. The levels of plasma insulin in these subjects were not significantly different (P > 0.05) when compared to each other. The C-peptide values were significantly higher (P < 0.05) in the patients than in normal subjects. These values were 7.5 2 1.5 rig/ml
70
GAMBHIR
ET AL.
(mean fr SD) for the diabetic patients with CRF, 3.7 F 0.6 rig/ml for the normal subjects, and 8.1 ? 2.5 rig/ml for the nondiabetic patients with CRF. C-Peptide clearance is lowered in CRF (21). Thus, the higher C-peptide values in CRF are feasible without noticeable hyperinsulinemia. It should be mentioned at this point that insulin binding to erythrocytes was not the lowest in that subject (EP-Table 1) whose serum insulin concentration was the highest among all the subjects studied. We did not see any relationship of insulin binding to endogenous insulin concentration (r = -0.32). when all the subjects were considered together or even when they were considered as three separate groups. The glycosylated hemoglobin levels were also not statistically different (P < 0.05) in these three groups. The diabetic patients with CRF had 9.2 ? 2.4 (mean t SD, percentage of total hemoglobins), the nondiabetic patients with CRF had 9.3 IT 1.9 (mean + SD, percentage of total hemoglobins) and the normal subjects had 7.3 + 1.3 (mean I~I SD, percentage of total hemoglobins). It should be noted here that one of the nondiabetic patients (WM-Table 1) with CRF had a higher glycosylated hemoglobin level in spite of the fact that this glucose level was not elevated (104 mg/ml). Although the exact reason for increased glycosylated hemoglobins level is not known, a possibility exists that isocyanate, which is known to be present in in vitro preparations of urea, may cause carbamylation of hemoglobin molecules (22) in the patients with CRF. These carbamylated hemoglobin molecules are eluted along with glycosylated hemoglobins in the procedure for the isolation of glycosylated hemoglobins. To investigate the possibility of a factor responsible for lowering insulin binding in the diabetic patients with CRF, the isolated and purified erythrocytes from a normal subject were preincubated in an equal volume of plasma from a diabetic patient with CRF at 37°C for I hr before the insulin radioreceptor assay. The ‘251-insulin binding to those erythrocytes remained unchanged. Similarly, the ‘251-insulin binding to erythrocytes from diabetic patients with CRF preexposed to normal plasma, did not change. However, preincubation of the erythrocytes from a diabetic CRF patient with serum containing anti-insulin receptor antibody lowered more than 90% of the insulin binding to these erythrocytes. DISCUSSION These studies demonstrate that there is a marked reduction in insulin binding to erythrocytes in the diabetic patients (4.7%) with CRF as compared to both the normal subjects (9.9%) and the nondiabetic patients with CRF (13.1%). A similar decrease in insulin binding to erythrocytes has been demonstrated in diabetic patients without renal failure (9) as well as in nondiabetic patients with renal failure who were not on dialysis (23). The diabetic patients without CRF (9) and nondiabetic patients with CRF
INSULIN
BINDING
TO RBC
IN CRF
71
but not on dialysis (23) had 5.9 and 7.9% specific insulin binding, respectively. However, diabetic patients with CRF in the study, showed a further decrease in insulin binding compared to the diabetic without CRF (9) and the uremic patients (23). A decrease in insulin binding has also been shown in monocytes (7), adipocytes (8), and placental membranes (24) of diabetic patients without CRF. Increased insulin binding to erythrocytes in nondiabetic patients with CRF who were undergoing hemodialysis cannot be explained at this time. There are reports showing increased insulin binding to younger erythrocytes than to older cells (25,26). One expects more younger cells in CRF patients. If increase in younger cells were to be the cause of increased insulin binding it should have also been evident in the nondiabetic patients with CRF who were not on dialysis. The Scatchard analyses indicated that the marked reduction in insulin binding observed in the diabetic patients with CRF was accompanied by a decrease in R,, while the average affinity constants, k, and &, remained almost the same as those of the normal subjects. Robinson et al. (9) also observed reduction in R, with little or no change in the average receptor affinities in erythrocytes from the diabetic patient without CRF. On the other hand, the nondiabetic patients with CRF had R, similar to the normal subjects but the average affinity constant, k, was increased (40%). In the acutely starved obese human subjects, Bar et al. (10) have reported variation in receptor affinities without any changes in R, compared to the unstarved obese subjects. Many investigators have reported the presence of reduced insulin sensitivity and down regulation of insulin receptors accompanied by hyperinsulinemia in diabetes (8,9), obesity (lo), acromegaly (1 I), and insulinoma (27). In the present investigations, such a relationship was not evident as shown by analyses of variance of endogenous insulin levels and insulin binding to erythrocytes (r = -0.32). A number of other studies also have been reported which do not support down regulation of insulin receptors (28,29). According to this study, the patient groups do not show any significant variations in levels of fasting glucose, insulin, and glycosylated hemoglobins as compared to those of normals. The C-peptide values for the patients with CRF were higher than normal as similarly shown by Jaspan et al. (21). The higher C-peptide levels in patients with CRF were due to the prolonged half life of endogenous C-peptide compared to that of nsulin and these increased C-peptide values did not constitute a hyperiniulinemia in patients with CRF (21). The only differentiating entity among ;he patients with CRF were higher than the normals as similarly shown by zrythrocytes. The metabolic studies by DeFronzo (6) have shown reduced glucose utilization in undialyzed patients with renal failure and return to almost normal following dialysis. We previously have demonstrated (23) that
72
GAMBHIR
ET AL
reduced insulin binding in the nondialyzed uremic patients was improved following chronic dialysis. In this investigation, we are reporting decreased insulin binding in the dialyzed diabetic patients with CRF and confirming the improved insulin binding after dialysis in the nondiabetic patients with CRF. The abnormal carbohydrate metabolism in the renal failure which has been related to reduced insulin sensitivity to peripheral tissues by very elaborate techniques and experiments (4-6) can also be evaluated as an insulin receptor defect by studying insulin binding to erythrocytes using the much simpler erythrocyte insulin receptor assay. Thus, if the receptor modulation acquires a status of treatment for diabetes, evaluation of insulin receptor status may become evident in the treatment of diabetes. In such a case, availability of representative cell type and a suitable receptor assay as well as measurement of receptor characteristics will be a necessity. The present investigation can be viewed as an attempt to satisfy these requirements. SUMMARY
1251-Insulin binding to most accessible and easily obtained circulating erythrocytes in 7 diabetic and 16 nondiabetic, nonobese patients with chronic renal failure (CRF) on maintenance hemodialysis was studied and compared with that of the 30 normal, nonobese volunteers. The percentage of 1251-insulin binding was 4.7 + 1.8 (mean + SD) in the diabetic and 13.1 + 2.4 in the nondiabetic patients with CRF while in the normal subjects it was 9.9 k 1.4. There was no statistical difference in the fasting levels of glucose, insulin, and glycosylated hemoglobins in all the subjects studied. No correlation (r = -0.32) between insulin binding and endogenous insulin was observed in these subjects. The diabetic patients with CRF had no circulating factor(s) that would reduce insulin binding to nornal erythrocytes. In comparison to the normal subjects, the reduction in insulin binding in the diabetic patients with CRF was accompanied by 47% reduction in insulin receptor sites (R,) but with no change in insulin receptor affinity, (R,, 0.46 x 108 M-I), while in the nondiabetic patients with CRF no change in insulin receptor sites (R,) of increased affinity (k,,, 0.63 X 108 M-l) was observed. These findings suggest that insulin bindin utilizing the much simpler erythrocyte insulin receptor assay can be use 1 to evaluate alteration in insulin receptor status in CRF. These investigai tions also provide a very useful assay and characteristic studies for insulin rei ceptors, if receptor modulation acquires a status of treatment for diabetes. REFERENCES 1. Linder, G. C., Hiller, A., and VanSlyke, D. D., J. Cfin. Invest. 1, 247 (1925). 2. DeFronzo, R. A., Andres, R., Edgar, P., and Walker, W. G., Medicine 52,469 (1973). 3. Simmons, R. G., and &hilling, K. .I., in “Rehabilitation: Diabetic vs. Non-Diabetiq Transplants” (M. Schesinger,R. E. Billingham, F. T. Rapaport, Eds.), Transplantal tion Today Vol. 3, p. 719. Grune & Stratton, New York, (1975).
INSULIN
BINDING
TO RBC IN CRF
73
4. Westervelt, F. B., J. Lab. Clin. Med. 74, 79 (1969). 5. Mondon, C. E., Dolkas, C. B., and Reaven, G. M., Diabetes 27, 571 (1978). 6. DeFronzo, R. A., Metabolism 27, 1866 (1978). 7. Goldstein, S., Blecher, M., Binder, R., Perrino, P. V., and Recant, L., Endocr. Res. Commun. 2, 367 (1975). 8. Olefsky, J. M., Diabetes 25, 1154 (1976). 9. Robinson, T. J., Archer, J. A., Gambhir, K. K., Hollis, V. W., Jr., Carter, L., and Bradley, C., Science 205, 200 (1979). IO. Bar, R. S., Gorden, P., Roth, J., Kahn, C. R., and DeMeyts, P., J. Clin. Invest. 58, 1123 (1976). Il. Muggeo, M., Bar, R. S., Roth, J., Kahn, C. R., and Gorden, P., J. Clin. Endocrinol. Med. 48, 17 (1979). 12. Kahn, C. R., Flier, J. S., Bar, R. S., Archer, J. A., Gorden, P., Martin, M. M., and Roth, J., N. Engl. J. Med. 294, 740 (1976). 13. Wachslicht-Rodbard, H., Gross, H. A., Rodbard, D., Ebert, M. H., and Roth, J., N. Engl.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
J. Med.
300, 882 (1979).
Soman, V., and Felig, P., Clin. Res. 27, 260A (1979). Gambhir, K. K., Archer, J. A., and Bradely, C., Diabetes 27, 701 (1978). Nerurkar, S. G., and Gambhir, K. K., Clin. Chem 25, 1672 (1979). Gambhir, K. K., Archer, J. A., and Carter, L., Clin. Chem 23, 1590 (1977). Morgan, C. R., and Lazarow, A., Diabetes 12, 1 I5 (1%3). Trivelli, L. A., Ranney, H. M., and Lai, H. T., N. Engf. J. Med. 284, 353 (1971). DeMeyts, P., and Roth, J., Rio&em. Biophys. Res. Commun. 66, 1118 (1975). Jaspan, J. B., Mako, M. E., Hideshi, K., Blix, P. M., Horowitz, D. L., and Rubenstein, A. H., J. Clin. Endocrinol. Metub. 45, 441 (1977). Lee, C. K., and Manning, J. M., J. Biol. Chem. 248, 5861 (1973). Gambhir, K. K., Archer, 1. A., Nerurkar, S. G., Cruz, I., and Sanders, M., Nephron, in press. Harrison, L. C., Billington, T., Clark, S.. Nichols, R., East, I., and Martin F. I. R., J. Clin. Endocrinol. Metab. 44, 206 (1977). Kosmakos, F. C., Nagulesparan, M., and Bennett, P. H., J. Clin. Endocrinof. Metab. 51, 46 (1980). Eng. J., Lee, L., and Yalow, R. S., Diabetes 29, 164 (1980). Bar, R. S., Gorden, P., Roth, J., and Siebert, C. W., J. Clin. Endocrinol. Metab. 44, 1210 (1977). Thorsson, A. V., and Hintz, R. L., N. Engl. J. Med. 297, 908 (1977). Misbin, R. I., O’Leary, J. P., and Pulkkinen, A., Science 205, 1003 (1979).