BIOCHEMICAL

MEDICINE

AND

METABOLIC

BIOLOGY

45, 133-153 (1991)

REVIEW Red Blood Cell Insulin Receptors in Health and Disease KANWAL Molecular

Endocrinology

K. GAMBHIR’

AND VEENA R. AGARWAL*

Laboratory, Department of Medicine, College University, Washington, DC 20059

of Medicine,

Howard

Received April 30, 1990, and in revised form August 24, 1990 Contents: Structure and characteristics of erythrocyte insulin receptor. Red blood cell age and insulin receptors. Insulin receptors in human disease states. Obesity. Chronic renal failure. Acanthosis nigricans. Miscellaneous disease states. Insulin receptors in children. Insulin receptors in women during pregnancy. Insulin binding and other hormones. Comparison of biosynthetic insulin, pancreatic human insulin and porcine insulin binding to erythrocytes. Effect of exercise on insulin binding to red blood cells of normal human volunteers. Miscellaneous insulin binding studies. Insulin internalization and degradation. Insulin and erythrocyte metabolism. Summary and conclusion. o 1991 Academic PIWS. IK.

INTRODUCTION Conventionally, hepatocytes, myocytes, and adipocytes are regarded as “target cells” for insulin. Insulin receptors have been identified in a variety of other human cells, viz., brain, placental, and kidney cells; lymphocytes; monocytes; and erythrocytes (1). Technical limitations make it difficult to evaluate insulin receptor status in human beings because the process requires either tissue biopsy or a large amount of blood. Discovery of specific insulin receptors in erythrocytes provided an extremely useful and easily accessible cellular model for study of insulin receptors in humans. Moreover, specific insulin receptors of erythrocytes with hormone binding characteristics, similar to those of other human cell types, provided a milieu whereby erythrocytes could be used in a variety of morbid and physiologic conditions (2). Extensive work on insulin binding to human erythrocyte insulin receptors ’ To whom all correspondence should be addressed. ’ This author compiled all the literature and drafted this review. She was an international trainee fellow and was supported by the grant from the Latham Trust Fund.

research

133 08854505/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(EIRs)~ has been done under multiple physiologic and pathologic conditions; nevertheless, there is no comprehensive reference to studies involving the red blood cell (RBC). This review provides a compilation from the literature describing insulin receptor studies involving human erythrocytes. STRUCTURE AND CHARACTERlSTlCS OF ERYTHROCYTE 1NSULlN RECEPTOR

Like hepatocytes and adipocytes, insulin receptors of erythrocytes have insulin binding (o-subunit) and tyrosine kinase (&subunit) components (3-5). Also, like other cells, EIR is a tyrosine-specific protein kinase and possesses sulfhydryl groups which are important for enzyme activity (6,7). Insulin receptor consists of three species of molecular weights approximately 295,000, 265,000, and 245, 000 containing two disulfide-linked subunits of molecular weights approximately 130,000 and of 95,000. To evaluate the location of receptors on the membrane, the insulin binding to sealed outside-in and inside-out human erythrocyte vesicles was studied (8). These studies suggested that receptors were present on both sides of the plasma membrane. Baumann et al. (9) found that binding sites were on the extracellular surface and that transmembrane mobility of the receptors from one side to the other was severely restricted. Recently, Kelleher et al. (10) confirmed the insulin internalization in human erythrocyte ghosts. When erythrocyte ghosts were incubated with insulin, the number of free surface insulin receptors decreased by 30-40%. The number of insulin receptors in solubilized ghosts was the same as that in insulin-treated or untreated ghosts. This finding suggested that erythrocyte ghost insulin receptors were internalized to a vesicular compartment in response to insulin treatment. Downregulation of insulin receptors also indicated that EIRs were, subsequently, recycled to the surface of the cell (11). On the other hand, Schulz et al. (12) could not find a downregulation of insulin receptors in their in vitro studies. Benson et al. (13) also concluded that the insulin binding to erythrocytes was an invalid model to study downregulation of insulin receptors. But, other characteristics of insulin binding sites of erythrocytes were similar to those of other insulin binding cells, viz., direct relation of specific insulin binding to cell concentration, an inverse relationship between specific binding and fasting plasma insulin level, pH and temperature dependence, and upward Scatchard plots (2,14-16,X3). Further, like liver and fat cells, there were two types of insulin receptors on erythrocytes (17). One population of receptors was inhibited by concanavalin A (a lectin which mimics insulin activity, leaving another population unaffected). Negative cooperativity was also shown by insulin receptors on erythrocytes (2). Gherzi et al. (19) reported that preincubation of mature RBCs with high con3 Abbreviations used: EIR, erythrocyte insulin receptor; HEIRA, human erythrocyte insulin receptor assay; NIDDM, non-insulin-dependent diabetes mellitus; IDDM, insulin-dependent diabetes mellitus; OGTT, oral glucose tolerance test; PCOD, polycystic ovarian disease; MD, myotonic dystrophy; FA, Friedreich’s ataxia; DMD, Duchenne muscular dystrophy; IDE, insulin degradation enzyme; and CRF, Chronic Renal Failure.

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RECEPTORS

centration of insulin or insulin with glucose had no effect on the number or the affinity of their insulin receptors. On the other hand, by comparing the insulin binding in different cells of humans, pigs, and rodents, Hjollund et al. (20) concluded that measurement of insulin binding to blood cells cannot replace studies of insulin binding to liver, fat, and muscle cells. On the basis of the above work with two points of view, we conclude that erythrocytes have insulin binding sites which are indistinguishable from insulin receptors of other insulin binding tissues. Thus, when human erythrocyte insulin receptor assay (HEIRA) is performed appropriately, the EIR can be tested invariably like insulin receptors in nucleated cells. Erythrocyte ghosts are preferable to the intact RBC for long-term insulin receptor studies (22,23) due to the fact that measurements of insulin binding in erythrocyte ghosts are similar to those in intact RBCs, allowing their storage without loss of binding characteristics (21). This also minimizes interassay variation in insulin binding to RBCs. RED BLOOD CELL AGE AND INSULIN RECEPTORS The relationship of insulin receptor numbers to erythrocyte age was shown by Kosmakos et al. (24). Having a half-life of 40 days, insulin receptor is an agedependent integral component of human erythrocyte membrane (24-31). ‘*‘IInsulin binding to RBCs, including reticulocytes, was found to decrease exponentially as a function of their mean cell age. A more rapid decline of insulin receptors occurred coincidently with reticulocyte maturation. Clinically, the highest binding was found in a patient with hereditary spherocytosis and very active hematopoiesis (32). Camagna et al. (32) characterized insulin receptors from young and old RBCs. Their results demonstrate that only young RBCs respond to dexamethasone and glucose ingestion; old RBCs gave no response. Some data reported on whole RBCs are not in agreement with those reported in cells like hepatocytes, adipocytes, and myocytes (28). Therefore, it is recommended that erythrocytes be separated by age before any insulin receptor studies are undertaken. Red cells may be an excellent cell model to understand the aging process. The life of the cell is 120 days. INSULIN

RECEPTORS

IN HUMAN DISEASE

STATES

Non-Insulin-Dependent Diabetes Mellitus

A number of groups observed reduced insulin binding to erythrocytes in nonobese, mature non-insulin-dependent diabetes mellitus (NIDDM, type II) patients in comparison to controls (32-36). This reduced binding due to a decreased number of receptors was inversely correlated with insulin concentration. Prager and Schernthaner (37) compared insulin binding in erythrocytes and monocytes in the newly diagnosed hyperinsulinemic type II diabetics. In both cell types, binding was significantly reduced; this reduction was due to a fewer number of receptors. However, on individual patient analyses, authors observed a marked discrepancy between erythrocyte and monocyte binding. Since monocytes have a much shorter turnover time (hours to days) in the circulation than erythrocytes

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(120 days), the two cell types might be affected differently by circulating factors affecting receptor activities. Kautzky et al. (38) found that insulin binding to erythrocytes was decreased in young patients with NIDDM. This decreased binding was due to decreased number of receptors rather than the reduced affinity. When insulin binding was followed in the circulating erythrocytes and monocytes of young third-generation NIDDM patients (39), binding was decreased in monocytes but no difference in insulin binding to erythrocytes was found. The effect of oral drug treatment on EIRs in type II diabetes has been evaluated in several studies. EIRs were evaluated in nonobese NIDDM before and after 14 and 90 days of chloropropamide (250 mg/day) treatment (40). Insulin binding was not affected by this drug treatment, but the normalization of the initially low number of receptors and an increased high affinity constant were found. Results showed that with chloropropamide treatment, improvement of glucose tolerance in NIDDM is associated with a greater insulin secretion and with a correlation in receptor parameters which could be partially related to proportionate changes in reticulocyte count. Using normal subjects, Hjollund et al. (41) noted that glibenclamide did not affect the insulin binding to erythrocytes (in vitro and in vivo experiments) but it increased insulin binding in monocytes. In contrast to these results, Agarwal observed improved insulin binding in erythrocytes of NIDDM patients after glibenclamide treatment which was due to an increased number of receptors rather than affinity (42). The reason for this difference was that Hjollund et al. used normal subjects who might not respond to improved insulin binding after treatment with glibenclamide. The effects of clofibrate are contradictory. One group found improved insulin binding to erythrocytes with an increased oral glucose tolerance test (OGTI’), while the other group showed no response (43,44). The same results were observed with the biguanide (metformin). In other studies, metformin either did (45) or did not (46) produce a rapid increase in binding affinity; even these studies showed improvement in glucose disposal rate (45,46). Rizkalla et al. (47) reported that metformin increased the number of receptors in obese type II diabetics. An increase in insulin binding to erythrocytes was found in type II diabetics after insulin therapy. This increase was correlated with the fall in fasting hyperglycemia (48). Nankervis et al. (49) did not find any change in insulin binding after insulin treatment in NIDDM patients nor did they observe any established difference in insulin binding in NIDDM patients as compared to normal controls. Moreover, during insulin infusion, downregulation of insulin binding to erythrocytes was similar in both normal subjects and NIDDM patients (50). In the Hjollund et al. study, a diet of low fat/high starch/high fiber did not affect insulin receptor binding in RBCs (51). Dietary regulation of insulin receptor binding in erythrocytes of NIDDM is abnormal (52), but it warrants further elucidation. One recent report showed that treatment of poorly controlled NIDDM subjects with granulated guar gum improved EIR binding (53).

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Insulin-Dependent Diabetes Mellitus

Erythrocytes from insulin-dependent diabetes mellitus (IDDM) patients with acute diabetes showed varying degree of insulin binding (56). Circadian profiles of insulin receptors have been studied in IDDM (type I) in normal and poor metabolically controlled subjects (57). In conventionally controlled diabetics and normal volunteers, binding of insulin to erythrocytes was found to be high in the early morning and low at daytime and to peak around midnight. Diabetics with poor metabolic control due to insulin deprivation had preserved a similar 24-hr rhythm of EIRs, while insulin receptor binding to monocytes decreased significantly during the daytime both in newly discovered diabetes and in healthy controls. Mechanisms responsible for these changes are unknown; however, in diabetics the interrelationship between erythrocyte insulin binding and plasma insulin concentration during the 24-hr period suggested that in these patients, insulin might be one of the factors determining the rapid insulin receptor regulation. In 1986, Hung et al. (58) studied insulin requirements during fasting and feeding variation and insulin binding to erythrocytes at different times of the day in IDDM patients accessed under glucose-controlled insulin infusion. The calorie and carbohydrate-related insulin demands after breakfast were highest and declined after lunch. The insulin tracer binding was higher at 0800 hr before breakfast than at 1200 hr before lunch. The increased binding could be more likely attributed to change in receptor concentration than to change in affinity. Further, Yasuda and Kitabchi (59) have reported increased insulin binding in diabetic ketoacidosis due to elevated receptor affinity which was reversible with insulin therapy. A study of drug treatment showed that short-term use of chlorpropamide, in addition to insulin, in IDDM patients did not affect insulin binding to erythrocytes (60). OBESITY

In comparison of insulin binding to erythrocytes and monocytes from obese and nonobese subjects, binding was decreased in both cells of the obese due to a decrease in receptor concentration and a 50% increase in monocyte receptor concentration (63). Later in 1985, Pav et al. (64) found that insulin binding was significantly lower in obese patients having NIDDM in comparison to control subjects, but no significant difference was observed in insulin-dependent nonobese NIDDM patients. Both groups showed lower numbers of receptors in insulindependent diabetes which seems to be counterbalanced partly by their high affinities. In a family experiment of insulin binding to erythrocytes, binding was decreased in the obese subjects in comparison to their normal-weight siblings and parents (65). The daily food intake was the same in the family subjects. The studies dealing with the influence of diet show that carbohydrates and high-fat diets lower insulin binding to erythrocytes due to reduced number of receptors. In a detailed study of the influence of diet on insulin binding, Rizkalla et al. (66) noted that hypocaloric diet in moderately obese subjects affected EIRs. Hypocaloric carbohydrate-free diet or diet supplemented with glucose or galactose produced a decrease in plasma insulin and glucose concomitant with an increase in the number

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of insulin receptors. No change in insulin receptors was found when diet was supplemented with fructose. Rizkalla et al. (66) also studied EIR changes before and after 8 days of continuous subcutaneous insulin infusion by a pump in uncontrolled obese NIDDM patients. The specific insulin binding was significantly increased and this increase was correlated with the fall in fasting hyperglycemia. In addition, the increase in binding correlated negatively with changes in fasting plasma insulin levels. CHRONIC RENAL FAILURE Our investigations have shown that chronic renal failure (CRF) is associated with altered insulin sensitivity (67,68). The patients with CRF showed a reduction in insulin binding and in the number of receptor sites with increased affinity. The binding and receptor concentration was found to be improved after chronic dialysis (69). Further, there was a significant decrease in insulin binding in CRF patients with diabetes in comparison to both normal subjects and nondiabetic CRF patients (67,69). This difference was due to decreased number of insulin binding sites with no change in its affinity. Milutinovic et al. (70,71) reported a 50% decrease in insulin binding in nondiabetic uremic patients when compared to healthy controls. During hemodialysis, insulin binding to erythrocytes steadily increased in a timedependent manner in proportion to the efficiency of hemodialysis as assessed by relative decrease in plasma urea and creatinine (69). It was not correlated with the changes in plasma insulin levels. These reports indicated the presence of dialysable inhibitors of insulin binding in uremic plasma. Later in 1987, Marttins et al. (72) confirmed that uremic plasma contains inhibitors of insulin binding to erythrocytes. No difference in insulin binding was found in purified uremic erythrocytes in comparison to that of normal subjects. Uremic plasma caused a significant decrease even in insulin binding on healthy erythrocytes. Hemodialysis treatment decreased the inhibition of insulin binding by uremic plasma. In contrast, Weisinger et al. (73) concluded that insulin resistance observed in uremia did not involve a defect in hormone binding or in the intracellular capacity to utilize glucose through glycolysis. In the author’s investigations, insulin degradation, a postreceptor event of insulin action, was also found to be lower in erythrocytes of CRF patients in comparison to those of normal subjects (74). Further, a linear correlation was observed between the duration of dialysis and maximum percentage of insulin degradation in the CRF patients. Insulin binding to erythrocytes was studied in pediatric renal transplant recipients (75). Ten of the thirteen patients had normal binding while three showed reduced binding of insulin. ACANTHOSIS NIGRICANS Two patients with obesity, acanthosis nigricans, acral hypertrophy, basal hyperinsulinism, and exaggerated insulin responses to oral glucose were studied (76). One of the patients, a diabetic female, showed features of virilization due to polycystic ovarian disease (PCOD) and underwent gonadectomy with some resolution of her androgenization and acanthosis nigricans. Insulin binding to erythrocytes was found to be decreased in both patients. Three years after a bilateral

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ovarian wedge resection in a 24-year-old patient with a triad of acanthosis nigricans, hirsutism associated with polycystic ovaries, and insulin resistance, extreme resistance to endogenous and exogenous insulin was found (77). Studies on insulin binding to RBCs showed abnormal inhibition-competition curves characterized by an increased percentage of insulin binding at higher unlabeled insulin levels, which was due to an apparent increase in the number of low-affinity receptors; binding of insulin to other insulin binding tissues appeared normal. In 1984, when Chaussain et al. (78) studied specific binding of insulin to erythrocytes in girls with acanthosis nigricans and hirsutism, they found significantly lower binding. Thus, they concluded that insulin resistance in these patients was due to primary defect of insulin receptors, while an earlier group suggested that this was due to postreceptor defect. In the absence of acanthosis nigricans in nonobese patients with PCOD, specific insulin binding was found to be decreased and attributed to decreased receptor affinity. These results established insulin resistance in the patients with PCOD (79). The type A syndrome of insulin resistance and acanthosis nigricans is characterized by severe insulin resistance due to cellular defect in insulin action (80). To understand the molecular nature of this defect, insulin binding to circulating monocytes, erythrocytes, Triton X-lOO-solubilized EIR, and insulin-stimulated receptor autophosphorylation were studied. The results showed that maximal stimulated phosphorylation was reduced linearly with insulin binding. MISCELLANEOUS DISEASE STATES In patients with anorexia nervosa, insulin binding was found to be increased due to increased number of receptors per cell with little change in affinity (81). This abnormality of binding was corrected by restoration of normal food intake and body weight. Friedreich’s ataxia (FA) is a neurological disorder associated with a high prevalence of diabetes mellitus. In 1979, Draper et al. (82) did the preliminary studies on insulin binding to erythrocytes of patients having FA. They observed a change in binding in diabetic FA, while the binding in the nondiabetic FA group was similar to that of the nondiabetic controls. A small group of the nondiabetic siblings showed normal insulin binding, while increased binding at low insulin concentration among diabetic family members was observed. Later, Kahn et al. (83) studied insulin binding in fractionated erythrocytes from FA patients and normal volunteers. Both groups showed highest insulin binding to the youngest erythrocytes, but no difference was observed between the two groups. Insulin binding to monocytes was significantly decreased in subjects with FA. In Klinefelter’s syndrome, insulin binding to erythrocytes was found to be decreased (84). In search of elucidation of mechanism and significance of insulin resistance in myotonic dystrophy (MD), researchers found that specific binding of insulin to erythrocytes was slightly reduced in MD patients (85). However, an earlier group demonstrated significantly impaired binding due to a low number of highand low-affinity binding sites (86). In contrast to this, Sakai et al. (87) demonstrated that decreased binding was due to decreased receptor affinity rather than receptor number. Thus, the insulin resistance of MD might be due to both receptor and postreceptor defects. In patients with Reye’s syndrome, insulin binding to RBCs

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was found to be decreased. Sex and age differences were observed in this group’s binding. Reduction in insulin binding may play a role in the acute disease if binding is shared by more traditional insulin binding cells (88). In evaluation of the pathogenesis of insulin resistance in Cushing’s syndrome, no significant changes in the parameters of insulin receptor interaction could be observed (89) although Lee et al. (90) observed higher insulin binding to erythrocytes in Cushing’s disease. Binding was found to be decreased after transphenoidal surgery with Cushing’s syndrome. But, in the patients with adrenal insufficiency, binding was lower in comparison to normal subjects. After glucocorticoid treatment, binding was increased (91). Patients with primary hyperparathyroidism showed a marked hyperinsulinemia and a significant lower insulin binding to monocytes and erythrocytes (92). This insulin-resistant state in primary hyperparathyroidism is caused partly by a downregulation of insulin receptors. In a 17year-old boy with growth retardation, marked hepatomegaly and sexual infantilism and despite high circulating insulin, insulin binding to erythrocytes and cultured fibroblasts was normal. However, the normal increase in thymidine incorporation in response to insulin, epidermal growth and fibroblast growth sectors, was blunted. These studies demonstrated a new form of post receptor defect (93). In a 3-month-old female leprechaun, insulin resistance existed with hyperinsulinemia. Decreased insulin binding to erythrocytes, cultured lymphocytes, and fibroblasts was observed, which was due to decreased concentration of insulin receptors (94). In 1987, Grigorescu et al. (95) studied patients with two inherited conditions of severe insulin resistance (leprechaunism and the type A syndrome of insulin resistance) and their families; insulin binding to erythrocytes in both cases was decreased. This was due to reduced receptors. Tyrosine kinase activity of the solubilized receptors from erythrocytes in these patients also was found to be decreased. Neither parents of the patients had clinical manifestations of leprechaunism or the type A syndrome. The father of the leprechaun had decreased insulin stimulated phosphorylation. Thus, alteration in insulin receptor kinase activity in these clinically normal parents indicated that these alterations may be useful genetic markers and more sensitive than insulin binding studies for examining the patterns of inheritance in these diseases. In hypertriglyceridemia, insulin binding was diminished and this diminution was due to a few number of receptors (96,97). Despite normalization of plasma triglyceride, obtained by diet alone, insulin binding to RBCs was unaltered (96). In severe hyperprolactinemia, downregulation of insulin receptors causes insulinresistant states (98), and insulin binding to monocytes and erythrocytes was reduced due to the low number of receptors. Insulin binding decreased in thyrotoxicosis, and returned to normal after treatment with antithyroid drug or after normalization of serum thyroid hormones (99). There was a reduction in average affinity of insulin receptors, when Suzuki and Fujino (100) studied the EIRs from hyperthyroid patients. No correlation was found between the number of insulin receptors and serum insulin. In 1987, Petrides et al. (101) studied insulin binding to erythrocytes in hyperinsulinemic patients with precirrhosis, hemochromatosis, and cirrhosis. They concluded that no correlation existed between insulin binding on erythrocytes and pathogenesis

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of insulin resistance in liver disease after observing no difference in binding in precirrhotic and cirrhotic patients. Nor was there a difference found in insulin receptor binding to erythrocytes in patients with cystic fibrosis, whereas binding was increased in monocytes (102). Insulin binding to erythrocytes was studied in Duchenne muscular dystrophy (DMD) (103). DMD is an X-linked inherited neuromuscular disease characterized by progressive weakness and severe muscle wasting. Alteration in carbohydrate metabolism is often associated with neuromuscular disorders. About 20-30% decreases were determined in insulin binding to erythrocytes of all DMD patients in comparison to normal males. This was due to lower affinity of insulin receptors. On the other hand, insulin binding to fibroblasts was the same in DMD patients and normal males. The mothers and sisters of male patients showed normal insulin binding. These results suggest that abnormal binding in DMD erythrocytes was an acquired rather than genetic abnormality. Pav et al. (104) studied insulin binding to erythrocyte receptors in acromegalic patients in relation to the activity of acromegaly and to concomitant diabetes mellitus. They observed decreased binding in active and inactive acromegalics in comparison to normal. A greater decrease was found in active acromegaly coupled with diabetes, while Hansen et al. (105) could not observe the difference in insulin binding either to monocytes or to erythrocytes in the acromegalic subjects. In myocardial infarction, no change in insulin binding was found but there may be a decrease in insulin receptors which can cause insulin resistance in the acute phase of the disease (106). In Hodgkin’s disease, hypoglycemia probably produces autoimmunity by producing an antibody to the insulin receptor (107,108). Hypoglycemia remitted after treatment with azathioprine and prednisone (108). A male infant with congenital generalized lipodystrophy had mildly reduced insulin binding with less involvement of the high-affinity receptors in erythrocytes, but not in fibroblasts (109). Insulin binding to erythrocytes was studied in patients with diabetes secondary to chronic pancreatitis or pancreatectomy and was compared to that in patients with type I diabetes and normal controls. Binding was increased with chronic hypoinsulinemia of the same degree (110). In two sisters having insulin resistance due to uncleaved insulin proreceptor, insulin binding to erythrocytes was found to be decreased as in fibroblasts and transformed lymphocytes (111). When insulin binding to erythrocytes was studied in type I diabetes with superimposed Addison’s disease, binding was low during Addisonian crisis but normalized during hydrocortisone treatment (112). Heterogeneity of the insulin receptor interaction in lipoatropic diabetes was found which was due to receptor and postreceptor abnormalities (113). Table I lists a number of other miscellaneous disease states in which insulin binding to erythrocytes has been described. INSULIN RECEPTORS IN CHILDREN

Insulin binding to erythrocytes from small volume of blood is very useful in children with carbohydrate and other metabolic disorders in which abnormal insulin receptor function may occur. Kappy and Plotnick (114) first characterized

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TABLE 1 Changes in Insulin Binding and Miscellaneous Disease States Disease state

Insulin binding

Reference

NIDDM IDDM Obesity CRF Acanthosis nigricans With acral hypertrophy With hirsutism With polycystic ovary With type A syndrome Anorexia nervosa Friedreich’s ataxia Khnefelter’s syndrome Myotonic dystrophy Reye’s syndrome Cushing’s syndrome Adrenal insufficiency Primary hyperparathyroidism A new form of insulin resistance with growth retardation, fatty liver, and hypogonadotropic hypogonadism Patients with syndrome of severe insulin resistance (leprechaun and type A syndrome) and their parents Hypertriglyceridemia Severe hyperprolactinaemia Hyperthyroid patients of different ages Hyperinsulinemic patients with precirrhotic hemochromatosis and cirrhosis Cystic fibrosis Duchenne muscular dystrophy Acromegaly Myocardial infarction Hypoglycemia with Hodgkin’s disease Congenital generalized hpodystrophy Pancreatogenic diabetes Insulin resistance due to uncleaved insulin proreceptor Type I diabetes with Addison’s disease Lipoatropic diabetes

Decreased Controversial Decreased Decreased

(32-55) (55-61) (62-66) (67-75)

Decreased Controversial Decreased Decreased

(76) (77,78) (79)

Controversial Decreased Decreased Decreased Controversial Controversial Decreased Constant

(8&W

(80) W) (85-87)

(88) (89,90) (90,91) (92) (93)

Decreased

(94,95)

Decreased Decreased Decreased Constant

(965’7)

Constant Decreased Controversial Constant Decreased Mildly reduced Increased Decreased Decreased Controversial

(98) (99,100)

(101) WQ) (103) (104,105)

VW (107,108) (109)

W) (111) (112) (113)

the insulin insulin-receptor binding in children. All properties were similar to other insulin binding cells. The erythrocytes isolated from children have greater numbers of insulin receptors in comparison to adults. Red blood cells from cord blood have more receptors even in comparison to children. Knip et al. (115) compared ?-insulin binding to erythrocytes in cord blood from the preterm and the term newborns. There is a negative correlation between insulin binding and gestational age. Preterm infants bound more insulin than term infants which bound more insulin than adults. The increased number of receptors caused high insulin binding in term infants, while high binding in preterm infants was due to both increased receptor concentration and affinity.

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Thus, from these studies the authors hypothesized that increased insulin binding in newborn may be a mechanism by which the growth stimulatory effect of insulin in fetal life was mediated. The authors also characterized the EIRs from human newborns (116) and confirmed above results. Lautala et al. (117) studied insulin binding to the erythrocytes of infants whose mothers had insulin-treated diabetes mellitus. Insulin binding in these erythrocytes was similar to that in normal infants in spite of hyperinsulinemia. This increase in insulin action may contribute to the tendency toward hypoglycemia and may be partly responsible for the macrosomy in infants with insulin-treated diabetes mellitus. The authors also observed a postnatal decrease in insulin binding, which is a possible explanation for the diminishing risk of hypoglycemia after the first few days of life despite persistent hyperinsulinemia. Like adults, insulin binding was also found to be increased in hypoglycemic children (118). During insulin binding studies in erythrocytes from children with type I diabetes, an increase in binding affinity was found, whereas receptor concentration showed an inverse behavior. However, in the first 2 years of the disease, an opposite relationship existed, showing an increase in receptor number and a decrease in affinity (119). In 1980, Kappy et al. (120) failed to demonstrate any change in insulin binding to erythrocytes in children and adolescents with type I diabetes. When biosynthetic human insulin was used in place of porcine insulin in children with IDDM, results were the same as with porcine insulin, i.e., binding affinity of insulin to its receptors was found to be increased while receptor concentration was decreased (121). As in cultured fibroblasts and transformed lymphocytes, decreased insulin binding was observed in erythrocytes, in a 5-year-old boy with clinical Rabson-Mendenhall syndrome. These results suggested that patients with extreme insulin resistance have a primary defect in insulin receptors (122). The insulin receptor studies in erythrocytes also explain the cause of insulin resistance in an infant with leprechaunism (123). In these cases with no other defect causing insulin resistance, erythrocyte receptors have very low affinity to insulin. Decreased insulin binding to the RBCs from the obese children and the adolescents was consistent with the data in the obese adults. Similarly, binding was inversely proportional to the fasting plasma insulin concentration (124,125). There are contrary results on the effect of prednisone on insulin binding to erythrocytes of children. Canivet et al. (126) reported an increased number of insulin receptors, whereas Kan et al. (127) observed no difference in insulin binding, affinity, and receptor concentration of erythrocytes. Human growth hormone can also affect insulin binding in erythrocytes in growth hormone-deficient children (128). Specific receptor binding for insulin-like growth factors has also been measured in young erythrocytes (129). INSULIN RECEPTORS IN WOMEN DURING PREGNANCY In women, insulin binding was higher to erythrocytes in the follicular phase in cornprison to the luteal phase (130,131), but in comparison to men, binding was low even in the follicular phase (131-134). These differences were due to the changes in receptor concentration but not to the binding affinity. No correlation was found between insulin binding to erythrocytes and sex hormones (17/3 estra-

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diol, progesterone, and 17a hydroxyprogesterone) while an inverse correlation existed in insulin binding to monocytes and the aforementioned sex hormones studied (131). Various studies on human pregnancy concluded that the insulin resistance of pregnancy was not accompanied by an impaired binding of insulin to its receptors in erythrocytes (130,135-138). These studies speculated that the defect of insulin action might lie at the postreceptor level. Moreover, in pregnant women, insulin binding was comparable to that seen in nonpregnant women during the luteal phase of the menstrual cycle (130). When binding was followed during the third trimester and in the postpartum period, insulin binding decreased after delivery (130,137) Ryan et al. (136) observed the same insulin binding to erythrocytes in nonpregnant, nondiabetic pregnant, and gestational-onset diabetic women. Insulin receptor binding was followed in women using oral contraceptive drugs. Binding was not altered by using oral contraceptives containing low doses of levonorgestrel-desogestrelor nortestosterone-derived progestogen (139). Earlier, Tribris et al. (140) reported that insulin binding was the same in four groups: (A) late pregnants, (B) users taking 50 g estrogen daily, (C) users taking 35 g estrogen daily, and (D) controls. During intake of low doses of oral contraceptives, no direct association has been demonstrated between glucose tolerance, plasma insulin levels, and insulin binding to erythrocytes and monocytes (141). In cases of gestational diabetes, an increase in insulin binding to erythrocytes was observed during dietary treatments when the patient’s diabetes was diagnosed early in pregnancy, although no similar change was observed in late-diagnosed patients. Postpartum insulin receptor binding to erythrocytes decreased in all women when compared to the time-of-diagnosis level (142). As stated earlier, the increased insulin resistance during pregnancy and during the luteal phase in nonpregnant women cannot be explained by the changes in insulin receptors. During the late pregnancy of obese women, reduction of insulin receptors might be one cause of the deterioration in carbohydrate metabolism (143). Cheney et al. (144) have concluded that pathophysiology of gestational diabetes differs between obese and lean patients. In obese gestational diabetics, glucose intolerance was characterized by insulin resistance, hyperinsulinemia, and decreased insulin binding to erythrocyte receptors. This is in contrast to gestational diabetes in lean subjects whose glucose tolerance could develop due to relative insulin deficiency. By insulin receptor assay in erythrocytes, it is possible to study various insulin receptor functions as well as the insulin receptor status throughout pregnancy and in the newborn of the diabetic mothers. In the first and third trimesters of pregnancy with diabetes, insulin binding to erythrocytes and monocytes was similar to that in normal pregnant women in spite of increased insulin requirement and concomitant hyperinsulinemia (145). These results suggested that insulin receptors were not involved in alterations of diabetic control during pregnancy. Insulin binding was found to be increased in infants of normal and diabetic women in comparison to their mothers. Thus, the enhanced neonatal glucose tolerance might be related not only to the hyperinsulinemia, but also to increased insulin sensitivity, mediated in part by increased insulin receptor binding.

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145

HORMONES

1251-Insulin binding to erythrocyte membrane was increased by incubating erythrocyte membrane with low concentration of prostaglandin El (146). Prostaglandins Al, A*, RI, RZ, NZ, F1,, and FZa or 6-ketoprostaglandin F1, did not affect insulin binding to erythrocytes, whereas prostaglandin E3, at a similar concentration, decreased the hormone binding. Schriock et al. (147) found a positive correlation between RBC insulin binding and serum dehydroepiandrosterone sulfate (DHEAS) and a negative correlation between insulin binding to erythrocytes and testosterone (T). They concluded that DHEAS may enhance insulin binding action, and that DHEAS and T have divergent functional relationships with basal plasma insulin concentration. Dexamethasone decreased insulin binding in erythrocytes due to a decrease in receptor affinity (148,149). Hydrocortisone produces a significant decrease in binding while cortisone shows a transient decrease. Prednisone also caused insulin resistance with an insignificant decrease in insulin binding(149). However, the studies of Dwenger’s et al. (150) have excluded the possibility that the diabetogenic effect of glucocorticoids is accompanied by an alteration of the insulin receptor status of erythrocytes. Further, Yasuda et al. (149) concluded that alteration in postreceptor events may also play a major role in the induction of insulin resistance in hypercortisolism in humans (149). Insulin binding was also observed to be significantly decreased by preincubation of RBCs with adrenalin or cortisol (151). Scatchard analysis showed that the decrease was due to reduction in insulin receptor concentration. After corticosteroid therapy in patients with insulin autoimmune syndrome, hypoglycemia disappeared with an improvement in the levels of plasma glucose. Following this, an increase in binding affinity and decrease in insulin receptor numbers on erythrocytes were also observed (152). COMPARISON OF BIOSYNTHETIC AND PORCINE INSULIN

INSULIN, BINDING

PANCREATIC HUMAN TO ERYTHROCYTES

INSULIN,

Binding characteristics of biosynthetic insulin were comparable to those of human pancreatic insulin and pork insulin in human cultured lymphocytes, rat fat cells, and human RBCs (153). Another group also noted in their experiments that binding affinity of EIRs from both normal and diabetic subjects were comparable for biosynthetic and pancreatic human insulin (155); however, in the normal as well as in the diabetic subjects, porcine insulin binding to erythrocytes at low insulin concentration was significantly decreased due to reduced receptor affinity. EFFECT

OF EXERCISE ON INSULIN BINDING TO RED BLOOD CELLS OF NORMAL HUMAN VOLUNTEERS

Insulin binding to erythrocytes of normal humans is sensitive to a small change in energy balance and especially to physical activity (156). Insulin binding to erythrocytes were studied after acute-mild and acute-moderate exercise. Binding was significantly increased immediately after acute-moderate exercise (5 1,157) and

146

GAMBHIR

AND AGARWAL

was the same after acute-mild exercise (157). When insulin binding was followed after 30 and 60 min, it decreased below the basal level. These changes were due to alteration in receptor affinity but not due to insulin receptor numbers. In untrained male volunteers after 15 min of exhaustive bicycle exercise, insulin receptor affinity was found to be decreased with no change in receptor numbers (158). Burstein et al. (159) studied the insulin-stimulated glucose disposal and insulin binding to erythrocytes. They found that increased insulin action in trained athletes was rapidly reversed after detraining. This effect was parallel to decreased insulin binding in young erythrocytes. These results show that alterations in insulin response in vivo to detraining may be partially mediated by changes in insulin receptor numbers. Physical exercise is known to improve glucose tolerance and diminish insulin requirement in patients with well-controlled diabetes mellitus. Improvement in insulin binding to erythrocytes was also observed when binding was studied after exercise in athletically untrained young men having insulin-treated diabetes. This increased insulin binding to erythrocytes was observed both during 3 hr of postprandial bicycle exercise and during 2 hr of fasting bicycle exercise (61). These results were similar to those for monocytes. Further, in contrast to increased binding to erythrocytes after exercise in normal subjects, insulin binding decreased in NIDDM patients after acute exercise, which was due to decreased affinity without any change in receptor numbers (54). These results conclude that the mechanism of insulin receptor affinity in response to exercise is different in NIDDM and normal subjects. Comi et al. (55) assayed insulin binding and insulin-stimulated tyrosine kinase activity and found a high degree of correlation between these activities in RBCs obtained from normal subjects and diabetics. In conclusion, RBCs like liver, fat, and muscle cells have reduced binding in NIDDM patients but the effect of drug treatment on insulin binding to RBCs is controversial. MISCELLANEOUS INSULIN BINDING STUDIES A significant increase in specific binding of ‘251-insulin to the insulin receptor due to alteration in receptor affinity was observed in the presence of higher concentrations of disulfide reducing agents, dithiothreitol, and 2-mercaptoethanol (160). This effect on binding properties persisted even after washing. Insulin binding to erythrocytes was found to be decreased by alkylating or oxidizing agents of free sulfhydryl groups. These studies indicated that alterations of sulfhydryl groups might be an important mechanism in modulating insulin receptor affinity. In spite of having its inhibitory effect on insulin binding in cultured human lymphocytes and Epstein-Barr virus transformed lymphocytes, heparin did not affect the insulin binding to isolated adipocytes, human erythrocytes, or intact hepatoma cells (161). McElduff et al. (162) in their studies have observed the increased insulin-insulin receptor binding in normal males after fasting. They reported that acute hyperinsulinemia associated with hypoglycemia did not result in downregulation of insulin receptors on erythrocytes, but resulted in an increased receptor binding

RED BLOOD

CELL INSULIN

RECEPTORS

147

(163). Thus, they concluded that insulin receptors may not play an active role in protecting the individual against acute hypoglycemia. INSULIN INTERNATIZATION

AND DEGRADATION

Insulin binding to human erythrocytes was studied using ‘251-insulin/unlabeled porcine insulin and mono- lz51-(Tyr-A14) insulin/unlabeled biosynthetic human insulin (164). Both systems exhibited regular thermodynamic behavior between 0 and 22”. From 22” to 37”, an irregular thermodynamic behavior was observed which could be partially explained by an increased insulin degradation during incubation and an additional time-dependent binding of the degradation products. These results are in agreement with author’s findings that at 1.5” insulin binding to human erythrocytes is similar to that of human adipocytes, fibroblasts, monocytes, and placental membranes (165). At 37”, insulin binding and degradation by human erythrocytes have a unique character. Intact erythrocytes had negligible degradation of the free 1251-insulin, but 56% of the lz51-insulin associated with the erythrocytes was degraded after 5 hr of incubation at 37”. The degradation of the bound insulin in erythrocytes was found to be an intracellular property and was not associated with their plasma membrane. In 1986, Marttinen (166) studied insulin degradation at 37” in relation to insulin binding in diabetic patients. He observed that intact erythrocytes generated 1641% degradation products of insulin and the remaining degradation was by cell free insulinase (related to leaky cells). The degradation rate was found to be correlated with the receptor number and to a lesser degree with affinity. These studies concluded that insulin binding is a regulatory step for the formation of insulin degradation products. Shii et al. (167) purified the insulin degradation enzyme (IDE) from human erythrocytes via the use of ammonium sulfate precipitation and column chromatography consisting of DEAE-Sephadex, pentylagarose, hydroxyapatite, chromatofocusing resins, and Ultrogel ACA-34. The properties of this enzyme were very similar to those reported for IDE from other tissues. These results are consistent with this laboratory’s studies on isolation of IDE (168). Gambhir also compared the insulin degradation in CRF patients with that of normal subjects (74). The average maximum degradation of insulin in the CRF patients was significantly lower than that of normal subjects. Further, a linear correlation was found between the duration of dialysis and the maximum percentage of insulin degradation in the CRF patients. INSULIN AND ERYTHROCYTE

METABOLISM

D-Glucose and u-galactose flux rates in human erythrocytes were studied using infinite-cis and zero-tram assay methods (169). When compared with controls, insulin decreased the infinite-& K,,, for both n-glucose and n-galactose influx and efflux. V,,, was not statistically changed. If insulin receptors were first downregulated and then influxed and efflux assays performed in the absence of exogenous insulin, a decrease in the infinite-& and zero-frans K,,, values were observed. These affinity changes were not due to persistent surface insulin receptor occupation by the insulin which was used to induce downregulation. These affinity

148

GAMBHIR

AND

AGARWAL

changes were comparable to those observed in non-downregulated cells in the presence of insulin. On linear regression analysis of insulin binding capability in human erythrocytes versus Mg, Ca, and Na/K adenosine triphosphatase (ATPase) activity, no significant relationship was observed (170). However, from our unpublished data, it is speculated that Na influx increases through an increased efflux of H by insulin when ATPase is blocked by ouabain.

SUMMARY

AND CONCLUSION

This review on insulin receptor studies in erythrocytes provides a survey of insulin receptor function in various morbid states and some comparisons to other insulin binding cells. The majority of the studies in different disease states concluded that insulin receptor on erythrocytes reflected insulin receptor in liver, muscle, adipocytes, and monocytes and, thus, established the usefulness of human erythrocyte insulin receptor assay. The assay made possible a longitudinal study in pregnant, pediatric, newborn, and anorexic patients. The erythrocytes isolated from the patients with NIDDM, obesity, insulin resistance, chronic renal failure before dialysis, Klinefelter’s syndrome, primary hyperthyroidism, hypertriglyceridemia, severe prolactinemia, Duchenne muscular dynstrophy, and type 1 Addison’s disease showed decrease insulin receptors. Furthermore, anorexia nervosa and pancreatogenic diabetes showed increased insulin receptors in erythrocytes. The RBC population is heterogenous with a life span of 120 days. The insulin receptor is an age-dependent integral component of human RBC membrane (2431). Reticulocytes have a higher insulin binding ability than mature erythrocytes. Consequently, reticulocytes and erythrocytes should be separated before conducting insulin binding studies. Studies of insulin binding to erythrocytes in states with increased erythrocyte turnover-where immature erythrocytes are present in the blood as in anemics (24) and in newborns (117)-should be interpretated with caution. At present, there is no simple way of isolating subpopulations of erythrocytes with well-defined mean cell age for routine receptor analyses. This review also indicates from the studies of insulin function in erythrocyte metabolism that the erythrocyte with its insulin receptor may provide a simpler and convenient model to study the insulin metabolism. Since there is no proven metabolic effect of insulin in RBC, even though all known receptor activities such as binding of insulin and tyrosine kinase activity are the same as in those tissues which show metabolic response to insulin, the question becomes acute as to whether the insulin receptor is any more than the transmembrane passage for insulin into the cell as proposed by Mohan et al. (175) and Bessman et al. (176). The mature red blood cell has no mitochondria which degenerates early in the course of cell growth. It is suggestive that the lack of metabolic effect of insulin on the RBC may possibly be explained by the discovery that the immediate and ultimate effect of insulin is on the mitochondrial Krebs cycle (176).

RED BLOOD

CELL INSULIN

RECEPTORS

149

ACKNOWLEDGMENTS Author’s studies referred in this review were financially supported by Latham Trust Fund, Clinical Grant 6-262, from the National Foundation of the March of Dimes, Washington, DC, the Affiliate American Diabetes Association, and Biomedical Research Support Grant 5S07RR05361 from the General Research Support Branch, Division of Research Resources, NIH.

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160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176.

RECEPTORS

153

JD, Guyda HJ, Posner BI. Diabetes

1985.

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