11 Insulin receptors in normal and disease states

11

Insulin Receptors in Normal and Disease States GEORGE GRUNBERGER S I M E O N I. T A Y L O R R O B E R T F. D O N S PHILLIP GORDEN

The first step in insulin action is binding of the hormone to the cell surface. Insulin receptors located in the plasma membrane are the structural components that bind the hormone and are defined in terms of their function: first, to recognize and bind insulin; secondly, to couple insulin binding to insulin action. HISTORICAL BACKGROUND The modern era of investigation of the insulin receptors began with the demonstration that insulin could be labelled with radioactive iodine without destroying its biological activity (Freychet, Roth and Neville, 1971a). Two key features of the interaction of [125I]insulin with ceils suggested that the receptor was responsible for mediating the bioactivity of insulin. First, the binding was saturable, specific, and involved a finite number of receptors on the cell surface. Secondly, the apparent affinity of the insulin receptor for various analogues of insulin correlated closely with the known bioactivity of the analogues (Cuatrecasas, 1971; Cuatrecasas, Desbuquois and Krug, 1971; Freychet, Roth and Neville, 1971a, b; Gammeltoft and Gliemann, 1973; Kahn et al, 1974). The earliest clinical investigations of the regulation of insulin receptors involved rodent models of insulin resistance. It was observed that many insulin-resistant states were associated with a decrease of the number of insulin receptors per cell. In the animal studies, the major focus was upon tissues recognized to be targets for insulin action (i.e., liver, adipose tissue and skeletal muscle) (Freychet et al, 1972; Kahn et aI, 1972; Kahn, Neville and Roth, 1973). The regulation of insulin receptors in these tissues was in parallel. Remarkably, similar observations were made with cell types ordinarily not considered to be targets for insulin action. For example, studies with thymic lymphocytes from ob/ob mice gave essentially identical results to those observed with liver membranes and isolated adipocytes (Soil Clinics in E n d o c r i n o l o g y a n d M e t a b o l i s m - - Vol. 12, No. 1, March 1983.

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$03.00 ©1983 W. B. Saunders Company Ltd.

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et al, 1974). It seemed likely that the regulation of insulin receptors might be similar in all tissues whether or not they were targets for insulin action. This was a curious sidelight in the animal systems. In human studies, however, this proved to be the key observation leading to explosive development over the past decade in the studies of the regulation of insulin receptors in various physiological and pathological states (Archer et al, 1973; Bar et al, 1976; Olefsky, 1976). Insulin binding by the receptor initiates insulin action. Binding of the hormone occurs to the specific plasma membrane receptor and is followed by a rapid internalization of the receptor--ligand complex. The internalized material is enclosed in membrane-bounded vesicles which then fuse with lysosomes. Internalization has been shown to be time and temperature dependent and appears to function as a degradative pathway regulating removal of the hormone from the cell surface. Hormone-induced receptor loss, or down-regulation, represents a new steady state of reduced binding (Gorden et al, 1980). As will be described below, this process seems to play an important role in many insulin-resistant states. The insulin receptor has been biosynthetically labelled and characterized. It is a complex glycoprotein of apparent minimum molecular weight of 450 000 daltons, composed of two a subunits (135 000 daltons each) and two /3 subunits (95 000 daltons each), held together by disulphide bonds (Pilch and Czech, 1979; Hedo et al, 1981; Van Obberghen et al, 1981). The subunits may be synthesized by proteolysis from a common precursor (Hedo, Kasuga, and Kahn, 1982). Both subunits appear to be essential for insulin action; they both react with anti-receptor antibodies and are reduced in 'down-regulated' cells. Major effort has been directed at defining steps occurring beyond binding of insulin to its cellular receptor that mediate the numerous bioeffects of the hormone. One of the candidates for an early insulin effect is phosphorylation of the receptor. Insulin has been shown to stimulate phosphorylation of the 95 000 dalton subunit of its receptor in human IM-9 lymphocytes (Kasuga, Karlsson and Kahn, 1982). Insulin binding also stimulates release from the plasma membrane of intracellular mediators. These insulin mediators are low molecular weight substances that affect enzyme function by dephosphorylation, (i.e., by activating phosphatases rather than inactivating kinases) (Lamer et al, 1979; Seals, McDonald and Jarett, 1979; Seals and Czech, 1981). Human studies: cell types Various cell types have been employed to study insulin receptors from humans. Most commonly, freshly obtained tissue such as peripheral blood cells, including erythrocytes and monocytes, has been employed (Bar et al, 1976; Dons et al, 1981). Adipocytes, although somewhat less readily available, have the advantage of allowing the study of the biological effects of insulin in parallel with studies of insulin binding (Olefsky, 1976). In some cases, especially for the study of genetic diseases in the human, tissue culture systems have been used. These have included cultured diploid skin fibroblasts (Rechler and Podskalny, 1976), B lymphocytes transformed

INSULINRECEPTORS

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with Epstein--Barr virus (Taylor et al, 1981, 1982b), and short-term cultures of mitogen-stimulated T lymphocytes (Helderman and Raskin, 1980). In special circumstances, other tissues (e.g. liver, placenta, etc.) have been available for study (Harrison et al, 1977; D'Ercole et al, 1979). METHODOLOGY Conditions for the radioreceptor assay

Several physical and chemical factors are important regulators of insulin binding in vitro. Insulin binding is exquisitely sensitive to alterations in pH, with optimal binding occurring at pH of approximately 7.8 (Kahn et al, 1974). Maximal binding is observed at temperatures in the range of 10 to 20 °C in most systems (Waelbroech, Van Obberghen and De Meyts, 1979). However, temperature may in addition have other complicated effects on the system. First, with many cell types, there may be considerable degradation of [125I]insulin which is more pronounced at higher temperatures (Kahn et al, 1974; Terris and Steiner, 1975; Kahn and Baird, 1978). In addition, it has been recognized that the hormone--receptor complex is internalized by the cell more rapidly and to greater extent at 37 °C than at lower temperatures (Gorden et al, 1980). Many other factors may also affect the receptor. For example, the concentration of albumin included in the incubation medium may influence the rate of degradation of insulin (Gammeltoft and Gliemann, 1973). With some cell types the choice of chemical substances employed as buffering species may affect the extent of internalization and/or down-regulation observed (Marshall, Green and Olefsky, 1981). Correlation between insulin receptor status of different cell types Circulating mononuclear cells and erythrocytes have been used in a majority of clinical studies assessing the status of insulin receptors. It has been established that the majority of insulin binding to cells of mononuclear fraction is to monocytes. These cells make only a brief appearance in the blood stream on their way from the bone marrow to the tissues. Erythrocytes, on the other hand, have a lifespan of about 120 days in the circulation. In addition, they lack the capacity to synthesize new insulin receptors and are thus unable to replace insulin receptors lost during ageing of the ceils. Consequently, the great majority of detectable erythrocyte insulin receptors are located in reticulocytes and other young cells (Dons, Corash and Gorden, 1981). Many factors, including previous phlebotomy, aswell as various haemolytic and other disease states, may affect the reticulocyte percentage and the mean erythrocyte age. Such confounding factors may complicate interpretation of insulin binding studies involving the erythrocytes. Several investigators have studied the status of insulin receptor in both monocytes and erythrocytes simultaneously in various clinical conditions. Spanheimer et al (1982) evaluated the status of insulin binding in cells of fed and fasted obese subjects in both types of circulating cells. They reported that during a 14-day fast the change of insulin binding to both cell types

194

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occurred in the same direction (increase) but the magnitude of the change was markedly different. The increase in insulin binding to monocytes was significantly greater than to the red cells. In addition, while there was about a 40 per cent increase in the number of receptors in the monocytes, there was no such increase found in the erythrocytes. The data indicate that the mechanism by which binding undergoes change may be different between the two cell types. Dons et al (1981) attempted to provide correlation of the insulin receptor status between monocytes and erythrocytes in various normal and disease states. Good correlation was found between the specific insulin binding to both cell types from a large number of subjects with a wide variety of physiological and pathological conditions whose metabolic status was not acutely perturbed. Correlation between the shapes of competition curves for the two cell types was weaker, while derived parameters, such as Scatchard plots or affinity profiles, used for analysis of insulin binding, did not correlate. In general, insulin binding to red cells changes more slowly than in monocytes and thus the magnitude of the red cell change is attenuated. In addition, unless the mean erythrocyte age of the subjects is standardized, one cannot make a meaningful analysis of studies involving the red ceils. The most valuable information comes from the simultaneous study of both monocytes and erythrocytes.

Analysis of insulin binding data In the most commonly employed experimental design, a competition curve is constructed. This is accomplished by incubating cells or subcellular components with a constant concentration (tracer) of [125I]insulin and increasing concentrations of unlabelled insulin. The percentage of bound iodoinsulin is plotted as a function of the concentration of insulin (Figure la). The percentage of bound insulin decreases as increasing concentrations of unlabelled insulin are employed. All results are corrected for non-specific binding by subtracting the percentage binding found at the highest insulin concentration from binding measured at all other concentrations. For some purposes, it is important to estimate the affinity of the insulin receptor as well as receptor concentration. The data from the competition--inhibition studies are commonly transformed to a Scatchard analysis (Scatchard, 1949) where the ratio of bound to free insulin is plotted as a function of bound hormone (B/F v s B) (Figure lb). A single class of non-interacting high-affinity binding sites results in a linear plot over the entire concentration range of binding. For insulin, however, the Scatchard plot is curvilinear with the concavity upward. This could result from two discrete populations of insulin-binding sites (Figure 2a) - - a high-affinity/lowcapacity site and a low-affinity/high-capacity site - - or from heterogeneous receptors (Klotz and Hunston, 1971). Some cells have receptors for both insulin and insulin-like growth factors. These types of receptors have distinct biological specificities and therefore probably represent distinct structural entities (Rechler et al, 1977; Kasuga et al, 1981; Massague and Czech, 1982). Nevertheless, insulin may bind to both types of receptors, although with greatly different affinities. Another alternative explanation for the curvilinearity of Scatchard plots involves negatively cooperative

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OCCUPANCY (percent) Figure 2. Two models to analyse the curvilinear Scatchard plot. (a) A two-site model in which there exist both a high-affinity/low-capacity site and a low-affinity/high-capacity site (Klotz and Hunston, 1971). (b) The negative cooperativity model of De Meyts and Roth (1975), in which apparent affinity is plotted as a function of receptor occupancy.

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interactions (De Meyts and Roth, 1975; De Meyts, Bianco and Roth, 1976; Rodbard, 1979). In this model, the affinity of the insulin receptor for insulin is regulated as a result of interactions among the receptor sites. The affinity of the receptor for insulin decreases with increasing occupancy of receptor sites (Figure 2b). This hypothesis was supported by the demonstration that the initial dissociation rate of labelled hormone was increased when excess insulin concentrations were added to tracer concentrations of insulin bound to cells (De Meyts, Bianco and Roth, 1976). The negative cooperativity model seems to provide an explanation for the curvilinearity of the Scatchard plot of insulin binding in cells (such as cultured human lymphocytes) which appear to have a single homogeneous class of insulin receptors. These models are not mutually exclusive, however, and some combination of the above may pertain for individual systems. It should be emphasized that only the results obtained from the competition--inhibition studies represent primary data. All other popular means of expressing results of insulin binding studies have used derived parameters. IDs0, or the insulin concentration required for displacement of one-half the maximum of [~25I]insulin bound, can be calculated from the competition curve and serves as a rough index of affinity. The Scatchard plot and affinity profile are the most widely used parameters to measure both the concentration and the affinity of the insulin receptor. The number of insulin receptors (R0) is determined from the extrapolated x-intercept of the Scatchard plot. As the lower limit of the bound/free ratio is reached, each point becomes less precise. The R0 in our laboratory is thus estimated from an extrapolation using an arbitrary cut-off point from the competition curve. We found that using a 200 ng/ml insulin concentration point for erythrocytes and 100 ng/ml for monocytes gives the most reproducible results and correlates well with a computer fit of the data. The average affinity profile expresses the relationship between the average affinity and the receptor occupancy. On the vertical axis the average affinity for insulin [(K = (B/F)/(Ro--B)] is plotted on a linear or log scale. On the horizontal axis the fractional occupancy of the receptor (B/Ro) is plotted on a log scale (Figure 2b). The higher affinity state exists at the lowest level of receptor occupancy (affinity of the empty receptor, Ke) and the lowest affinity state at highest level of receptor occupancy (affinity of the filled receptor Ks). If one uses this type of analysis, it must be remembered that the R0 value is determined from the Scatchard analysis; the affinity profile, therefore, represents a further derivative of the primary data.

[IZSlllnsulin Monoiodinated insulin of varying purity has been used by different laboratories in clinical investigations of insulin binding (Gliemann et al, 1979; Keefer, Piron and De Meyts, 1981). Recently, a number of advantages of using more highly purified (by t h e method of high-pressure liquid chromatography) monoiodoinsulin, especially (tyr-A-14)-monoiodoinsulin,

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have been emphasized. Such a preparation has a greater affinity for the insulin receptor, much higher specific activity, greater stability, and a longer shelf-life. Another recent advance has been the demonstration by De Meyts (Keefer, Michiels-Place and De Meyts, 1980) that certain insulin analogues (such as turkey desalanine--desasparagine insulin) fail to induce negatives cooperafivity and thus give rise to a linear Scatchard plot of insulin binding. It is hoped that these analogues, in combination with porcine insulin, can be used to obtain more accurate estimates of the binding parameters. STUDIES OF INSULIN RECEPTORS IN HEALTH AND DISEASE Physiological states

Diurnal variation of insulin binding. Beck-Nielsen and Pedersen (1978) showed that when insulin binding to peripheral monocytes from healthy subjects was studied, daytime insulin binding was lower (nadir at 2 p.m.) and a peak occurred at 10 p.m. Higher binding persisted through the early morning hours. Essential role of diet in producing this pattern is likely since total fasting abolished these dfu(nal changes. Diet. Target tissues possess only a limited repertoire of responses to alterations of ambient insulin levels. As the plasma insulin concentration changes, the insulin binding to cells can be altered by changes of affinity of the receptor for insulin, the receptor concentration, or both. The insulin receptor appears to be an important site for modulation of tissue insulin sensitivity. Marked changes in receptor affinity have been observed within seconds to a few hours. Two to three hours after the ingestion of 100 g oral glucose load in normal subjects, insulin receptor affinity increases in both monocytes and erythrocytes (Muggeo, Bar and Roth, 1977; Bhathena et al, 1981). In at least some patients with reactive hypoglycaemia, this effect may be exaggerated (Muggeo et al, 1980). Changes in the receptor concentration occur over a longer period of time. Beck-Nielsen, Pedersen and Sorensen (1978) investigated the effects of diet on monocyte binding of insulin in normal subjects over two weeks. They noted that excessive intake of both sucrose and fat induced a reduction in insulin binding, while an isocaloric low-sucrose diet led to increased insulin binding to monocytes. They further presented evidence that changes in insulin sensitivity after a high-caloric high-sucrose diet may be mediated through alteration of insulin receptor status. Acute caloric restriction in obese hyperinsulinaemic subjects results in lowered circulating insulin and normalized insulin binding (Bar et al, 1976). The rising insulin binding was accounted for by increased receptor affinity without any change in the receptor number. Insulin binding decreased on refeeding, while chronic diet was found to restore insulin binding to normal because of increased receptor concentration without altered affinity of the receptor. More recently Spanheimer eta I (1982) showed that a 14-day fast of obese patients increased insulin binding to both circulating monocytes and erythrocytes. This increase in insulin binding was due to a pure affinity

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change for the red cells, while in the monocytes it was thought to occur secondarily to an increase both of affinity and of concentration of the insulin receptor. It can thus be concluded that total caloric reduction in obese individuals modulates insulin receptor function rapidly by changing receptor affinity and more chronically by changing receptor number. The change in receptor number is inversely correlated with the ambient insulin concentration. Receptor modulation related to dietary composition independent of total calories is less comp!etely understood, but carbohydrates appear to play the mostimportantrole~ +' ~ '

Exercise. The marked suppression of insulin secretion with exercise in normal subjects is assOciated with a concomitant increase in receptor affinity (Soman et al, 1'979). A similar effect was observed in patients with insulin-dependent diabetes mellitus (Pedersen, Beck-Nielsen and Heding, 1980). However, there was no correlation between free insulin levels and insulin receptor binding in these patients. Technical difficulties with free insulin assays or the influence of insulin antibodies in the diabetic patients might be responsible for this finding. In any case, it appears that exercise increases insulin binding of both normal and diabetic subj ects.

Menstrual cycle. De Pirro etal (1978) and Bertoli et al (1980) reported that sex hormones can serve as additional modulators of insulin receptors. Insulin binding to both monocytes and erythrocytes from women in the follicular phase of the menstrual cycle was higher than in the luteal phase. No such fluctuation of insulin binding was observed either in postmenopausal women or in men. In addition, insulin binding to erythrocytes from normal men was higher than that in women during any part of the menstrual cycle. Changes in insulin receptor concentration rather than affinity appear to account for the variation in binding results. It has been suggested that the decreased insulin binding during the luteal phase could be associated with the reduced glucose tolerance seen during this part of the cycle.

Age. The status of the insulin receptor in fetal cells and during infancy is of considerable interest because of the influence of insulin on growth. A significant increase in the concentration of high-affinity insulin receptors was found on monocytes from placental-cord blood of normal newborns (Thorsson and Hintz, 1977). The binding was markedly elevated due to an increase in both the receptor affinity and the concentration when compared with normal adult values. This finding is thus consistent with the concept of importance of insulin in intrauterine growth and development. While there is general agreement that insulin binding and sensitivity is increased in the newborn and young child, there is no consensus in the elderly. Though hyperinsulinaemia and some degree of insulin resistance are common in older subjects, insulin-binding data varied with the tissue studied. In the adipocytes the binding has been reported to be decreased (Pagano et al, 1981), in the monocytes unchanged (Rowe, Minaker and Flier, 1981) and in the fibroblasts increased (Rosenbloom, Goldstein and Yip, 1976).

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Pregnancy. Glucose tolerance and insulin sensitivity become gradually impaired during pregnancy. Insulin-binding studies have been conflicting. For example, Beck-Nielsen et al (1979) reported that insulin binding to circulating mon0cytes of healthy pregnant women was decreased by about 35 per cent owing to a decreased number of insulin receptors, while Tsibris et al (1980) found no significant change, and Puavilai et al (1982) observed that insulin binding might be modestly increased.

Effect of drugsOn insulin binding Oral contraceptives. Sex steroids contained in the oral contraceptives have been reported to alter the insulin receptor (De Pirro et al, 1981). These results are interesting in view of:previous reports of altered glucose tolerance and insulin sensitivity in women taking these drugs. Oral contraceptives appear to abolish the normal variation in insulin binding during the menstrual cycle. Insulin binding to circulating cells remains equal to that of normal women during their luteal phase and is thus lower than in the follicular phase. Reduced receptor concentration was found to account for the changes in binding. While the effects of sex steroids are small, interpretation of insulinbinding data should take into account the use of these drugs as well as the stage of the menstrual cycle. Oral hypoglyeaemics. In type II or non-insulin-dependent diabetes it has been shown that treatment with chlorpropamide (Olefsky and Reaven, 1976) or glibenclamide (Beck-Nielsen, Pedersen and Lindskov, 1979) will increase insulin binding over periods of 10 days to three months. Under these conditions, hyperglycaemia is reduced, with lower ambient insulin concentrations. In contrast, in type I or insulin-dependent diabetes there is neither an effect on insulin binding nor an improvement in level of glucose control (Grunberger, Ryan and Gorden, 1982). Taken together with the data that fail to demonstrate a direct effect of sulphonylureas on insulin binding in vitro, these studies suggest that the effect of these agents on insulin binding is indirect and mediated primarily by the ability of these drugs to increase insulin secretion acutely (Grunberger, Ryan and Gorden, 1982; Vigneri et al, 1982). Another antidiabetic drug, met formin, reportedly increases insulin binding in erythrocytes from healthy volunteers (Holle et al, 1981). This increased binding was associated with an increased number of low-affinity insulin receptors. It remains unclear, however, how the increased insulin receptor concentration can be achieved in these enucleated cells. Glucocorticoids. Results of four clinical studies investigating the effect of exogenous corticosteroids on insulin binding differ markedly. De Pirro et al (1980) found a significant decrease in insulin binding to monocytes after a 24to 72-hour administration of dexamethasone or cortisone to normal males. This decrease was due to reduced insulin receptor affinity. Yasuda and Kitabchi (1980) reported also a decrease in insulin binding, this time to erythrocytes, following administration of either dexamethasone or prednisone to healthy subjects. In contrast, Beck-Nielsen, De Pirro and Pedersen (1980) found that prednisone raised the number of insulin receptors

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in circulating monocytes in a time- and concentration-dependent fashion in normal volunteers. Finally, Fantus et al (1981) confirmed that a state of moderate insulin resistance, but without any change in insulin binding to monocytes, was induced after a three-day administration of 40 mg prednisone to healthy females. Interestingly, in vitro an 18-hour incubation of cultured human lymphocytes with hydrocortisone increased insulin binding, owing to an increased number of insulin receptors. Corticosteroids have multiple actions; they alter the kinetics of blood cells in the circulation, increase insulin secretion and induce protein synthesis. These multiple competing effects undoubtedly contribute to the ambiguity of the various studies. Further, corticosteroid administration may represent a situation where blood receptor studies do not reflect the status of other cellular insulin receptors (Fantus et al, 1981). DISEASE STATES Insulin Resistance

Insulin resistance is a state of either decreased insulin sensitivity (with a normal response to a maximal concentration of insulin but impaired response to lower insulin levels), decreased insulin responsiveness (with an impaired maximum insulin effect), or both. Characteristically, all insulin-resistant patients have hyperinsulinaemia. In addition, they are resistant to exogenously injected insulin. Obesity The ambient insulin concentration appears to be the most important modulator of insulin receptor status. Obesity is associated with insulin resistance and hyperinsulinaemia. Insulin sensitivity of obese humans appears to b e heterogeneous and corresponds to the degree of hyperinsulinaemia. Chronic hyperinsulinaemia is postulated to lead to downregulation of the receptor. Thus, obese subjects without hyperinsulinaemia have no evidence of insulin resistance and show normal insulin binding and normal concentration of insulin receptors on either monocytes or adipocytes (Bar et al, 1976; Kolterman et al, 1980). Obese patients with only a mild hyperinsulinaemia have a small degree of insulin resistance due to decreased insulin sensitivity resulting from a decrease in the number of insulin receptors. Obese subjects with a more pronounced hyperinsulinaemia demonstrate greater insulin resistance due apparently to both receptor and postreceptor defects. Insulin binding to cells from these patients is lowest and the loss of cellular receptors most marked. It should be pointed out that the reduced insulin binding in obese humans can be explained entirely by a decreased number of receptors. The remaining receptors display a normal affinity for insulin (Bar et al, 1976). Treatment of elevated insulin levels by diet, streptozotocin or diazoxide corrects the binding abnormalities despite the persistence of significant obese, non-insulin dependent and insulin resistant. Insulin binding to cells has been proposed that a cycle of 'hyperinsulinemia -* down-regulation of

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insulin receptor concentration ~ decreased insulin sensitivity -~ hyperglycaemia' exists and that it is not obesity per se which is responsible for the altered insulin receptor concentration.

Diabetes mellitus (DM)

Non-insulin-dependent (type II) DM. The majority of diabetic patients are obese, non-insulin dependent and insulin resistant. Insulin binding to cells of these patients is decreased due to a reduced receptor concentration, with the same inverse relationship between the number of insulin receptors and basal plasma insulin levels as seen in obese non-diabetic persons (Bar et al, 1976; Olefsky and Reaven, 1977). As in obese type II patients, about a 50 per cent decrease in insulin binding to peripheral cells has been shown in non-obese diabetics with fasting hyperglycaemia (Olefsky and Reaven, 1977). This reduction in binding seems again to be due to a reduction of the number of insulin receptors and it correlates inversely with basal plasma insulin levels. The remaining receptors have an entirely normal affinity for insulin. This binding defect has been found to correlate well with the degree of insulin resistance as measured by the steady-state plasma glucose. Interestingly, patients with impaired glucose tolerance (abnormal response to oral glucose tolerance test) display a reduction in the number of insulin receptors that is quantitatively identical to that seen in subjects with fasting hyperglycaemia (about 50 per cent) (Olefsky and Reaven, 1977). It is not completely clear why non-obese type II patients show the binding defect. It seems unlikely that either overeating or increased stimulated insulin secretion can be implicated. Constant hyperglycaemia, however, does modestly increase basal insulin concentration. Thus, there may be some degree of receptor down-regulation (Kahn, 1979). As mentioned above, both dietary and drug therapy of obese type II diabetic patients can lead to improved glucose control, insulin sensitivity, and insulin binding to circulating cells.

Insulin-dependent (type I) DM. The ambient insulin concentration is believed to act as the most important modulator of the membrane insulin receptors. One would then expect increased insulin binding to the target cells in insulin-dependent diabetics. Indeed, this is the case in the hypoinsulinaemic animal models (Hepp et al, 1975; Davidson and Kaplan, 1977). Studies of insulin-dependent patients are less clear. Most subjects have already been treated with insulin and circulating insulin concentrations have been variable. When insulin binding to placental receptors from insulindependent patients was studied, a marked decrease in the number of receptors was observed, suggesting a role of exogenous insulin in downregulation of the insulin receptor (Harrison et al, 1977). However, when binding to circulating monocytes of type I subjects has been assessed, the results have been heterogeneous (Pedersen, Beck-Nielsen and Heding, 1978; Fantus, Ryan and Gorden, 1981). Some of the poorly controlled and newly diagnosed patients had increased binding, while some showed normal or even decreased binding. With appropriate insulin therapy

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there is a tendency for the binding results to normalize. For the group as a whole, the initially slightly elevated binding falls to normal on optimal therapy, but the results of individual patients are not distinguishable from those of normal subjects. In an attempt to delineate specific factors seen in vivo in diabetes that could alter receptor binding, hyperglycaemia, acidosis, hypertonicity and ketosis were simulated in vitro. None of these factors had a significant effect on binding once cells were restored to a normal buffered environment. Thus, cells do not appear to have a 'memory' for these effects similar to other circumstances that lead to down-regulation (Fantus et al, 1981). On the other hand, in severe ketoacidosis the low plasma pH might contribute to low insulin binding and the well-known insulin refractoriness characteristic of this condition (Roth and Muggeo, 1981). Acromegaly

Moderate insulin resistance, accompanied by hyperinsulinaemia, with or without hyperglycaemia is seen in patients with acromegaly. When insulin binding has been characterized in acromegalic subjects, two alterations have been seen: (1) inverse relationship between fasting insulin levels and the number of insulin receptors on monocytes, and (2) increase of the affinity of the empty receptor (high-affinity), without a change in the affinity of the filled receptor (low-affinity) (Muggeo et al, 1979). In most acromegalic patients, the combination of increased affinity and reduced receptor number resulted in normal insulin binding at basal insulin levels, but decreased binding at stimulated insulin concentrations. Since incubation in vitro of either growth hormone or acromegalic plasma had no effect on insulin binding to normal monocytes, it seems unlikely that insulin resistance of acromegaly is caused solely by the changes of the hormone--receptor interaction. Interestingly, patients with hyperglycaemia failed to develop the affinity shift. Glucocorticoid excess

Hypercortisolism is another state associated with moderate insulin resistance. It can result from endogenous corticosteroid overproduction (Cushing's syndrome) or from exogenous glucocorticoid administration. A 50 to 60 per cent decrease of insulin binding was observed in rat liver membranes from animals with hypercortisolaemia (Kahn et al, 1978). The binding changes could be accounted for entirely by a marked decrease of the receptor affinity for insulin. In general, decreased insulin binding has also been observed in rodent and cultured 3T3L1 adipocytes exposed in vivo to glucocorticoids (Fantus et al, 1981; Grunfeld et al, 1981). The conflicting studies in the human using blood cells have been discussed previously in the section dealing with drugs. Other insulin-resistant states

Insulin binding in a number of other diseases associated with moderate insulin resistance, such as ataxia telangiectasia, uraemia, myotonic dystrophy, cirrhosis, Werner's syndrome, etc., has been investigated. A summary of essential findings can be found in Table 1.

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Syndromes of Extreme Insulin Resistance Differential diagnosis of extreme insulin resistance T h e h a l l m a r k o f insulin resistance is a q u a n t i t a t i v e l y decreased b i o l o g i c a l r e s p o n s e to a n o r m a l o r i n c r e a s e d q u a n t i t y o f insulin. M u l t i p l e p a t h o p h y s i o l o g i c a l m e c h a n i s m s give rise to e x t r e m e insulin resistance ( T a b l e 2).

Table 2. Syndromes o f extreme insulin resistance associated with aeanthosis nigricans Syndrome I. Autoantibodies to theinsulin receptor (type B extreme insulin resistance) II. Primary (? genetic) target cell resistance 1. Lipoatrophic diabetes 2. Type A extreme insulin resistance

3. Leprechaunism

4.

Rabson--Mendenhall syndrome

Reference Flier et al, 1976 Kahn et al, 1976 Bar et al, 1980 Wachslicht-Rodbard et al, 1981 Rossini and Cahill, 1979 Kahn et al, 1976 Barnes et al, 1974 Flier et al, 1980 Scarlett et al, 1982a, b Bar et al, 1980 Donohue and Uchida, 1954 Rosenberg et al, 1980 D'Ercole et al, 1979 Kobayashi et al, 1978 Schilling et al, 1979 Taylor et al, 1981, 1982a, b Podskalny and Kahn, 1982a, b Knight et al, 1981 Rabson and Mendenhall, 1956 West, Lloyd and Turner, 1975 West and Leonard, 1980 Perez Corral et al, 1980 Taylor et al, 1983

F o r e x a m p l e , circulating anti-insulin a n t i b o d i e s m a y interfere with delivery o f insulin to the target tissue ( K a h n a n d R o s e n t h a l , 1979). In i n s u l i n - t r e a t e d diabetics, there m a y be s u b c u t a n e o u s d e g r a d a t i o n o f insulin p r i o r to a b s o r p t i o n i n t o the b l o o d s t r e a m ( F r i e d e n b e r g et al, 1981). Genetic defects in insulin b i o s y n t h e s i s m a y give rise to elevated levels o f p l a s m a i m m u n o reactive insulin, a n d t h e r e f o r e m a y be c o n f u s e d with insulin resistance ( G a b b a y et al, 1979; Given et al, 1980; R o b b i n s et al, 1981; H a n e d a et al, 1982). H o w e v e r , these p a t i e n t s are t y p i c a l l y n o t resistant to e x o g e n o u s insulin a d m i n i s t r a t i o n . T h e focus in this section is on the clinical c o n d i t i o n s in which there is extreme resistance to insulin at the cellular level despite the delivery o f a n o r m a l or i n c r e a s e d q u a n t i t y o f b i o l o g i c a l l y active insulin.

Autoantibodies to the insulin receptor (type B extreme insulin resistance) In 1976, Flier et al d e s c r i b e d a s y n d r o m e o f e x t r e m e insulin resistance associated with a c a n t h o s i s nigricans which was a t t r i b u t a b l e to a u t o a n t i b o d i e s directed a g a i n s t the insulin r e c e p t o r . T y p i c a l l y , these patients have o t h e r

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G. GRUNBERGER,S. LTAYLOR, R.F. DONS AND P. GORDEN

serological and/or clinical evidence of autoimmunity. In vitro, their antibodies have insulin-like effects on most tissues that have been studied. The clinical correlate of this insulinomimetic effect may be the hypoglycaemia which is observed in some patients with anti-receptor antibodies (Flier et al, 1978; Taylor et al, 1982c). In vitro, the effect of these anti-receptor antibodies in inhibiting insulin action occurs only after a prolonged exposure of cells to the anti-receptor antibody. We do not completely understand what determines the balance between the insulinomimetic and insulin-antagonistic effects of anti-receptor antibodies in different clinical circumstances. When anti-receptor antibodies are present, however, insulin binding to mono~ cytes, erythrocytes, and adipocytes is markedly reduced and this represents the most sensitive indicator of the presence of such antibodies (Flier et al, 1976; Pedersen et al, 1981). Primary (? genetic) syndromes of extreme insulin resistance There are multiple syndromes associated with extreme insulin resistance where~the defect appears to reside primarily at the level of the target tissue. These syndromes may be genetic in aetiology. Each syndrome of extreme insulin resistance has distinctive features, for example, atrophy of subcutaneous fat in lipoatrophic diabetes or intrauterine growth retardation in leprechaunism. However, there are certain clinical features (i.e., acanthosis nigricans and masculinization of females) which are commonly observed in most, if not all, of'the syndromes. Markedly elevated plasma insulin levels which are usually sufficient to maintain the fasting plasma glucose level in the normal range occur in all of these patients.~:Thu~,~while fasting hyperinsulinaemia is common, fasting hyperglycaemia is relatively rare. Interestingly, typical microvascular complications of diabetes such as diabetic retinopathy have been observed in these syndromes. Pathophysiological consequences of extreme insulin resistance In most cases, extreme insulin resistance is accompanied by compensatory hyperinsulinaemia. While target cells are resistant to biological effects of insulin which are ordinarily mediated through the insulin receptor, it is possible that these pathologically elevated levels of plasma insulin may interact with receptors for other insulin-like peptides (i.e., 'specificity spillover'). For example, both acanthosis nigricans and masculinization of female patients have been described in all of the syndromes of extreme insulin resistance, regardless of whether this resistance results from antireceptor antibodies or from a genetic defect. Assuming a unitary cause of acanthosis nigricans or masculinization of female patients in all of these syndromes, a hypothesis for a causal role of hyperinsulinaemia in the development of these clinical features can be proposed. In the case of acanthosis nigricans, it seems likely that the elevated levels of insulin directly affect the skin. Similarly, we have suggested that hyperinsulinaemia may have a direct effect on the ovary in premenopausal women to promote the synthesis and secretion of testosterone (Taylor, et al, 1982d).

INSULIN RECEPTORS

209

Biochemical causes of extreme insulin resistance

There is marked heterogeneity in the biochemical defects that give rise to the primary syndromes of extreme insulin resistance. This was first described in type A extreme insulin resistance. The first patients described with this syndrome were noted to have a marked decrease in the number of insulin receptors per cell (Kahn et al, 1976; Bar et al, 1980; Taylor et al, 1982a). However, subsequently a clinically identical patient was described in whom the number of receptors appeared to be normal (Bar et al, 1978; ImperatoMcGinley et al, 1978). Similar degrees of heterogeneity have been observed in other syndromes. It is possible to divide these biochemical defects into three categories:

Quantitative receptor defects. In some patients with extreme insulin resistance (e.g., leprechaunism, Rabson--Mendenhall syndrome, and type A extreme insulin resistance), the patient's cells appear to have a decrease in the number of insulin receptors. However, the residual receptors appear to be normal in all respects. Because the quantitative defect in receptor number persists in cells cultured in vitro, it seems likely that this defect is primary, possibly genetic (Kahn et al., 1976; Bar et al, 1980; Taylor et al, 1982a). Qualitative receptor defects. The best-studied example of a qualitative defect in the insulin receptor is one patient with leprechaunism (leprechaun/ Ark-l). In this patient, the number of insulin receptors per cell appears to be normal; however, these receptors have clearly abnormal binding properties. In particular, insulin binding to receptors from leprechaun/ Ark-1 are abnormally insensitive to changes in temperature and pH (Taylor et al, 1981, 1982a,b). Curiously, these abnormal receptors bind an abnormally large amount of insulin under physiological conditions of temperature and pH. It seems likely that the insensitivity of this receptor to changes in temperature and pH provides a marker for a structural abnormality in the receptor. This abnormality probably causes a severe compromise in the ability of the receptor to couple insulin binding to insulin bioactivity. Probable post receptor defects. Many, possibly most, patients with extreme resistance appear to have insulin receptors which are normal, both quantitatively and qualitatively. The molecular mechanism of insulin resistance in these patients is not understood. Although the possibility has not been ruled out that there may be an abnormality in the insulin receptor which interferes with the coupling of insulin binding to insulin action, it seems most likely that the biochemical defects in these patients reside at a step distal to the hormone receptor. Lipoatrophic diabetes

The patients with this syndrome exhibit severe insulin resistance, lipodystrophy, hyperinsulinaemia, impaired glucose tolerance and hyperglycaemia without signs of autoimmunity. The lipoatrophy may involve

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G. GRUNBERGER, S. I. TAYLOR, R. F. DONS AND P. GORDEN

the entire body or may spare certain areas. When a spectrum of these patients was studied, heterogeneous insulin binding was found. The binding to their monocytes and erythrocytes was low in about half of the patients, but others had normal or even elevated results. The values were heterogeneous even within a family (Wachslicht-Rodbard et al, 1981). Since some patients have entirely normal insulin binding and binding in cultured fibroblasts has also been normal, the pathogenesis of lipoatrophic diabetes probably involves both receptor and postreceptor abnormalities.

Insulin Hypersensitivity Growth hormone deficiency Growth hormone deficiency in the rodent produces supersensitivity to insulin, associated with an increase in receptor concentration and reduction in receptor affinity for insulin (Kahn et al, 1978). Administration of growth hormone partially corrects the defect. While hypersensitivity to insulin occurs in growth hormone-deficient children and is corrected by growth hormone administration, no consistent changes in insulin binding have been demonstrated in this condition (Lippe et al, 1981; Rosenfeld et al, 1982). Glucocorticoid deficiency In the rodent, adrenalectomy increases insulin sensitivity and is associated with increased insulin binding to purified rat liver membranes (Kahn et al, 1978). The elevated binding is secondary to increased affinity of the liver insuIin receptor. There have been so far no comparable studies performed in humans. Insulin deficiency Insulin deficiency of insulin-dependent DM was considered above. Other insulin-deficient states both in experimental animals and in humans, such as in pancreatitis or after pancreatectomy, are associated with increased binding due to increased concentration of insulin receptors (Hepp et al, 1975; Davidson and Kaplan, 1977; Nosadini et al, 1982). Thus, in the latter situations insulin seems to play a major role in regulating its own receptors. Further, Nosadini et al (1982) showed that both insulin binding and insulin sensitivity were increased in pancreatogenic diabetes compared with type I, insulin-dependent, diabetes. Anorexia nervosa Concentrations of both basal plasma glucose and insulin are low in women with anorexia nervosa, suggesting a state of insulin supersensitivity. Insulin binding to erythrocytes from these patients was found to be elevated secondarily to an increase in receptor concentration, without any affinity changes (Wachslicht-Rodbard et al, 1979). Furthermore, normalization of eating patterns and body weight was associated with a return to normal insulin binding and normal insulin sensitivity. In addition to the altered plasma insulin levels in these subjects there are many dietary as well as

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endocrine abnormalities, each of which could be responsible, alone or in combination, for the changes in insulin binding. It is not entirely clear which factor predominates in this clinical disorder.

Hypoglycaemic States Insulinoma The inverse correlation between the fasting insulin levels and the number of insulin receptors on circulating monocytes found in patients with obesity, type II DM and acromegaly hold true for subjects with insulin-secreting tumours. In addition to the altered receptor concentration, increased affinity for insulin is encountered in some patients (Bar et al, 1977). In patients with the most severe hypoglycaemia there is a tendency toward increased receptor affinity. This could be in part related to the more frequent food intake in these subjects, as shown previously (Muggeo, Bar and Roth, 1977). On the other hand, some patients with hyperinsulinaemia, but normal or subnormal affinities for insulin, seem to be protected from fasting hypoglycaemia for longer periods of time by the decreased receptor number. This example stresses the existence of multiple modulators of insulin receptor affinity providing complex and sensitive adjustments of the receptor in various pathophysiological states. Infants of diabetic mothers Insulin concentrations are elevated in utero in fetuses of diabetic mothers owing to stimulation of the fetal beta cells by maternal hyperglycaemia and hyperaminoacidaemia. These infants often present at birth with hypoglycaemia associated with hyperinsulinaemia. It is postulated that the chronic fetal hyperinsulinaemia results in the metabolic effects, that is, hypoglycaemia and excess of stored protein, glycogen, and fat mediated through cross-reaction with growth factor receptors (Van Obberghen, De Pablo and Roth, 1981). Insulin binding is increased in these infants when compared with both normal newborns and adults, despite significant elevations of plasma insulin (Neufeld et al, 1978). This increased binding seems to be due to greatly increased receptor concentration. Up-regulation of the monocyte insulin receptor occurs in this setting, contributing to the hypersensitivity to insulin in such infants. Earlier findings using placentae of infants of diabetic mothers, however, revealed either no change or an actual decrease in the number of insulin receptors (Posner, 1974; Harrison et al, 1977). Different response of the two tissues to hyperinsulinaemia has to be postulated to reconcile the above observations. Hypoglycaemia in childhood A study of seven infants and children with symptomatic hypoglycaemia (three with nesidioblastosis after a 90 per cent pancreatectomy, two with leucine sensitivity, and two with glycogen storage disease type I) revealed a

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G. GRUNBERGER, S. I. TAYLOR, R. F. DONS AND P. GORDEN

significant increase of insulin binding to circulating erythrocytes (Chrousos et al, 1981). All patients had normal basal plasma insulin levels; their hypoglycaemia thus correlated better with the receptor binding than with the ambient insulin concentration. In addition, in the cases where hypoglycaemia was successfully treated, some decrease of receptor concentration was noted. SUMMARY

The binding of insulin to its receptor has been studied under various physiological and pathological conditions. Quantitative studies have involved h u m a n circulating cells such as monocytes and erythrocytes, adipocytes, placental cells, and cultured cells such as fibroblasts and transformed lymphocytes. In animals, other target tissues such as liver and muscle have been studied and correlated with the h u m a n studies. Various physiological conditions such as diurnal rhythm, diet, age, exercise and the menstrual cycle affect insulin binding; in addition, m a n y drugs perturb the receptor interaction. Disease affecting the insulin receptor can be divided into five general categories: (1) Receptor regulation - - this involves diseases characterized by hyper- or hypoinsulinaemia. Hyperinsulinaemia in the basal state usually leads to receptor 'down' regulation as seen in obesity, type II diabetes, acromegaly and islet cell turnouts. Hypoinsulinaemia such as seen in anorexia nervosa or type I diabetes may lead to elevated binding. (2) Antireceptor antibodies - - these immunoglobulins bind to the receptor and competitively inhibit insulin binding. They may act as agonists, antagonists or partial agonists. (3) Genetic diseases which produce fixed alterations in both freshly isolated and cultured cells. (4) Diseases of receptor specificity where insulin may bind with different affinity to its own receptor or related receptors such as receptors for insulin-like growth factors. (5) Disease of affinity modulation where physical factors such as pH, temperature, ions, etc. may modify binding. In this review, we have considered primarily abnormality in insulin receptor binding. There are numerous other functions of the receptor such as coupling and transmission of the biological signal. These mechanisms are frequently referred to as postreceptor events, but more properly should be referred to as postbinding events since the receptor subserves other functions in addition to recognition and binding of insulin. ACKNOWLEDGEMENTS

We wish to thank Ms Laurie Tuchman for her excellent secretarial skills in preparing this manuscript. REFERENCES

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