Klinische Wochenschrift

Klin Wochenschr (t987) 65:949-954

~,~ Springer-Verlag 1987

[Jbersicht Insulin Receptor Binding to Blood Cells: An Outdated Concept for Clinical Studies on Insulin Resistance ? M.J. Mfiller Medizinische Hochschule Hannover, Abteilung Klinische Endokrinologie

Summary. Clinical data on insulin binding to blood cells contribute to our present knowledge of insulin resistance. Binding data have been frequently presented to indicate alterations in insulin action. It is now evident that insulin binding data are of limited value for our understanding of insulin resistance. This is mainly due to the tissue-specific binding of insulin as well as the "pleiotypic" nature of insulin action. As insulin action is determined at (a) the "pre-"receptor level, (b) the receptor, a n d / o r (c) different postbinding sites, it is unlikely that receptor data alone can explain defective insulin action. As knowledge of the molecular biology of insulin receptor morphology and function as well as of the action of insulin on intermediary metabolism increases, clinicians should not be further overloaded with binding data. Nevertheless, binding data on blood cells may be still of some value in investigating patients with severe insulin resistance due to genetic disorders of the insulin receptor or insulin receptor autoantibodies. Key words: Insulin receptor - Insulin resistance - Insulin action

Insulin resistance is a characteristic feature of obesity and noninsulin-dependent diabetes mellitus. Insulin resistance can be defined as a state of reduced insulin action, and severe insulin resistance exists when 100 to 1000 times the normal level of insulin is necessary to produce effects on intermediary metabolism [~8]. Insulin binding to specific cell-surface receptors is the first step in insulin action. With respect to the pathogenesis of insulinresistant states, numerous clinical studies have been done on insulin receptors. As tissue sampling

from liver or skeletal muscle is (a) practically difficult and (b) not justified for ethical reasons in man, most of these studies have been done on erythrocytes, mononuclear cells, or lymphocytes. The concept of blood cells as indicators of the insulin receptor state as well as of insulin action is now considered outdated [28], putting in question the value of many studies which have been done in the past or are presently under way. In fact, clinicians have been overloaded with insulin receptor data on blood cells, some of which are unnecessary or confusing. The explosion of research on insulin receptors may be considered in connection with the relevance of these studies to the pathophysiology of certain diseases. The need for a careful consideration of the current concept is obvious. Significance of Blood Cell Metabolism and Insulin Binding

It is evident that blood cells are not the main target cells for insulin action: In the postabsorptive resting state erythrocytes utilize 0.06 mg glucose/kg × rain, which is about 3% of the total glucose uptake (i.e., 2.2 mg/kg x rain; see [9]). The percentage contribution by blood cells to the total body glucose disposal may further decrease after glucose loading, where skeletal muscle and liver markedly increase their glucose utilization. On the other hand, hyperinsulinemia downregulates insulin binding to human monocytes or T lymphocytes [4, 15] and insulin has been shown to increase glucose uptake in these cell types [5, 14]. Yet even their contribution to total glucose utilization is small, monocytes and lymphocytes seem to have similar adaptive mechanisms to hyperinsulinemia as the main insulin responsive tissues. In addition, there is an apparent relationship between insulin

950 receptor structure in blood cells and the main target tissues for insulin [31], which provides a further basis for the use of blood cells. From a teleological point of view, blood cell insulin binding may have implications for the hormonal modulation of immune response or cellular differentiation. With respect to quantitative terms, the human body contains about 1,500 g of lymphocytes from which 1,300 g are present in the tissues in general (e.g., 100 g are present in the lymphatic tissues, 70 g are present in the bone marrow), whereas only 3 g are present in the circulating blood (see [1]). Thus, it is evident that only a minor proportion of total body lymphocytes is present in the blood. As lymphocytes in the tissues are in dynamic equilibrium with circulating blood cells, observations on blood lymphocytes may reflect a greater proportion of cells than previously has been assumed. For a long time lymphocyte intermediary metabolism has been considered to depend primarily on glucose, implying that these cells are metabolically uninteresting. This idea has been now replaced by a more sophisticated view, e.g., energy metabolism of lymphocytes depends on an interaction of different substrates, such as glutamine, free fatty acids, ketones, and glucose [1]. It is evident that, although lymphocyte immune response has been studied in detail, its metabolic basis is still far from clear. Thus, misconceptions and ignorance may contribute to our present view of blood cell insulin binding and/or of the action of insulin on blood cells. Taken together, nucleated blood cells have some of the biochemical capabilities of muscle and liver cells. This does not automatically mean that these cells are a realistic model for other nucleated cells in the body.

Heterogeneity of Insulin Binding It is generally accepted that binding data on skeletal muscle or on hepatocytes would be of great value for our understanding of the disturbances in glucose metabolism in man. There are good reasons for why we do not know much about insulin binding to these cell types in man. Nevertheless, one group of authors have studied liver cell insulin receptors in nonobese and obese subjects and have shown that obesity is associated with a loss of hepatic insulin receptors [2]. This finding is compatible with a decreased hepatic insulin sensitivity in obese subjects, which has also been shown recently [25], and is similar to data reported previously for peripheral cells [20]. To overcome the problems of tissue sampling in man, it has been suggested that insulin binding to human adipocytes or fibro-

M.J. Miiller: Insulin Receptor Bindingto Blood Cells blasts mirrors changes in insulin receptors in the main target tissues. Unfortunately, adipose tissue and fibroblasts contribute to only 1% or even less than 1% of total body glucose utilization [6, 9]. The tacit assumption that adipose tissue behaves like skeletal muscle may not be justified unless sufficient data are provided. However, up to now insulin binding to myocytes and adipocytes has not been measured in parallel in the same subjects. Comparing insulin binding to different cell types shows considerable differences within the same individual (e.g., blood cells vs adipocytes, see [24, 28]). It is now evident that insulin receptor binding is heterogeneous [24]. But this phenomenon is not only due to blood cells, where erythrocyte insulin binding may differ from monocyte data within the same subject (see [28]). Insulin receptor number results from the de novo synthesis as well as the internalization, the recycling, or the intracellular degradation of the receptor protein. With respect to insulin-mediated cycling, differences in various aspects of this process are apparent among different tissues and cells, e.g., energy depletion blocked insulin-mediated receptor internalization in monocytes, whereas depletion of ATP provoked insulinreceptor internalization in fat cells [23]. Thus, differences in receptor cycling complement the differences reported in the arrangement of the insulin receptors on the cell surface found between different cell types. Even more confusing, insulin binding can differ within the same organ: adipocytes isolated from different sites of the body exhibit different binding characteristics [8], suggesting that adipose tissue by itself is heterogeneous with respect to insulin binding. Furthermore, insulin action on different types of skeletal muscle shows considerable differences with respect to the kinetic data as well as the net response [17]. Thus, insulin binding as well as its action are obviously tissue-specific. This limits any generalization from data obtained in singlecell types (i.e., blood cells as well as adipocytes or even muscle and liver cells). Regarding insulin binding and its action, there is no single cell type in the body which mirrors changes in all target tissues. The insulin receptor state of the body obviously is not homogeneous. This idea limits the clinical value of receptor data, but does not exclude the possibility that different cell types may be affected in unison.

Insulin Binding and Insulin Action It has been assumed that insulin receptor binding is the main regulatory step in insulin action. A

M.J. M/iller: Insulin Receptor Binding to Blood Cells

rise or fall in cellular insulin binding has been thought to reflect a rise or a fall in insulin action. This idea may still be proven true for severe insulin resistance due to genetic alterations of the binding site, where defective binding results in severe disturbances in glucose metabolism. Assuming an intact insulin molecule, insulin binding is the first step in insulin action. Although the insulin receptor has been purified and its gene cloned [29], we still do not understand the molecular mechanism(s) of insulin action. The insulin receptor is an integral membrane protein, which is a tetrameric glucoprotein complex consisting of two a- and two b-subunits linked together by disulfide bonds. Insulin binds to the a-subunit of the insulin receptor, which again activates the b-subunit by phosphorylation. A defect of the receptor beyond the binding site (i.e., a defective b-subunit) results in a dissociation between insulin binding and insulin action, i.e., normal insulin binding and defective action of the hormone. Normal binding but a defect in the phosphorylation of the b-subunit has been described as a possible cause of the type A syndrome of insulin resistance [13]. However, the severe insulin resistance of these patients may result from a heterogeneity of defects, where a loss of receptor number and/or a reduction in the affinity constant leads to a fall in both, receptor binding and phosphorylation, and a linear coupling between receptor occupancy and phosphorylation is preserved. In contrast to this, a specific defect of the b-subunit only results in decreased phosphorylation [19]. All these possible defects are expressed in erythrocytes, which makes these cells a suitable model for studying severe insulin resistance [13]. Defective coupling between the a- and the b-subunit is not only known for genetic disorders of the insulin receptor: At equal numbers of insulin receptors, insulin-stimulated kinase activity is decreased by about 50% in adipocytes isolated from noninsulin-dependent diabetic subjects, indicating a decreased coupling efficiency [11]. From all these data, the importance of transmembrane signalling becomes evident. The expression of the insulin signal results from the sum of transmembrane and intracellular signalling mechanisms, which are part of the complex interactions between processes triggered by cellsurface receptors. A detailed biochemical description of the receptor-mediated control of intermediary metabolism is beyond the scope of this paper and the reader is therefore referred to a recent review article [16]. Briefly, insulin exerts its action by at least three different mechanisms: (a) The activation of the tyrosine kinase, (b) endocytosis of

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the insulin receptor complex, and/or (c) the involvement of different guanine nucleotide regulatory proteins, which again may activate different second-messenger systems involving cyclic-AMP turnover and/or polyphosphatidylinositol turnover and/or less characterized "mediator" molecules (see [16]). The involvement of different signaltransducing mechanisms is evident, as the coupling of insulin binding to glucose transport seems to be independent of insulin receptor phosphorylation [27]. Furthermore, the existence of different signaling systems may have implications for the pathophysiology of insulin-resistant states: Glucagon and/or catecholamines may induce selective insulin resistance in rat hepatocytes by blocking insulin interaction only with the guanine regulatory protein system [16]. As a net result of insulin binding and the subsequent transmembrane and intracellular signaling mechanisms, a series of key enzymes involved in different metabolic pathways (e.g., glycogen, synthase, and pyruvatedehydrogenase in skeletal muscle) and consequently intracellular glucose metabolism are increased (e.g., accelerated synthesis of glycogen as well as glucose oxidation in the muscle cell). In contrast, the stimulatory action of insulin on the activity of glycogensynthase and pyruvatedehydrogenase is reduced in adipocytes isolated from type II diabetics, which is consistent with their decreased rate of oxidative and nonoxidative glucose metabolism [21]. Considering the different insulin-dependent metabolic events, it becomes evident that selective defects contribute to glucose intolerance to a different extent, e.g., defects in glucose transport can at least explain 25%-50% of the variability observed in the glucose disposal rate. Thus, selective defects in glucose transport or in the oxidative and/or nonoxidative metabolism of glucose will result in different degrees of insulin resistance. Insulin binding initiates a cascade of regulatory events, where insulin action obviously is not only determined at the receptor level, but also at a postreceptor level. Insulin resistance is not only accounted for by a reduction in insulin binding to its receptor, but the major component is now attributed to possible postbinding defects. Thus, some insulin resistance may be due to decreased binding, but postbinding events are more likely to explain defective insulin action. Comparing receptor and postreceptor abnormalities in obese subjects with normal glucose tolerance suggests that receptor and postreceptor alterations reflect different degrees of insulin resistance: Diminished insulin binding explains diminished insulin sensitivity

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observed in moderately obese subjects compared to lean controls, whereas diminished insulin sensitivity of severely obese subjects is mainly explained by an alteration at a postbinding site (i.e., decreased maximal response; [19]). In cultured rat adipocytes, insulin-induced insulin resistance may be progressive and results from a sequential fall in coupling efficiency between receptor occupancy and glucose transport, postreceptor defects, and receptor down-regulation [t2]. These findings implicate that insulin resistance is a dynamic concept, where different defects may contribute to different stages of this syndrome. With regard to insulin-induced alterations in intermediary metabolism in single adipocytes, it is obvious that the antilipolytic action of the hormone occurs at lower insulin concentrations when compared to insulin-induced stimulation of glucose oxidation [7]. Thus, lipolysis seems to be more sensitive to insulin than glucose metabolism. From a biochemical point of view, this finding may be explained by the different second messenger systems involved in insulin action. This result is of particular interest with respect to the pathogenesis of insulin resistant states: The sensitivity of glucose metabolism to insulin, but not the sensitivity of antilipolysis to insulin nor insulin binding, may decrease with increasing abnormalities of glucose tolerance in nondiabetic subjects [10]. Thus, decreased insulin binding may be associated with alterations in glucose metabolism at concomitantly suppressed lipolysis. Insulin action obviously is "pleiotypic" : Alterations in the insulin receptor state alone can rarely explain the pathogenesis of insulin resistance. It is evident that even t h e " best" (i.e. muscle or liver) receptor data alone cannot reflect insulin action. But again, this problem is not only true for bloodcell insulin binding. "Pleiotypic" Nature of Insulin Action

The "pleiotypic" nature of insulin action is further exemplified by a recent clinical study. Hyperthyroidism is generally thought to antagonize insulin action (see [22]). As expected, insulin binding to human monocytes, cultured-mitogen activated T lymphocytes, as well as to adipocytes, is decreased, whereas insulin binding to erythrocytes remains unchanged [3, 22]. On the other hand, at euglycemia insulin-induced glucose oxidation is increased in thyrotoxic patients in vivo [26], as well as in adipocytes isolated from hyperthyroid patients in vitro [3]. These discrepancies may be due to a direct effect of thyroid hormones on glucose oxidation,

M.J. M/iller: Insulin Receptor Binding to Blood Cells

which again is independent of the effect of insulin. However, during hyperglycemia, thyroid hormoneinduced insulin resistance becomes unequivocal [22]. Even more confusing, in untreated hyperthyroidism there is a decreased sensitivity of human adipocytes to the antilipolytic effect of insulin, which is normalized after antithyroid therapy [32]. Thus, insulin resistance may or may not become obvious in hyperthyroidism, depending on the experimental design of the study. It is obvious that in hyperthyroid patients decreased insulin binding to blood cells and adipocytes does not tell us the whole story. The regulation of different metabolic pathways by insulin differs even within the same cell type (e.g., adipocytes). This complex metabolic event cannot be mirrored by the "best" receptor data. Nevertheless, the finding that hyperthyroidism decreases insulin binding to human T lymphocytes is of particular interest, as defective binding (a) contributes to thyroid hormone-induced alterations in intermediary metabolism and (b) gives some insight into thyroid hormone action at the cellular level. In fact, physiological amounts of T3 directly decrease the number of insulin receptors of cultured human T lymphocytes [30]; future studies will further clarifiy the pathophysiology of this event. It is evident that human T lymphocytes represent an ideal system for studying insulin receptor regulation, because they represent human cells which possess high affinity insulin receptors and can be chronically manipulated in tissue culture, where the kinetics of insulin-receptor loss and regeneration can be studied in detail. Conclusion

Assuming an intact insulin molecule, insulin binding is the first step of insulin action, which is determined at (a) a '° pre-"receptor level, (b) the receptor itself, as well as (c) at different postbinding sites. Some insulin resistance may be due to decreased binding. As postbinding events are the major component of insulin action, most insulin resistance is now attributed to postbinding defects. Insulin binding is heterogeneous, thus, decreases in insulin binding to blood cells do not necessarily reflect a reduced insulin binding to skeletal muscle. On the other hand, normal binding to blood cells may be associated with a decreased binding of insulin in the main target tissues. But this problem comes true for all cell types of the body, where insulin binding to adipocytes may not reflect skeletal muscle receptor states, which again may be different from hepatocyte insulin binding within the same individual. If, however, decreases

M.J. Mfiller: Insulin Receptor Binding to Blood Cells in b l o o d cell i n s u l i n b i n d i n g a r e a c c o m p a n i e d b y d i s t u r b a n c e s in g l u c o s e m e t a b o l i s m (e.g., i m p a i r e d g l u c o s e t o l e r a n c e ) , t h e r e is e v i d e n c e t h a t d e f e c t i v e insulin binding may contribute to the alterations o b s e r v e d . H o w e v e r , this f i n d i n g d o e s n o t e x c l u d e f u r t h e r p o s t r e c e p t o r defects. A l t h o u g h p r e v i o u s l y p u b l i s h e d i n s u l i n b i n d i n g d a t a c o n t r i b u t e to o u r present knowledge of insulin resistance, future s t u d i e s b a s e d o n b l o o d cell o r e v e n a d i p o c y t e i n s u lin b i n d i n g a l o n e a r e o f l i m i t e d v a l u e w i t h r e s p e c t to c l i n i c a l s t u d i e s o n i n s u l i n r e s i s t a n c e . I t is n o w k n o w n t h a t n o n i n s u l i n - d e p e n d e n t d i a b e t e s is n o t a s i n g l e d i s e a s e e n t i t y , w h e r e h e t e r o g e n e o u s d e f e c t s (e.g., in i n s u l i n s e c r e t i o n a n d / o r hepatic or peripheral insulin action) result in hyperg l y c e m i a . I t is o b v i o u s t h a t e v e n c e l l u l a r i n s u l i n r e s i s t a n c e is h e t e r o g e n e o u s a n d i n s u l i n b i n d i n g is o n l y o n e s t e p in a l o n g c h a i n o f e v e n t s . N e v e r t h e less, i n s u l i n r e c e p t o r d a t a o n b l o o d cells a r e o f v a l u e in i n v e s t i g a t i n g p a t i e n t s w i t h e x t r e m e i n s u l i n r e s i s t a n c e d u e t o (a) g e n e t i c d e f e c t s o f t h e r e c e p t o r a n d (b) i n s u l i n r e c e p t o r a u t o a n t i b o d i e s . W i t h regard to the fascinating investigations of the molecular biology of insulin receptor morphology and f u n c t i o n , f u t u r e s t u d i e s in t h i s a r e a s h o u l d a v o i d further overloading with insulin receptor information and/or misinformation.

Acknowledgement. Financial support for our own data described in this text was provided by generous grants from the Deutsche Forschungsgemeinschaft (M/.i 714 1-1) and F6rdermittel des Landes Niedersachsen (Ba 1016). References 1. Ardawi MJM, Newsholme EA (1985) Metabolism of lymphocytes and its importance in the immune response. Essays Biochem 21 : 1-44 2. Arner P, Einarsson K, Bachman L, Nilsell L, Lerea KM, Livingston JN (1983) Studies of liver insulin receptors in non-obese and obese human subjects. J Clin Invest 72:1729-1736 3. Arner P, Bolinder J, Wennlund A, Oestman J (1984) Influence of thyroid hormone level on insulin action in human adipose tissue. Diabetes 33:369-375 4. Baker B, Mandarino C, Brick B, Rizza R, Gerich J (1984) Influences of changes in insulin receptor binding during insulin infusions on the shape of the insulin dose-response curve for glucose disposal in man. J Clin Endocrin Metab 58: 392-396 5. Beck-Nilson H, Pederson O (1979) Insulin binding, insulin degradation and glucose metabolism in human monocytes. Diabetologia 17:7%84 6. Bjrrntop P, Sjrstrom L (1978) Carbohydrate storage in man: speculations and some quantitative considerations. Metabolism 27:1853-1863 7. Cheng JS, Kalant F (1970) Effect of insulin and growth hormone on the flux rates of plasma glucose and plasma free fatty acids in man. J Clin Endocrin Metab 31 : 647-653 8. Engfeldt P, Bolinder J, Oestman J, Arner P (1985) Effect

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Received: March 2, 1987 Accepted:August 3, 1987

Priv.-Doz. Dr. Manfred J. Miiller Medizinische Hochschule Hannover Abteilung Klinische Endokrinotogie Konstanty-Guttschow-Str. 8 D-3000 Hmmover 61