Insulin receptors in human mononuclear leucocytes. II. Binding and degradation of insulin in obese subjects.

The binding of insulin to the receptors on circulating mononuclear cells of obese subjects is significantly decreased when compared to the binding in normal subjects. This fenomenon is due to the reduction of the number of insulin receptors rather than reduction in affinity. The insulin degradation is also reduced, but a very strong correlation, similar to that demonstrated in normal subjects exist between insulin binding to its receptors and insulin degradation.     

Insulin and analogue effects on protein degradation in different cell types. Dissociation between binding and activity

In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation. Cellular protein degradation is under the control of multiple systems, including lysosomes, proteasomes, calpains, and giant protease. Insulin has been shown to alter proteasome activity in vitro and in vivo. We examined the inhibition of protein degradation by insulin and insulin analogues (Lys(B28),Pro(B29)-insulin (LysPro), Asp(B10)-insulin (B10), and Glu(B4),Gln(B16),Phe(B17)-insulin (EQF)) in H4, HepG2, and L6 cells. These effects were compared with receptor binding. Protein degradation was examined by release of trichloroacetic acid-soluble radioactivity from cells previously labeled with [(3)H]leucine. Short- and intermediate-lived proteins were examined. H4 cells bound insulin with an EC(50) of 4.6 x 10(-9) m. LysPro was similar. The affinity of B10 was increased 2-fold; that of EQF decreased 15-fold. Protein degradation inhibition in H4 cells was highly sensitive to insulin (EC(50) = 4.2 x 10(-11) and 1.6 x 10(-10) m, short- and intermediate-lived protein degradation, respectively) and analogues. Despite similar binding, LysPro was 11- to 18-fold more potent than insulin at inhibiting protein degradation. Conversely, although EQF showed lower binding to H4 cells than insulin, its action was similar. The relative binding potencies of analogues in HepG2 cells were similar to those in H4 cells. Examination of protein degradation showed insulin, LysPro, and B10 were equivalent while EQF was less potent. L6 cells showed no difference in the binding of the analogues compared with insulin, but their effect on protein degradation was similar to that seen in HepG2 cells except B10 inhibited intermediate-lived protein degradation better than insulin. These studies illustrate the complexities of cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The differences seen in the action of the analogues cannot be attributed to binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.     

Regulatory role of insulin in the degradation of low density lipoprotein by cultured human skin fibroblasts.

The degradation of 125I-labeled low density lipoprotein by cultured human skin fibroblasts was enhanced 25% by preincubation of cells with insulin. This effect of insulin appeared to be mediated via stimulation of low density lipoprotein binding to its cell surface receptor, since binding and subsequent internalization of low density lipoprotein were stimulated to a similar extent as was degradation. In addition, insulin enhanced binding of low density lipoprotein at 4 degrees C, at which temperature internalization of the lipoprotein does not occur. A similar effect of insulin on the interaction of very low density lipoprotein with cultured fibroblasts was observed. Insulin-induced changes in the degradation of low density lipoprotein and very low density lipoprotein appeared to be a function of the change in lipoprotein binding. Thus, insulin may play a role in the regulation of low density lipoprotein and very low density lipoprotein degradation by peripheral cells by influencing the receptor-mediated transport of these lipoproteins.     

Differences in degradation processes for insulin and its receptor in cultured foetal hepatocytes.

Binding and degradation of 125I-labelled insulin were studied in cultured foetal hepatocytes after exposure to the protein-synthesis inhibitors tunicamycin and cycloheximide. Tunicamycin (1 microgram/ml) induced a steady decrease of insulin binding, which was decreased by 50% after 13 h. As the total number of binding sites per hepatocyte was 20000, the rate of the receptor degradation could not exceed 13 sites/min per hepatocyte. Cycloheximide (2.8 micrograms/ml) increased insulin binding by 30% within 6 h, an effect that persisted for up to 25 h. This drug had a specific inhibitory effect on the degradation of proteins prelabelled for 10 h with [14C]glucosamine, without affecting the degradation of total proteins. Chronic exposure to 10 nM-insulin neither decreased insulin binding nor modified the effect of the drugs. The absence of down-regulation of insulin receptors cannot be attributed to rapid receptor biosynthesis in foetal hepatocytes. Cellular insulin degradation, which is exclusively receptor-mediated, was determined by two different parameters. First, the rate of release of degraded insulin into the medium was 600 molecules/min per hepatocyte with 1 nM labelled hormone, and increased (preincubation with cycloheximide) or decreased (tunicamycin) as a function of the amount of cell-bound insulin. Secondly, the percentage of cell-bound insulin degraded was not changed by the presence of protein-synthesis inhibitors (25-30%). The stability of insulin degradation suggested that this process was dependent on long-life proteinase systems. Such differences in degradation rates and cycloheximide sensitivity imply that hormone- and receptor-degradation processes utilize distinct pathways.     

Chloroquine stimulation of insulin binding to IM-9 lymphocytes: Evidence for action at a nonlysosomal site

Chloroquine has been shown to both increase insulin binding and decrease intracellular insulin degradation in several target cells. However, whether this increased binding is a consequence of decreased degradation is unclear. Accordingly, we studied the effects of chloroquine on insulin binding to IM-9 cultured lymphocytes, a cell type that does not degrade insulin in the cell interior. In these cells, chloroquine enhanced insulin binding in a dose dependent manner over the concentration range of 100 μM to 1 mM; at 1 mM binding was increased by 70%. Scatchard analysis indicated that chloroquine acted to increase receptor affinity. These studies indicate, therefore, that chloroquine can enhance the binding of insulin to its receptor via a mechanism that is independent of effects on intracellular insulin degradation.     

Bacitracin enhances intracellular accumulation of insulin in rat adipocytes

Bacitracin (1 mg/ml) markedly increased (approx. 75%) the cell-associated specifically bound 125I-labelled insulin without altering the affinity of the binding sites. Bacitracin also exerted a modest inhibitory effect on the degradation of insulin in the incubation medium determined as radioactivity not precipitated by trichloroacetic acid (from 9.6 to 4.8%). The effect on insulin binding was about 5-times as sensitive as the effect on degradation. The increased binding was due to intracellular accumulation of radioactivity which could not be removed by treating the cells with trypsin. This increase was not seen when the internalization process was reduced by ATP-depletion or low temperature. Since the trypsin-sensitive fraction of cell-associated radioactivity was apparently not altered, it is suggested that bacitracin, in addition to its well-known inhibition of extracellular degradation, also inhibits the intracellular degradation of insulin.     

Absence of down-regulation of the insulin receptor by insulin. A possible mechanism of insulin resistance in the rat.

Insulin resistance occurs in rat adipocytes during pregnancy and lactation despite increased or normal insulin binding respectively; this suggests that a post-receptor defect exists. The possibility has been examined that, although insulin binding occurs normally, internalization of insulin or its receptor may be impaired in these states. Insulin produced a dose-dependent reduction in the number of insulin receptors on adipocytes from virgin rats maintained in culture medium, probably due to internalization of the hormone-receptor complex. In contrast, adipocytes from pregnant and lactating rats did not exhibit this 'down-regulation' phenomenon. Down regulation was, however, apparent in all groups when the experiments were performed in Tris buffer (where receptor recycling is inhibited), suggesting that in pregnant and lactating rats insulin receptors are rapidly recycled back to the plasma membrane, whereas in virgin rats this recycling process is less effective. Internalization of insulin was also determined by using 125I-labelled insulin. Adipocytes from pregnant and lactating rats appeared to internalize similar amounts of insulin to virgin rats. In the presence of the lysosomal inhibitor chloroquine, adipocytes from pregnant rats internalized more insulin than virgin or lactating rats. These results suggest that adipocytes from pregnant and lactating rats internalize insulin and its receptor normally, whereas intracellular processing of the insulin receptor may differ from that in virgin rats. In addition the rate of lysosomal degradation of insulin may be altered in adipocytes from pregnant rats.     

Insulin processing and signal transduction in rat adipocytes

A glycine-HCl buffer (glycine, 50 mM/NaCl, 0.15 M/HCl, pH 3.5) was used to strip insulin bound to adipocyte cell surfaces. Adipocytes retained their integrity in the glycine buffer and their binding capacity for [125I]iodoinsulin could be completely recovered on transfer of the cells to physiological media. At 37 degrees C, [125I]iodoinsulin binds rapidly to plasma membrane receptors; maximal binding occurs within 10 min. At this temperature, the initial binding is followed by rapid internalization, degradation of the hormone and subsequent loss of label. Insulin treatment, at 37 degrees C, induced internalization of 37% of the plasma membrane insulin receptors. Phenylarsine oxide (PAO), a confirmed inhibitor of protein internalization, allowed insulin binding but completely inhibited degradation of the hormone. Monensin, a carboxylic ionophore which impairs uncoupling hormone-receptor complexes, effectively restricted insulin degradation over short time periods (less than 30 min). Addition of monensin to insulin-stimulated cells did not impair D-glucose uptake. It has previously been reported that PAO inhibits hexose transport through the direct interaction with the glucose transporters and low concentrations of PAO (1 microM) transiently inhibit insulin-stimulated glucose uptake. This recovery phenomenon was again observed when PAO was added to insulin-stimulated, monensin-treated adipocytes. The data suggests that lysosomal degradation of insulin is not requisite for signal transduction.     

Participation of cellular thiol/disulphide groups in the uptake, degradation and bioactivity of insulin in primary cultures of rat hepatocytes.

The effects on the uptake (cell-associated 125I) and degradation (125I-labelled products released into the medium) of 125I-insulin and bioactivity (protein, glycogen and lipid synthesis) of insulin caused by altering the cellular thiol/disulphide status in primary cultures of rat hepatocytes were studied. Incubation of hepatocyte cultures with various exogenous thiol compounds (reduced glutathione, 2-mercaptoethanol, cysteamine, dithiothreitol) resulted in increased insulin binding, but markedly decreased degradation and bioactivity. These effects could be reversed by washing or by the addition of oxidized glutathione, which alone had no effect. When cultures were exposed to certain thiol-modifying reagents (N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzenesulphonate, iodoacetamide, iodoacetate), some decreases in bioactivity were evident, but the pronounced decrease in insulin degradation observed with the thiol-containing compounds was not observed with this class of compounds. None of the thiol-containing or -modifying agents tested had any significant effect on cellular ATP concentrations, indicating that the effects observed were due to perturbation of the thiol/disulphide status. Depletion of intracellular glutathione by DL-buthionine SR-sulphoximine (a specific inhibitor of glutathionine biosynthesis) decreased the syntheses of glycogen and lipid by about one-half, while having essentially no effect on protein synthesis, ATP concentrations or on the binding and degradation of insulin. The data presented here indicate that although intracellular thiol (glutathione) concentrations may be important for the maintenance of full expression of certain biological activities (glycogen and lipid synthesis), the thiol/disulphide groups on the cell surface and those immediately inside the cell membrane may be more critical in the mediation of insulin action, including the degradation and bioactivity of insulin in primary cultures of rat hepatocytes.     

Effect of internalization and degradation of insulin on rat adipocyte insulin receptor binding kinetics

We have assessed the influence of nondisplaceable (internalized) insulin and insulin degradation during binding reactions at 37 degrees C on the numbers and affinities of insulin binding sites on isolated rat adipocytes. Corrections for nondisplaceable insulin caused a 33% reduction in the number of the high affinity sites (p less than 0.01) and a 24% reduction (p less than 0.01) in the number of the low affinity sites which was associated with a 20% increase (p less than 0.01) in affinity when a two-site model was applied. With a one-site model, the number of insulin receptors decreased by approximately 33% (p less than 0.01), but the affinity did not change. These results indicate that the internalization and degradation of insulin that occurs during the binding reaction can significantly affect the estimation of insulin binding kinetics. Potential variations in internalization and degradation of insulin by cells obtained under various physiological or pathologic conditions should, therefore, be taken into consideration in the interpretation of insulin binding data.     

Insulin inhibition of protein degradation in cell monolayers

Protein degradation has been measured in confluent monolayers of eleven lines of contact-inhibited cells and ten transformed lines as the rate of release of trichloroacetic acid-soluble radioactivity after prelabeling cell protein with [³H]leucine. Insulin, at concentrations from 10^?12 M to 10^?6 M, has been added at the beginning of the 4-hour degradation period to detect selective effects of this hormone as an inhibitor of the inducible proteolysis occurring in serumfree medium. In addition insulin binding measurements have been performed on selected cell lines in an attempt to relate receptor properties to insulin action. Substantial effects of insulin are found in most cells with a selective inhibition at low insulin concentrations noted in several of the transformed lines. The difference in insulin sensitivity is not entirely definitive because temperature-sensitive transformation mutants of NRK cells are not more sensitive to insulin at a temperature where they show the transformed phenotype. Although insulin receptors on different cell lines have similar binding properties, two of the hepatomas used, H35 and MH₁C₁, show inhibition of protein degradation at insulin concentrations where receptor occupancy is extremely low. Calvarial osteoblast-like cells have a high rate of protein degradation which can be reduced by growth factors but not by insulin. The lack of an insulin response is a consequence of poor insulin binding to the cells. Insulin binds to the osteogenic sarcoma cells in substantial amounts. However, its normal action to inhibit the induced proteolysis is restricted because with these cells no increase of proteolysis occurs in serum-free medium. Generally higher rates of protein degradation are observed in the contact-inhibited lines than the transformed cells. We suggest that this difference may provide a selective growth advantage to transformed cells.     

Binding and degradation of 125I-insulin by rat hepatocytes.

The binding and the velocity of degradation of 125I-insulin in the absence or presence of varying concentrations of native procline insulin were studied using isolated rat hepatocytes. At insulin concentrations ranging from 5 X 10(-11) to 10(-6) M, insulin degradation velocity showed a first order dependence on the total concentration of insulin bound at steady state. The overall reaction had an apparent rate constant of 0.030 +/- 0.011 min-1. Furthermore, the degradation of a given amount of 125I-insulin bound to cells was more rapid and extensive than the degradation of the same amount of insulin which had been newly exposed to fresh cells. Mid pretreatment of isolated hepatocytes with trypsin or chymotrypsin at concentrations of 5 to 20 mug/ml depressed to the same degree the amount of 125-I-insulin bound at steady state and the 125I-insulin degradation velocity. Peptide or protein hormones unrelated to insulin, including the oxidized A and B chains of insulin, failed to depress the amount of insulin bound or the velocity of insulin degradation when present at concentrations of 10-5 or 10-6 M. Over a wide range of concentrations, various synthetic insulin analogues and naturally occurring insulins depressed to the same degree the amount of 125I-insulin bound at steady state and the 125I-insulin degradation velocity. These observations suggest that insulin bound to hepatocyte plasma membranes is the substrate for insulin degradation by the liver.     

Mode of uptake and degradation of 125I-labelled insulin by isolated hepatocytes and H4 hepatoma cells.

The effects of various agents on the binding and degradation of 125I-labelled insulin by isolated rat hepatocytes and cultured H4 hepatoma cells were studied. Various lysosomotropic agents, including chloroquine, ammonium chloride, and the topical anesthetics, lidocaine and procaine inhibited insulin degradation by H4 hepatoma cells but had little effect on the binding of the hormone. Similarly, tosyl-L-lysyl chloromethyl ketone selectively inhibited the degradation of 125I-labelled insulin by isolated hepatocytes, as did the sulfhydryl reagents, p-hydroxy- and p-chloromercuriphenyl sulfonic acid. Inhibitors of energy production, including sodium fluoride, sodium azide, and dinitrophenol, also selectively inhibited the degradation of insulin by hepatocytes, although cyanide had no effect under the conditions used. Lectins and antimicrotubular agents, which are known to affect the mobility of plasma membrane proteins or of intracytoplasmic vesicles, selectively inhibited insulin degradation by hepatocytes to varying degrees, whereas agents which inhibit the function of microfilaments had no effect. At temperatures below 20 degrees C, insulin degradation was negligible but rose rapidly between 20 and 37 degrees C, suggesting that a membrane-related step is rate limiting in the overall degradative process. These results are all consistent with a model of insulin uptake by target tissue involving pinocytosis of receptor-bound hormone followed by intralysosomal degradation.     

Binding and degradation of insulin by human peripheral granulocytes. Demonstration of specific receptors with high affinity.

The interaction of insulin with human circulating granulocytes was studied with the use of 125I-insulin. Human granulocytes, isolated from blood by the B?yum technique, showed high insulin-degrading activity in vitro which almost obscured the presence of specific, high affinity binding sites. Degradation, measured by trichloroacetic acid precipitation and by binding to well characterized insulin receptors on cultured human lymphocytes (IM-9 line), was due to extracellular as well as cell-bound enzymes. Degradation was enhanced by Ca2+ and thiols and inhibited by various protease inhibitors and sulfhydryl-blocking reagents. Phenylmethylsulfonyl fluoride (5 X 10(-4) M), a serine protease inhibitor, was the most potent and inhibited 125I-insulin degradation by 80 to 90%. Tert-butyl hydroperoxide (2 X 10(-3) M), a glutathione-oxidizing reagent, inhibited degradation by 35 to 50%, possibly due to an effect on a glutathione-insulin transhydrogenase. Neither of the inhibitors affected cell viability. In the presence of inhibitors of degradation, binding sites for insulin with high affinity were detected, which by multiple criteria were true insulin receptors. Binding to these sites was rapid, saturable, and reversible with about 1000 sites/cell. The Hill coefficient for binding was 0.7, and the Scatchard plot of B/F versus B was curvilinear, due to site-site interactions of the negative cooperative type; the latter were demonstrated directly by kinetic studies. As shown previously for all other insulin receptors, binding was highly pH-dependent, and insulin analogues had affinities for these sites that closely correlated with their biological potencies.     

Insulin-binding peptide. Design and characterization

The design and characterization of a six-amino acid-containing peptide that binds insulin is described. The amino acid sequence of the insulin-binding peptide (IBP) was determined from the strand of DNA complementary to the strand of DNA coding for the insulin molecule in the domain of the insulin monomer believed to interact with the insulin receptor. The IBP (Cys-Val-Glu-Glu-Ala-Ser) binds specifically to insulin in a saturable manner with a Kd of 3 nM. This binding process is time dependent and slightly temperature dependent, and the peptide appears to interact with insulin near the carboxyl terminus of the B-chain of insulin. Incubation of insulin with the peptide decreases insulin binding to the insulin receptor by 50%, with no effect on the affinity of insulin for the receptor and no effect on cellular insulin-stimulated deoxyglucose uptake. A polyclonal antibody produced against the IBP will inhibit specific insulin binding to intact cells by approximately 50%, with no effects on insulin-stimulated glucose uptake. From this data, we suggest that there are at least two domains of the insulin molecule through which it interacts with its receptor, the "binding region" of insulin, which is the domain blocked by the IBP, and the "message region" of insulin, through which insulin not only binds to the receptor, but also generates the cellular signal.     

A short incubation time in insulin binding experiments leads to disappearance of negative cooperativity in H35 hepatoma cells

The effects of different periods of incubation (8 min vs 20 min) on insulin binding kinetics were examined in a H35 hepatoma cell line. Scatchard plots from cells incubated for 8 min were linear (r = 0.987 +/- 0.006), in contrast to curvilinear Scatchard plots from cells incubated for 20 min. Hill plots showed a slope of 1.006 +/- 0.024 for the 8 min incubation, whereas the slope was 0.827 +/- 0.0026 (p less than 0.0005) for the 20 min incubation. TCA precipitation of the medium showed minimal insulin degradation products at 8 min with a significant increase at 20 min (1.38 +/- 0.11% vs. 3.06 +/- 0.37%, p less than 0.0005). Internalized insulin was also significantly increased at 20 min as compared to 8 min incubation (48.9 +/- 5.6% vs. 32.4 +/- 3.0%, p less than 0.0005) These data indicate that after 8 min of incubation no appreciable cooperativity of insulin binding was present, while negative cooperativity was present after 20 min of incubation. As significantly more insulin degradation has taken place after prolonged incubation these data support the hypothesis that insulin degradation leads to negative cooperativity of insulin receptors.     

Phytohemagglutinin (PHA) activated human T-lymphocytes: concomitant appearance of insulin binding, degradation and insulin-mediated activation of pyruvate dehydrogenase (PDH)

Binding and degradation of A14125I-Insulin as well as the effect of insulin on pyruvate dehydrogenase (PDH) activation were studied in non-stimulated and phytohemagglutinin (PHA)-stimulated thymic-derived lymphocytes (T-lymphocytes) of man under varying conditions of time, temperature, and cell concentration. The nonstimulated viable T-lymphocytes exhibited neither binding, degradation, nor PDH activation in response to insulin. With PHA stimulation, a time and temperature-dependent binding was noted in T-lymphocytes which paralleled the appearance of cell-associated insulin degrading activity. Concomitant with the emergence of insulin binding and degrading activities in these cells, PDH activation was observed which was responsive to as little as 5.0 microU/ml of insulin. We conclude that in PHA-activated T-lymphocytes of man the process of insulin binding and degradation is closely related to insulin sensitive activation of PDH. These activated cells may serve as a useful model in which to study insulin binding and processing, as well as effects of insulin on postreceptor events.     

The fate of insulin in cardiac muscle. Studies on isolated muscle cells from adult rat heart.

Isolated muscle cells from adult rat heart were used to study myocardial degradation of insulin and the reactions after the initial binding event. After 60 min of association at 37 degrees C, 90% of specifically bound insulin could be dissociated from the cells; this fraction remained unaltered under steady-state conditions (up to 180 min). To assess the nature of cell-associated radioactivity, cardiocytes were solubilized and filtered on Sephadex G-50. After 5 min of association only intact insulin was observed, whereas under steady-state conditions 4% of 125I-labelled insulin bound to the cells was degraded to iodotyrosine-containing fragments. The Km for insulin degradation by isolated heart cells was estimated to be 1.75 x 10(-7)M. Receptor-mediated insulin degradation was studied by examination of the nature of radioactivity released by the cells after different times of association. After 5 min 83% of dissociating material consisted of intact insulin, whereas this fraction decreased to 50% under steady-state conditions. Treatment of cells with the lysosomotropic agent chloroquine (0.1 mM) significantly decreased the fraction that was eluted at the internal column volume. This study demonstrates that insulin degradation by the heart cell occurs by a receptor-independent and a receptor-dependent mechanism. The latter may involve internalization and a lysosomal pathway.     

Regulation of insulin degradation: expression of an evolutionarily conserved insulin-degrading enzyme increases degradation via an intracellular pathway.

The insulin-degrading enzyme (IDE) is an evolutionarily conserved enzyme that has been implicated in cellular insulin degradation, but its site of action and importance in regulating insulin degradation have not been clearly established. We addressed this question by examining the effects of overexpressing IDE on insulin degradation in COS cells, using both human IDE (hIDE) and its Drosophila homolog (dIDE). The dIDE, which was recently cloned in our laboratory, has 46% amino acid identity with hIDE, degrades insulin with comparable efficiency, and is readily expressed in mammalian cells. Transient expression of dIDE or hIDE in COS monkey kidney cells led to a 5- to 7-fold increase in the rate of degradation of extracellular insulin, indicating that IDE can regulate cellular insulin degradation. Insulin-degrading activity in the medium was very low and could not account for the difference between transfected and control cells. To further localize the site of IDE action, the fate of insulin after receptor binding was examined. The dIDE-transfected cells displayed increased degradation of prebound insulin compared to control cells. This increase in degradation was observed even when excess unlabeled insulin was added to block reuptake or extracellular degradation. These results indicate that IDE acts at least in part within the cell. The lysosomotropic agents chloroquine and NH4Cl did not affect the increase in insulin degradation produced by transfection with dIDE, indicating that the lysosomal and IDE-mediated pathways of insulin degradation are independent. The results demonstrate that IDE can regulate the degradation of insulin by intact cells via an intracellular pathway.     

Inhibition of insulin receptor binding by dimethyl sulfoxide.

Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured human lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of 125-I-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of 125I-labeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor was affected to a much lesser extent.