Insulin Binding to Adipocytes Evidence for Functionally Distinct Receptors JerroldM. Olefsky, M.D., and Helen Chang, M.S., Stanford SUMMARY Dissociation of m I-insulin from adipocyte insulin receptors was studied as a function of receptor occupancy. When cells were equilibrated with a tracer amount of 125I-insulin and the insulinreceptor complexes allowed to dissociate in diluted l25I-insulin-free buffer, in the presence or absence of unlabeled insulin, the unlabeled insulin led to acceleration of m I-insulin dissociation rates. Thus, the unlabeled insulin (dilution + insulin) led to higher fractional receptor occupancy and a clear-cut acceleration of m I insulin dissociation rates, a phenomenon consistent with negatively cooperative site-site interactions. This effect was dependent on the total insulin concentration employed and was also influenced by the temperature of the incubations. The insulin derivatives, desalanine desasparagine and desoctapeptide insulin, did not accelerate dissociation of m I-insulin. Therefore, we find evidence that adipocyte insulin receptors exhibit similar properties to those originally described by De Meyts et al. (J. Biol. Chem. 251:1877, 1976) for other insulin receptor systems. However, we also find that, under conditions where the accelerating effect of native insulin is either not appreciable or is maximal, 125I-insulin does not dissociate from its receptors as a first order process. This suggests that, even in the absence of negatively cooperative effects, adipocyte insulin receptors do not behave as a kinetically homogeneous population. Furthermore, when m I-insulin was allowed to associate with cells at high initial levels of receptor occupancy, the subsequent dissociation of the m I-insulin was faster than when only the negatively cooperative effect was determined. Therefore, something in addi-

Insulin receptors have been studied in a variety of tissues, and most workers do not find a homogeneous population of receptors with respect to binding affinA portion of this work was presented at the Thirtyseventh Annual Meeting of the American Diabetes Association held in St. Louis, Mo., June 1977. From the Department of Medicine, Stanford University Medical Center, and the Palo Alto Veterans Administration Hospital, Palo Alto, California. Dr. Olefsky's present address is University of Colorado Medical School, Department of Medicine, 4200 E. 9th Avenue, Denver, Colorado 80262. Accepted for publication April 11, 1978.

946

tion to the cooperative effect led to further acceleration of insulin dissociation. If receptors exist with functionally distinct binding characteristics such that one group of receptors has a high affinity and a low capacity while another group has a lower affinity and a high capacity, then, when cells are associated with m I-insulin plus high concentrations of unlabeled insulin, most of the m I-insulin binds to the low affinity (fast-dissociating) sites. Therefore, dissociation rates are faster when high insulin concentrations are used in the association phase, since negative cooperativity as well as rapid dissociation from a functionally lower affinity receptor are being determined. When the material that dissociates from the cells was examined, it was found that the rapidly dissociating material was intact insulin while the more slowly dissociating material contained a significant proportion of degraded material. The rate of dissociation of intact insulin could be accelerated, whereas the rate of dissociation of degraded insulin could not be accelerated in the presence of unlabeled insulin in the buffer during the dissociation phase. Thus, negatively cooperative site-site interactions do not account for all the kinetic characteristics that were observed, and the data are best explained by a model consisting of functionally heterogeneous binding sites, i.e. low affinity, high capacity sites, which are susceptible to the cooperative effect but don't degrade insulin, and high affinity, low capacity sites, which participate in the degradative process but are not susceptible to the cooperative effect. DIABETES 27:946-58, September, 1978.

ity. 1 Thus, Scatchard plots of insulin-binding data are typically curvilinear and Hill plots yield coefficients less than one. At first, these curvilinear Scatchard plots were interpreted as evidence for two distinct populations of insulin receptors: a high affinity, low capacity site and a low affinity, high capacity site. 2 More recently, De Meyts et al. 3 ' 6 found that the dissociation of 125 I-insulin from receptors can be progressively accelerated by performing the dissociation studies in the presence of increasing concentrations of unlabeled insulin. Their interpretation is that, as the fractional receptor occupancy increases, the affinity of the receptors decreases (faster dissociation rate) due to DIABETES, VOL. 2 7 , NO. 9

JERROLD M. OLEFSKY, M.D., AND HELEN CHANG, M.S.

site-site interactions, a phenomenon termed negative cooperativity. If receptor affinity decreases as fractional occupancy increases, this could provide an explanation for a curvilinear Scatchard plot. On the other hand, not all workers have been able to demonstrate that high concentrations of insulin can accelerate the dissociation rate of bound insulin, 7 and others interpreted this phenomenon as evidence for ligand-ligand interactions rather than site-site interactions. 8 ' 9 Furthermore, Gliemann recently found evidence for two distinct, adipocyte, insulin receptors based, on evidence independent of binding isotherms. 10 Therefore, the possibility that negatively cooperative sitesite interactions do not explain all the kinetic properties of insulin binding must be considered, at least for adipocytes. Negative cooperativity has not yet been characterized in adipocytes, and, therefore, the purpose of this study was (1) to determine if insulin dissociates from adipocyte receptors faster as receptor occupancy increases and (2) to see if this phenomenon explains all the kinetic features of the binding interaction and if it can entirely account for curvilinear Scatchard plots. MATERIALS AND METHODS Materials. Porcine monocomponent insulin was generously supplied by Dr. Ronald Chance of Eli Lilly Company. Highly purified ( > 90 per cent) desalanine desasparagine (DAA) insulin and desoctapeptide (DOP) insulin were generously supplied by Dr. Edward Arquilla, University of California Medical School, Irvine. Na 1 2 5 I and 14 C-inulin were purchased from New England Nuclear, bovine serum albumin (fraction V) from Armour, and collagenase from Worthington Biochemicals. Preparation of isolated adipocytes. Male SpragueDawley rats weighing 140 to 160 gm. were used for all experiments. All studies were begun between 8 and 9 a.m. Animals were stunned by a blow to the head and decapitated and epididymal fat pads were removed. Isolated fat cells were prepared by shaking at 37° C. for 60 minutes in Krebs-Ringer bicarbonate buffer containing collagenase (3 mg./ml.) and albumin (40 mg./ml.), according to the method of Rodbell. 11 Cells were then filtered through nylon mesh (250 /-iM), centrifuged at 400 r.p.m. for four minutes, and washed twice in buffer. Adipocyte counts were performed according to a modification of method III of Hirsch and Gallian, 12 in which the cells were fixed in 2 per cent osmium tetroxide in 0.05 M collidine buffer (made isotonic with saline) for 24 hours SEPTEMBER, 1978

at 37° C. and then taken up in a known volume of 0.154 M NaCl for counting. Counting was performed with a model ZB Coulter Counter with a 400-/u,M aperture. lodination of insulin. 125 I-Insulin was prepared at a specific activity of 100 to 150 yu,Ci. per microgram according to Freychet et al.' modification13 of the method of Hunter and Greenwood14 as previously described. 15 Binding studies. Isolated fat cells were suspended in a buffer containing 35 mM tris, 120 raM NaCl, 1.2 mM MgSO4, 2.5 mM KC1, 10 mM glucose, 1 mM EDTA, and 1 per cent BSA16 (pH 7.6) and incubated with 125 I-insulin and unlabeled insulin in plastic flasks in a 24° C. shaking water bath as previously described. 17 ' 18 At 24° C , optimal steady statebinding conditions are achieved after 90 minutes of incubation. In previous reports 18 ' 19 we found that binding reached steady state conditions after 45 minutes. However, the time of plateau binding is somewhat variable depending on the batch of collagenase used, but we find that steady state conditions are reached by 90 minutes regardless of the lot of collagenase. The incubations were terminated as described by Gammeltoft and Gliemann 20 by removing aliquots (200 fx\.) from the cell suspension and rapidly centrifuging the cells in plastic microtubes to which 100 fx\. of silicone oil had been added. Silicone oil has a specific gravity intermediate between buffer and cells; therefore, after centrifugation, three layers result: cells on top, oil in the middle, and buffer on the bottom. The cells were then removed and the radioactivity was determined. All studies were done in triplicate. Dissociation studies. Two types of dissociation studies were performed. In the first, adipocytes were allowed to reach steady state—binding conditions (90 minutes) with a tracer amount of 125 I-insulin (0.1 to 0.6 ng. per milliliter or 1.67 X 10"11 M to 10"10 M) in a total volume of 3 ml. at 24° C. At this point the cells were centrifuged (three minutes at 400 r.p.m.) and the buffer was removed and replaced with an equal amount of iced insulin-free media. The cells were then distributed in aliquots (100 /xl.) into tubes containing 1.9 ml. of buffer (24° C.) with added unlabeled insulin (dilution + insulin) or with no added insulin (dilution only). The cells in these tubes were then centrifuged through 0.5 ml. of silicone oil at the indicated time points, and the infranates were removed. The radioactivity remaining in the cell layer was determined directly in these tubes. Since the cells tend to stick to the sides of the tubes, it was necessary 947

INSULIN BINDING TO ADIPOCYTES

to use a counter with a three-inch crystal so that the radioactivity associated with the cells on the sides of the tubes was counted at the same efficiency as at the bottom of the tubes. The amount of specific binding before the initial centrifugation and at the time the aliquots (100 fii.) were distributed was comparable. The amount bound at time zero is the amount of specific binding in an aliquot (100 /Ltl.) of the cell suspension obtained just as the aliquots were distributed into the dissociation tubes. Using 14 C-inulin to assess extracellular water space, 21 it was found that the original extracellular medium is diluted ~ 2,000-fold* in the final dissociation incubations. Methodologic experiments have shown that this degree of dilution is more than enough to assure that essentially no unbound 125 I-insulin is carried through from the original, binding incubation and the rebinding of 125 I-insulin that dissociated from the cells does not occur. In this way, dissociation rates of 1 2 5 Iinsulin are compared at different levels of receptor occupancy and at different medium insulin concentrations. Adipocytes remained fully intact and functional throughout these maneuvers, as evidenced by the facts that (a) cell counts were the same before and after these procedures (indicating no cell rupture) and (b) when cells were treated as described above (except for the addition of insulin) they were fully able to transport and metabolize glucose compared with unincubated cells. In the second type of dissociation study, the cells were allowed to reach steady state—binding conditions with the tracer amount of 125 I-insulin plus the indicated amounts of unlabeled insulin. In this way the degree of receptor occupancy can be increased in the association phase. After steady state—binding conditions were reached, the cells were washed and diluted into 125I-insulin—free medium, and dissociation was monitored as described above. Thus, the dissociation of the bound 125 I-insulin can be compared at different levels of fractional receptor occupancy but at an essentially zero concentration of media insulin. Nonspecific binding. In these experiments, nonspecific binding is defined as the amount of 1 2 5 Iinsulin remaining in the cell layer in the presence of a large excess (200 /xg./ml.) of unlabeled insulin. When *When the original 3-ml. incubation is centrifuged, 1 per cent or 30 fxl. of buffer is trapped above the oil phase in the cell layer. The cells are reconstituted to 3 ml. with insulin-free buffer, and aliquots (100 fi\.) are added to the 1.9-ml. insulin-free buffer in the dissociation tubes. Each of these aliquots (100 fil.) contains 1 jU.1. of the original insulin-containing buffer; thus, the final dilution is 1:2,000.

948

cells equilibrate with a tracer concentration of 1 2 5 Iinsulin (0.1 to 0.6 ng./ml.), only 3 to 7 per cent of the bound insulin represents nonspecific binding. As previously demonstrated, 18 ' 22 much of this is accounted for by "trapped" buffer, and nonspecifically bound insulin dissociates rapidly from the cells 6 ' 23 during the dissociation phase of the experiment. Therefore, although correction for nonspecific binding has been carried out, this represents only a trivial difference under these conditions. On the other hand, at higher levels of initial receptor occupancy (second type of dissociation experiment), nonspecific binding is appreciable (up to 30 per cent of the total binding at time zero). Therefore, in the latter experiments, it is important that parallel tubes for measurement of nonspecific binding are treated indentically and the data at each time point are corrected for the amount of insulin nonspecifically bound. Thus, all data represent specific insulin binding. Insulin degradation. Degradation of insulin was assessed by adding aliquots (50 /Ltl.) of buffer containing radioactive material to 1 ml. of Krebs-Ringer phosphate buffer with 15 per cent trichloroacetic acid (TCA). One milliliter of Krebs-Ringer phosphate buffer with 10 mg. per milliliter of bovine serum albumin was added as carrier protein. 15 In selected experiments, insulin degradation was determined by measuring the ability of the radioactive material to precipitate with excess anti-insulin antibody as previously described.15 RESULTS Figure 1 summarizes the ability of unlabeled insulin to accelerate the dissociation rate of 125 I-insulin from isolated adipocytes. The experimental design is analogous to that originally reported by De Meyts et al., 3 and the results are entirely consistent with their findings. Adipocytes and the tracer amount of radiolabeled insulin are first allowed to reach steady state—binding conditions (association phase). Next the cells are diluted into buffer free of radiolabeled insulin, and dissociation of the previously bound 1 2 5 Iinsulin is determined in the presence and absence of unlabeled insulin (dissociation phase). This approach allows the measurement of 125 I-insulin dissociation rates at different levels of receptor occupancy and at different medium insulin concentrations. As can be seen, the previously bound 125 I-insulin dissociates more rapidly when unlabeled insulin is present in the incubation medium. Several points concerning the shape of the curves are worth noting. First, essentially DIABETES, VOL. 2 7 , NO. 9

JERROLD M. OLEFSKY, M.D., AND HELEN CHANG, M.S.

B.

DISSOCIATION PHASE

DISSOCIATION PHASE

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FIG. 1. Effect of unlabeled insulin on the dissociation of 125 l-insulin. (A.) 12s l-lnsulin (0.1 to 0.6 ng. per milliliter) is allowed to associate with isolated adipocytes (4 to 8 x 10 s per milliliter) for 90 minutes. At this point (arrow) the buffer is removed and cells are resuspended in insulin-free media (6° C.) and aliquoted (100 Ail.) into a series of test tubes containing 1.9 ml. buffer (24° C.) with 100 ng. per milliliter (dilution + insulin) or without (dilution only) insulin. At the indicated times, these tubes are centrifuged and the 125 l-insulin remaining bound to the cells is determined. For measurement of nonspecific binding (lower curves), parallel tubes are treated identically except for the presence of 200 /xg. per milliliter unlabeled insulin throughout the experiment. As can be seen, nonspecific binding is minimal and equal in both sets of tubes throughout the dissociation phase. In the association studies the tracer concentration of 125 l-insulin ranged from 0.1 to 0.6 ng. per milliliter, which leads to occupancy of only 0.2 to 1.2 per cent of the receptors. This level of occupancy is too low to exert cooperative effects by itself, 4 ' 6 ' 24 and we have found that the dissociation curves are identical when any 125 l-insulin tracer concentration over this range is used. Data represent the mean ( ± S.E.) of 13 separate experiments. (B.) Data from the dissociation phase replotted on a semi-log graph.

all the increase in dissociation is seen during the initial five to 10 minutes. Secondly, neither curve is monoexponential (figure IB, note log scale). That is, with dilution alone, the dissociation curve of the 125 I-insulin is not a single-order process. Similarly, at an insulin level of 100 ng. per milliliter, where the fractional receptor occupancy is about 70 per cent and cooperative effects are maximum (all receptors are in a low affinity state 4 ' 6 ) the dissociation curve is still multiexponential. The argument can be raised that the above results are due to rebinding of dissociated 125 I-insulin from an unstirred layer that surrounds the cells and acts as a diffusion barrier. In this case the added unlabeled insulin would serve to dilute the dissociated labeled insulin. This possibility is unlikely for cultured lymphocytes, as discussed by De Meyts. 6 To examine this alternative with adipocytes, two unlabeled insulin derivatives—desalanine desasparagine insulin and desoctapeptide insulin—which have been reported not to induce the accelerating effect,3 were employed. Thus, instead of native insulin, these analogues were added to the dissociation phase of the experiment at concentrations that produced equivalent binding to 100 ng. per milliliter of native insulin. As is seen from table 1, the ability of both these derivatives to inhibit 125 I-insulin binding was about 1 per cent that of native insulin. Consequently, 10 /xg. per milliliter of desalanine desasparagine and desoctapeptide insulin was used in the dissociation studies. Figure 2 shows SEPTEMBER, 1978

that neither analogue produced any acceleration of 125 I-insulin dissociation. In fact, both analogues appear to slightly slow the dissociation of 125 I-insulin. This latter finding was highly reproducible in five separate experiments and is currently unexplained. Nevertheless, the results in figure 2 argue strongly against unstirred layers as an explanation for the accelerating effect of native insulin, since, at the concentrations employed, these analogues would exert the same diluting effect as native insulin if such a layer existed. The increase in dissociation rate is dependent on the concentration of unlabeled native insulin used in the dissociation phase, and this is demonstrated in figure 3. In this plot the ability of unlabeled insulin to accelTABLE l Ability of DOP and DAA insulin to inhibit Added hormone concentration (ng./ml.) 0* 1 10 100 1,000 10,000

I25

I-insulin binding

% '"Mnsulin bound DAA DOP Native insulin insulin insulin 100 82 48 23 11 7

100 100 100 96 70 31

100 100 100 97 61 18

•Studies were done by incubating cells and 0.2 ng. per milliliter I-insulin plus the indicated concentration of unlabeled hormone for 90 minutes at 24°. Data are presented as a per cent of the amount bound in the absence of added unlabeled hormone. l25

949

INSULIN BINDING TO ADIPOCYTES

100

FIGURE 2 Effect of the insulin derivatives, desalanine desasparagine ( X ) and desoctapeptide (A) insulin on the dissociation of '"l-insulin. Experiments were performed as described in the methods section of the legend to figure 1. The lower two curves represent dissociation in dilution only (•) and dilution plus 100 ng. per milliliter native insulin (o). The concentration of the insulin analogues was 10 fig. per milliliter in the dissociation phase since competition studies (table 1) demonstrated that this gave a level of receptor occupancy equivalent to that produced by 100 ng. per milliliter native insulin. All data represent the mean of five separate experiments.

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erate the dissociation of 125 I-insulin is expressed as a per cent of the maximal effect at each insulin concentration. The absolute effect is calculated by dividing the amount of 125 I-insulin that dissociates from the cells at 10 minutes in the presence of insulin by the corresponding value in the absence of insulin (dilution

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FIG. 3A.

Effect of insulin concentration (dilution + insulin) to accelerate the dissociation of bound 125 l-insulin at 24° C. The difference between the amount of '"l-insulin dissociating from the cells in the presence of dilution alone versus dilution plus insulin is expressed as a per cent of the maximal effect and plotted as a function of the insulin concentration in the dissociation medium (dilution + insulin). The absolute effect at 10 minutes of dissociation was calculated by (

950

amount dissociated with dilution + insulin - D amount dissociated with dilution alone

100'

only) and multiplying by 100. Maximal acceleration is reached at an insulin concentration of about 100 ng. per milliliter, and, in absolute terms, is equal to a 40 per cent increase in dissociation. Appreciable effects are seen at insulin concentrations as low as 5 ng. per milliliter, while the effect noticeably decreases (24° C.) at the highest insulin concentration (10 //-g. per milliliter). Again, these findings are analogous to those reported by De Meyts et al. , 3>6 and these workers have attributed the decline of the negatively cooperative effect to the formation of insulin dimers that occurs at these high insulin concentrations. Temperature also influences insulin dissociation rates. In figure 4, 125 I-insulin is allowed to reach steady state—binding conditions at 24° C ; dissociation is then studied at 4°, 24°, and 37° C. As can be seen, the greater the temperature the faster the dissociation. However, at 37° C , the effect of insulin to accelerate dissociation (maximal effect = 40 per cent) is comparable to that seen at 24° C , and the dose response curve retains a similar configuration (figure 3B). This differs from the results of De Meyts, who found that, while an increase in temperature accelerated insulin dissociation from cultured lymphocytes, the extent of the cooperative effect diminished. 6 Thus far, the effects of unlabeled insulin on acceleration of the dissociation of previously bound 1 2 5 Iinsulin are distinct when dissociation is measured at DIABETES, VOL. 2 7 , NO. 9

JERROLD M. OLEFSKY, M.D., A N D HELEN CHANG, M.S.

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FIG. 3B.

Effect of insulin concentration to accelerate the dissociation of m l-insulin at 37° C.

different levels of receptor occupancy and different media insulin concentrations. As receptor occupancy increases, the insulin receptor has an apparent, lower affinity for insulin (faster dissociation rate), and this effect depends on the insulin concentration and the temperature. However, with this experimental design, both receptor occupancy and media insulin concentration are altered; to avoid this latter variable, a different experimental technique was employed. Adipocytes were allowed to reach steady statebinding conditions with either 0.6 ng. per milliliter 125 I-insulin or 3 ng. per milliliter 125 I-insulin plus 97 ng. per milliliter unlabeled insulin. At this point, both groups of cells were diluted into insulin-free buffer and the 125 I-insulin dissociation was determined (figure 5). The data are corrected for nonspecific binding (see legend to figure 5) and, therefore, represent 125 I-insulin specifically bound to cells. In this way, the dissociation of 125 I-insulin can be determined at different levels of receptor occupancy and at an essentially zero media insulin concentration.f As can be seen, 125 I-insulin dissociation is faster at the higher level of receptor occupancy.

the presence of 3 ng. per milliliter 125 I-insulin plus 97 ng. per milliliter unlabeled insulin (figure 6, middle two curves). In these two situations—association at 0.6 ng. per milliliter insulin and dissociation at 100 ng. per milliliter versus association at 100 ng. per milliliter insulin and dissociation in dilution only— the fractional receptor occupancy during the initial part of the experiment should be essentially identical, and yet the insulin dissociation rate is faster in the latter situation. Thus, something in addition to increased receptor occupancy and subsequent site-site interactions must be causing these accelerated dissociation rates. Since unlabeled insulin is not present in the dissociation media with the latter experimental design, the fractional receptor occupancy decreases as the insulin dissociates from the cell. Thus, one might expect that if site-site interactions occur, they would decrease as the fractional occupancy decreased. Evidence for this is seen in figure 6, since at the latter time points the

Furthermore, when these data are compared with experiments performed as described for figure 1—dilution plus insulin (100 ng. per milliliter)—the initial dissociation rate is faster when the cells are allowed to reach steady state—binding conditions in fit can be calculated that, when binding is performed at 100 ng. per milliliter and if all the insulin dissociates from the cells in the aliquot (100 fil.), this would produce a media insulin concentration of only 0.01 ng. per milliliter. At this media insulin concentration, less than 0.03 per cent of the receptors would be occupied and binding would be undetectable. Therefore, appreciable rebinding of dissociated insulin cannot occur. SEPTEMBER, 1978

50

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Time (min.) FIG. 4.

Effect of temperature on 125 l-insulin dissociation. In these studies the association phase was conducted at 24° C. and the dissociation phase was conducted at the indicated temperature.

951

INSULIN BINDING TO ADIPOCYTES

FIGURE 5 Dissociation of 1 2 5 l-insulin into insulin-free buffer (dilution only) after association at high and low levels of receptor occupancy. Cells were a l lowed to equilibrate at 24° C. with either 0.6 ng. per milliliter 135 l-insulin alone or 3 ng. per milliliter 125 l-insulin plus 9 7 ng. per milliliter unlabeled insulin. After 90 minutes both groups of cells were washed and allowed to dissociate into insulin-free buffer. The cells equilibrated with the low concentration of insulin (0.6 ng. per milliliter) had a low steady state level of receptor occupancy (~ 1 per cent) while the cells equilibrated with the high insulin concentration (100 ng. per milliliter) had a high steady state level of receptor occupancy (~- 70 per cent) before the dissociation study. Thus, dissociation can be determined at different levels of receptor occupancy but in the absence of insulin in the buffer. Nonspecific insulin binding is determined by assaying sets of tubes treated identically except for the presence of 200 fig. per milliliter of unlabeled insulin throughout the experiment. All data are corrected for nonspecific insulin binding and thus represent insulin specifically bound to cells. Data represent the mean ( ± S.E.) of nine separate experiments.

'I

40-

dissociation curves of the two types of experiments converge (middle two curves). To see if preventing this decrease in receptor occupancy prevented this apparent loss of the cooperative effect, experiments as described in figure 5 were carried out, with the modification that unlabeled insulin (100 ng. per milliliter) was present in the dissociation phase instead of allowing the insulin to dissociate into insulin-free media. As seen in figure 6 (lower curve), this resulted in even further acceleration of the insulin dissociation rate, and the curves no longer converge at the later time points. Thus, loss of the cooperative effect is prevented by maintaining high levels of receptor occupancy, and this leads to the fastest dissociation rates of any of the experimental conditions studied. The above data can be explained if binding of insulin to adipocyte receptors includes interactions with functionally distinct, binding sites (a high affinity, low capacity site and a low affinity, high capacity site) in addition to exhibiting negatively cooperative sitesite interactions. In this case, as the total insulin concentration in the association phase of the experiment increases, the proportion of the 125 I-insulin bound to the low affinity, high capacity sites increases due to 952

saturation of the high affinity, low capacity sites. Thus, in the dissociation phase the curve is steeper since the insulin is predominantly dissociating from a low affinity (fast-dissociating) site. This effect, plus the cooperative effect resulting from the high fractional receptor occupancy, leads to faster dissociation rates than those seen with dilution plus insulin (figure 1). Alternatively, it is possible that the difference between the two curves is due to a time-dependent development of the cooperative effect. In other words, in the experiment depicted in figure 1, high fractional occupancy exists for only a short time before dissociation is measured, while in figure 5 high receptor occupancy exists for 90 minutes before dissociation is measured. If the cooperative effect takes time to fully develop, then this could account for the faster dissociation rates in the latter experiment. To test this possibility we studied 125 I-insulin dissociation at various times after cells were associated with 100 ng. per milliliter insulin (3 ng. per milliliter 125 I-insulin plus 97 ng. per milliliter unlabeled insulin). As can be seen in figure 7, at this high insulin concentration, binding is rapid and near steady state—binding conditions are reached by three to five minutes. FurtherDIABETES, VOL. 2 7 , NO. 9

JERROLD M. OLEFSKY, M.D., AND HELEN CHANG, M.S.

FIG. 6. Comparison of 12s l-insulin dissociation when equally high levels of receptor occupancy are achieved by having 100 ng. per milliliter insulin present in the association phase (100 ng. per milliliter association + dilution, see legend to figure 5) or in the dissociation phase (dilution + 100 ng. per milliliter insulin, see legend to figure 1). The lower curve represents data obtained by studying dissociation of 125 l-insulin into buffer containing 100 ng. per milliliter unlabeled insulin after association with 100 ng. per milliliter insulin (100 ng. per milliliter insulin in association, then dilution + 100 ng. per milliliter insulin). All data are corrected for nonspecific insulin binding and represent the mean ( ± S.E.) of 11 separate experiments.

more, dissociation rates do not become faster when dissociation is studied at later time points, indicating that the cooperative effect is not time dependent and is maximum as soon as occupancy is achieved. Additional evidence that the cooperative effect develops rapidly is provided in figure 8. In this experiment dissociation is studied as described in figure 1. Five minutes after dissociation begins, unlabeled insulin (final concentration 100 ng. per milliliter) is added to a parallel set of tubes. As can be seen, the full accelerating effect is basically achieved by two minutes and is maintained throughout the entire experiment. Furthermore, when the two original curves (dilution versus dilution plus insulin) are compared, essentially all the accelerating effect is achieved by five minutes, t Thus, it is highly unlikely that a time-dependent development of the negatively cooperative effect extlt should be noted that the ordinate is a log scale, and equal differences appear smaller at early time points. SEPTEMBER, 1978

plains the results presented in figure 6. The experiments presented in figure 8 also indicate that little, if any, acceleration of dissociation occurs when unlabeled insulin is added to the cells after 35 minutes of dissociation. This suggests that, after five minutes of dissociation, the 125 I-insulin is dissociating from receptors that can exhibit the accelerating effect of unlabeled insulin, while after 35 minutes, the 1 2 5 Iinsulin is predominantly dissociating from receptors that do not display this effect. Additional evidence for functional heterogeneity is provided by examining the nature of the radioactivity that dissociates from the cells (figure 9)- Using solubility in 15 per cent TCA as an index of degradation, it was found that the radioactive material that dissociated by 10 minutes was essentially 100 per cent intact, while the material dissociating at later time points consisted of large amounts of degraded insulin. Since the plot for the appearance of degraded insulin in figure 9 represents the average amount of TCAsoluble material accumulated up to each time point, this actually underestimates the proportion of the material dissociating at any given point in time that is degraded. For example, in figure 9A, of the radioactivity that dissociates between 10 and 30, 30 and 60, and 60 and 120 minutes, 9 per cent, 25 per cent, and 35 per cent is soluble in TCA, respectively. These results are consistent with the concept that the rapidly appearing radioactive material predominantly comes from low affinity (fast-dissociating) receptors that do not degrade insulin, whereas the more slowly dissociating radioactivity originates from high affinity (slow-dissociating) receptors that somehow participate

dilution

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Time (min.)

FIG. 7. Dissociation of 12s l-insulin from cells after association with 100 ng. per milliliter insulin (3 ng. per milliliter 1 2 S I insulin + 97 ng. per milliliter unlabeled insulin; see legend to figure 5 and the METHODS section for details) for various periods of time. All data are corrected for nonspecific insulin binding.

953

INSULIN BINDING TO ADIPOCYTES

•k

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FIG. 8. Time course of onset of the cooperative effect. The experiment was performed as described for figure 1, except that after five minutes and 35 minutes of dissociation, insulin (100 ng. per milliliter final concentration) was added to a parallel set of tubes (arrows) and the rate of onset of the accelerating effect was determined.

A. 24°

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in the degradation process. Of the total radioactivity that dissociates from the cells, the bulk of this material (80 to 90 per cent) is intact insulin, and the rate of dissociation of intact insulin is accelerated in the presence of unlabeled insulin in the buffer. Furthermore, the shape of the dissociation curve of the intact insulin is qualitatively the same as the dissociation curve for the total radioactive material as seen in figure 1. On the other hand, the proportion of TCA-soluble material that dissociates from the cells progressively increases with time, and the rate of dissociation of the degraded material is not faster in the presence of unlabeled insulin. Qualitatively identical results are seen at 37° C. (figure 9B), although absolute rates of dissociation and degradation are faster. Thus, the sites engaged in insulin degradation are not susceptible to the accelerating effect of unlabeled insulin, whereas the other sites are. These results are not due simply to a progression of the degradative process with time, since identical results are seen when cells are allowed to associate with 125 I-insulin for 150 minutes (instead of 90) before the dissociation phase is initiated. Furthermore, the appearance of TCA-soluble material is not due to the presence of proteolytic activity in the buffer, since the infranates from the dissociation phase were without degradative activity. Comparable results FIGURE 9A Ability of the radioactive material that dissociates from the cells at the indicated time points to precipitate in 15 per cent TCA. The unlabeled dissociation curves are similar to those presented in previous figures and represent 125 l-insulin dissociation in dilution only (•—•) and in dilution plus 100 ng. per milliliter unlabeled native insulin (o—o). Broken lines represent the appearance of TCA-precipitable material in the dilution only (•—•) and dilution plus 100 ng. per milliliter insulin (o—o) tubes. The lower, solid lines represent the appearance of TCAsoluble radioactivity under both conditions; the dilution only (•) and dilution plus insulin (o) points are virtually superimposable at each time. Studies were performed at 24° and all results are expressed as a per cent of the amount of 125 l-insulin bound to the cells at time zero. Recovery of counts appearing in the media is quantitative in these experiments, as evidenced by the fact that in each situation the radioactivity in the three fractions (bound to cells + TCA precipitable + TCA soluble) is ~ 100 per cent of the zero time value at each time point. Data represent the mean of three separate experiments.

DIABETES, VOL. 27, NO. 9

JERROLD M. OLEFSKY, M.D., AND HELEN CHANG, M.S.

As the insulin concentration in the association or binding phase increases, the acceleration of the dissociation rate increases in a linear fashion (figure 10, upper curve). If functionally separate low affinity, high capacity sites exist, then as the unlabeled insulin concentration in the association phase increases, a progressively greater proportion of the labeled insulin will bind to these sites resulting in dissociation rates that also progressively increase and are faster than those observed when only the cooperative effect is measured (figure 10, lower curve).

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DISCUSSION 40

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Time (min.) FIG. 9B.

Identical experiments to those in figure 9A but performed at 37° C.

to those obtained with TCA precipitation were found when precipitation with excess anti-insulin antibody was used as the index of degradation, except that the calculated proportion of degraded material was 4 to 8 per cent higher. To characterize further the kinetic differences between the two different kinds of dissociation experiments, studies were done in which cells were allowed to reach steady state—binding conditions at a variety of medium insulin concentrations, after which dissociation was determined in insulin-free buffer (figure 10).

|

9>

100-

80

20

1000

10,000

insulin concentration (ng/ml)

FIG. 10.

Ability of an increasing insulin concentration in the association phase to accelerate the subsequent dissociation of 125 l-insulin. Experiments are performed as described in the legend to figure 5 and the METHODS section, except that the unlabeled insulin concentration in the association phase was varied as indicated. The accelerating effect is expressed as the absolute effect calculated by the formula given in the legend to figure 3. All data are corrected for nonspecific insulin binding and represent the mean of three experiments. The lower curve (dilution + insulin) represents the data from figure 3 replotted in absolute terms for comparison.

SEPTEMBER, 1978

Insulin-binding data typically yield nonlinear Scatchard plots, indicating that insulin receptors do not function as a single population of binding sites of homogeneous affinity. De Meyts and co-workers3"6 have directly demonstrated that increasing media concentrations of unlabeled insulin progressively accelerate the dissociation of previously bound 125 I-insulin from receptors. This phenomenon is felt to represent interactions between occupied insulin receptors such that the average affinity of a population of receptors decreases as the fractional occupancy increases, until a limiting lower affinity is reached.4 This process has been termed negative cooperativity, and the data presented in these studies are in agreement with the work of De Meyts et al. 3 - 6 However, although much of the data is comparable to that reported by De Meyts et al. 3 ~ 6>24 for other insulin receptor systems, we believe that, for the adipocyte, the results are best explained by a model incorporating negatively cooperative site-site interactions plus at least two functionally distinct, binding sites (a low capacity, high affinity site and a high capacity, low affinity site). The major evidence for this formulation is as follows: (1) When adipocytes are equilibrated with a tracer concentration of 125 I-insulin ( 3 3 X 10' 1 1 M), 0.3 per cent of the available receptors are occupied, and this degree of receptor occupancy is too low to exert any appreciable cooperative effects 4 ' 6 ' 24 (figure 3)- In this situation the 1 2 5 Iinsulin does not dissociate from its receptors as a first order process (dilution only), indicating that, despite the absence of cooperative effects, the receptors are not behaving as a kinetically homogeneous population. This is consistent with the idea that at the tracer concentration of 125 I-insulin, the hormone can bind to at least two functionally different receptors with respect to binding affinity. Furthermore, when the 125 I-insulin is allowed to dissociate into media con955

INSULIN BINDING TO ADIPOCYTES

taining enough unlabeled insulin to exert maximal cooperative effects—100 ng. per milliliter 3 ' 6 ' 24 (figure 3)—the dissociation curve is still multiexponential (figure 1). If negative cooperativity were the only factor, then all receptors should be in the "low" affinity or "fast"-dissociating state 4 ' 6 and dissociation should be a first order process at this level of receptor occupancy. § This is not the case for adipocytes, again indicating more than one kinetically distinct, receptor population. (2) When cells are equilibrated with a mixture of 125 I-insulin and unlabeled insulin at a total insulin concentration of 100 ng. per milliliter and dissociation is studied in insulin-free medium, the receptor occupancy at the beginning of the dissociation phase is the same as when the cells are equilibrated with a tracer amount of 125 I-insulin and dissociation is studied in media free of 125 I-insulin but containing unlabeled insulin, 100 ng. per milliliter. Thus, if negatively cooperative site-site interactions were the only factor to accelerate insulin dissociation, then the dissociation rates in these two experiments should be equal. However, as seen in figure 6, initial rates of dissociation were clearly faster when the cells were equilibrated in the association phase with the high, total insulin concentration. These results could be explained if distinct, high affinity, low capacity, and low affinity, high capacity—binding sites exist. Thus, when cells are equilibrated at the high, total insulin concentration, the high affinity receptors are relatively saturated so a greater proportion of the 125 I-insulin binds to the low affinity, high capacity sites. Therefore, during the dissociation phase (dilution alone), the 125 I-insulin dissociation rate is accelerated because the 125 I-insulin is predominantly dissociating from low affinity (fast-dissociating) binding sites, and this effect plus cooperativity leads to faster dissociation rates than those observed when only the cooperative effect is measured. (3) When cells are equilibrated at high insulin concentrations and dissociation is studied in insulin-free media (dilution only), the receptor occupancy will decrease as dissociation proceeds, and the cooperative effect should diminish (provided the termination of site-site interactions is rapid). Evidence for this is seen in figure 6, since at later time points, when most of the insulin has dissociated from the cell, the dissociation curves converge. (4) If the above formulation is correct, then §Ic should be noted that binding of HGH to cultured lymphocytes yields a linear Scatchard plot, but the hormone still does not dissociate from the cells as a first order process.3-25 This phenomenon has been pointed out previously3 but remains unexplained.

956

the addition of unlabeled insulin (100 ng. per milliliter) to media in the dissociation phase will maintain the high, fractional receptor occupancy and prevent the loss of the cooperative effect. In this situation (figure 6, lower curve), insulin dissociation was further accelerated and the resulting curve did not converge at later time points. (5) When the characteristics of the radioactivity dissociating from the cells was examined, it was found that the rapidly dissociating material was intact insulin whereas a substantial component of the slowly dissociating material was degraded insulin. Furthermore, all the accelerating effect of unlabeled insulin was accounted for by enhanced dissociation of intact 125 I-insulin, while the dissociation rate of the degraded material was the same in the presence or absence of unlabeled insulin added to the dissociation phase. (6) Finally, in a recent report Gliemann 10 provided evidence—independent of binding isotherms—that two distinct receptors exist on isolated adipocytes. These workers equilibrated adipocytes with 125 I-insulin, resuspended the cells in 125I-insulin-free buffer, and measured the appearance of radioactivity into the buffer. The previously bound 125 I-insulin dissociated as both TCAsoluble and TCA-precipitable material, and the appearance curves of these two components displayed different kinetic characteristics. These data are certainly consistent with the data presented in our studies. Several points deserve emphasis regarding our conclusion that faster dissociation rates are seen when cells are equilibrated with high concentrations of insulin during the association phase. First, it could be argued that when cells are equilibrated with 100 ng. per milliliter insulin in the association phase, the maximal occupancy achieved at this insulin concentration exists from the beginning of the dissociation study. On the other hand, when experiments are done as described in figure 1, receptor occupancy is low at the onset of dissociation and takes time to reach maximal levels. With this formulation, it could be reasoned that the studies presented in figure 6 were not done at the same fractional receptor occupancies, and, thus, the faster dissociation rates of the two lower curves are due simply to a higher level of receptor occupancy. This line of reasoning is unlikely to be correct, since at an insulin concentration of 100 ng. per milliliter, the half-time of association is ~ 30 seconds and is 86 per cent of maximum by two minutes (figure 7). Furthermore, when the dose response curves of these two types of experiments are compared (figure 10) at a media insulin concentration of 50 ng. DIABETES, VOL. 2 7 , NO. 9

JERROLD M. OLEFSKY, M.D., AND HELEN CHANG, M.S.

per milliliter (arrow, upper curve, figure 10), dissociation rates are still much faster than when only cooperativity is measured at an insulin concentration of 100 ng. per milliliter (arrow, lower curve). From the association data it can be calculated that at a media insulin concentration of 100 ng. per milliliter, fractional receptor occupancy at the end of one minute of association is greater than the steady state level of occupancy reached at a media insulin concentration of 50 ng. per milliliter. One could also argue that, since high receptor occupancy exists for different times during the two types of dissociation experiments, this could account for our results if the cooperative effect takes a relatively long time to develop. However, the development of the full cooperative effect is quite rapid (figure 8) and, yet, large differences in dissociation rates between the experiments presented in figure 6 (middle two curves) are seen at relatively late time points (up to 30 minutes). Additionally, when the curves in figure 1 are inspected, it can be seen that the full accelerating effect is reached by five minutes, since the absolute differences between the two curves are the same at five and 60 minutes. For these reasons, the results presented in figures 6 and 10 cannot be explained by different levels of fractional receptor occupancy at the onset of the dissociation phase of the experiments or by a time-dependent development of the negatively cooperative effect. Lastly, the data presented also indicate that not all receptors undergo site-site interactions. If the 1 2 5 Iinsulin that dissociates during the initial part of the dissociation experiment is predominantly coming from low affinity (fast-dissociating) sites, then since the full cooperative effect is reached quite early in the experiment, one can infer that only the low affinity site is capable of the site-site interactions. More direct evidence for this idea is provided in figure 8, which shows that the full accelerating effect of added, unlabeled insulin can be produced five minutes after dissociation begins, i.e., when the 125 I-insulin would be predominantly dissociating from the low affinity site. In contrast, only a minimal accelerating effect of added, unlabeled insulin is seen during the later phase of the experiment when the 125 I-insulin is predominantly dissociating from the high affinity (slowdissociating) site. Furthermore, figure 9 demonstrates that, although previously bound 125 I-insulin dissociates as both intact and degraded material, the degraded material dissociates slowly and this rate is not accelerated in the presence of unlabeled insulin. Qualitatively, the results obtained with adipocytes are quite similar to those seen with cultured lymphoSEPTEMBER, 1978

cytes. 3 ' 6 However, certain quantitative differences are evident, i.e., with adipocytes the absolute magnitude of the accelerating effect is less, and higher temperatures do not diminish this effect. We believe, however, that these quantitative differences are outweighed by the many similarities, especially the findings that the accelerating effect of native insulin diminishes at high concentrations, and the analogues desoctapeptide and desalanine desasparagine insulin do not elicit this phenomenon. Therefore, the process whereby unlabeled insulin accelerates the dissociation of previously bound 125 I-insulin is most likely the same for the two cell types. De Meyts and coworkers 3 ' 6 ' 24 have ascribed this effect to negatively cooperative interactions between receptor sites, and our results are consistent with this model. On the other hand, Pollet et al. 8 have attributed the accelerating effect of unlabeled insulin to an exchange reaction mediated through ligand-ligand interactions. The data presented in our studies do not allow a choice between these two possibilities, but at this point we believe our data are most consistent with the model of negative cooperativity plus functionally heterogeneous receptor sites. Regardless of which model is correct, one still must conclude that, at least for adipocytes, only the low affinity (fast-dissociating) receptor sites participate in this accelerating process. In summary, we have obtained results consistent with the extensive studies of De Meyts et al. 3 " 6 ' 2 4 in other cell systems and, thus, find evidence for an accelerating effect of unlabeled insulin on the dissociation rate of previously bound 125 I-insulin from adipocyte insulin receptors. This is consistent with negatively cooperative site-site interactions, but this effect alone cannot explain all the kinetic properties of the adipocyte insulin receptor. The data can be explained, however, by postulating that in addition to negatively cooperative phenomenon, adipocyte insulin receptors functionally behave as high affinity, low capacity, and low affinity, high capacity, binding sites. Clearly this is a functional definition, and several physicochemical situations can exist to satisfy this definition. One obvious explanation is that adipocytes could possess at least two structurally distinct receptors of differing affinity and capacity. On the other hand, it is equally possible that only one structurally homogeneous receptor population exists, but for a variety of reasons this receptor can behave either as a high affinity (slow-dissociating) or low affinity (fast-dissociating) binding site. For example, subsequent to insulin binding, the insulin receptor complex may undergo translocation within the plasma membrane, and the 957

INSULIN BINDING TO ADIPOCYTES

insulin dissociation rate and binding affinity of the receptor could change as a result of this process. This translocation may also relate to other functional aspects of the receptor, and, consistent with the work'of Gliemann, 10 we found that the 125 I-insulin which dissociates from the low affinity site is relatively undegraded compared with that which dissociates from the high affinity site. An analogous concept has previously been suggested by Steiner, 26 ' 27 and Kono et al. 28 recently published data indicating translocation of insulin receptor complexes within the plasma membrane. ACKNOWLEDGMENTS This work was supported by funds from the Medical Research Service of the Veterans Administration, by a grant from the American Diabetes Association, and by NIH-NIAMDD grant AM-19905. Dr. Olefsky was a Clinical Investigator with the Veterans Administration during the course of this work. REFERENCES 'Roth, J., Kahn, C. R., Lesniak, M. A., Gorden, P., De Meyts, P., Megyesi, K., Neville, D. M., Jr., Gavin, J. R., Ill, Soil, A. H., Freychet, P., Goldfine, I. D . , Bar, R. S., and Archer, J. A.: Receptors for insulin, NSILA-s, and growth hormone: applications to disease states in man. Recent Prog. Horm. Res. 31:95-159, 1976. 2 Kahn, C. R., Freychet, P., Roth, J . , and Neville, D. M., Jr.: Quantitative aspects of the insulin-receptor interaction in liver plasma membranes. J. Biol. Chem. 249:2249-57, 1974. 3 De Meyts, P., Roth, J., Neville, D. M., Jr., Gavin, J. R., Ill, and Lesniak, M. A.: Insulin's interactions with its receptors: experimental evidence for negative cooperativity. Biochem. Biophys. Res. Commun. 55:154-61, 1973. 4 De Meyts, P., and Roth, J.: Cooperativity in ligand binding: a new graphic analysis. Biochem. Biophys. Res. Commun. 66:1118-26, 1975. 5 De Meyts, P.: Cooperative properties of hormone receptors in cell membranes. J. Supramol. Struct. 4:241-58, 1976. 6 De Meyts, P., Bianco, A. R., and Roth, J.: Site-site interactions among insulin receptors. Characterization of the negative cooperativity. J. Biol. Chem. 252:1877-88. 1976. 7 Gliemann, J., Gammeltoft, S., and Vinten, J.: Time course of insulin-receptor binding and insulin-induced lipogenesis in isolated rat fat cells. J. Biol. Chem. 250:3368-74, 1975. 8 Pollet, R. J., Standaert, M. L., and Haase, B. R.: Insulin binding to the human lymphocyte receptor: evaluation of the negative cooperativity model. J. Biol. Chem. 252:5828, 1977. 9 Cuatrecasas, P., and Hollenberg, M. D.: Binding of insulin and other hormones to non-receptor materials: saturability, specificity and apparent "negative cooperativity." Biochem. Biophys. Res. Commun. 62:31-41, 1975.

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10 Gliemann, J.: Two groups of insulin "receptors" in rat adipocytes, one of them related to insulin degradation. Abstracts of the 12th Annual Meeting of the European Association for the Study of Diabetes, Helsinki, Finland. No. 98, page 393, 1976. n Rodbell, M.: Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J. Biol. Chem. 239:375-80, 1964. 12 Hirsch, J., and Gallian, E.: Methods for the determination of adipose cell size in man and animals. J. Lipid Res. 9:110-19, 1968. 13 Freychet, P., Roth, J., and Neville, D. M., Jr.: Monoiodoinsulin: demonstration of its biological activity and binding to fat cells and liver membranes. Biochem. Biophys. Res. Commun. 43:400-08, 1971. 14 Hunter, W. M., and Greenwood, F. C : Preparation of iodine-131 labeled human growth hormone of high specific activity. Nature (London) 294:495-96, 1962. 15 Olefsky, J., and Reaven, G. M.: The human lymphocyte: a model for the study of insulin-receptor interaction. J. Clin. Endocrinol. Metab. 38:554-60, 1974. 18 Gavin, J. R., Ill, Gorden, P., Roth, J., Archer, J. A., and Buell, D. N . : Characteristics of the human lymphocyte insulin receptor. J. Biol. Chem. 248:2202-07, 1973. 17 Olefsky, J. M., Jen, P., and Reaven, G. M.: Insulin binding to isolated human adipocytes. Diabetes 23:565-71, 1974. 18 Olefsky, J. M., and Reaven, G. M.: Effects of age and obesity in 125 I-insulin binding to isolated adipocytes. Endocrinology 96:1486-89, 1975. 19 Olefsky, J. M., Johnson, J., Lui, F., Jen, P., and Reaven, G. M.: The effects of acute and chronic dexamethasone administra : tion on insulin binding to isolated rat hepatocytes and adipocytes. Metab. Clin. Exp. 24:517-27, 1975. 20 Gammeltoft, S., and Gliemann, J.: Binding and degradation of 125 I-labelled insulin by isolated rat fat cells. Biochim. Biophys. Acta 320:16-32, 1973. 21 Gliemann, J., Osterlind, K., Vinten, J., and Gammeltoft, S.: A procedure for measurement of distribution spaces in isolated fat cells. Biochim. Biophys. Acta 286:1-9, 1972. 22 Olefsky, J. M.: Effect of dexamethasone on insulin binding, glucose transport, and glucose oxidation of isolated rat adipocytes. J. Clin. Invest. 56:1499-1508, 1975. 23 Olefsky, J. M., Johnson, J., Lui, F., Edwards, P., and Baur, S.: A comparison of 125 I-insulin binding to isolated rat hepatocytes and liver membranes. Diabetes 24:801-10, 1975. 24 Soll, A. H., Kahn, C. R., and Neville, D. M., Jr.: Insulin binding to liver plasma membranes in the obese hyperglycemic (ob/ob) mouse. Demonstration of a decreased number of functionally normal receptors. J. Biol. Chem. 250:4702-07, 1975. 25 Lesniak,M. A., Gorden, P., R o t h , J . , and Gavin, J. R., Ill: Binding of 125I—human growth hormone to specific receptors in human cultured lymphocytes. Characterization of the interaction and a sensitive radioreceptor assay. J. Biol. Chem. 249:1661-67, 1974. 26 Steiner, D. F.: Insulin today. Diabetes 26:322-40, 1977. 27 Terris, S., and Steiner, D. F.: Binding and degradation of 125 I-insulin by rat hepatocytes. J. Biol. Chem. 250:8389-98, 1975. 28 Kono, T., Robinson, F. W . , Sarver, J. W . , Vega, I. V., and Pointer, R. H.: Actions of insulin in fat cells. Effects of low temperature, uncouplers of oxidative phosphorylation, and respiratory inhibitors. J. Biol. Chem. 252:2226-33, 1977.

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