Fluctuations in the affinity and concentration of receptors on circulating monocytes of obese patients: effects of starvation, refeeding, and dieting.

R S Bar, … , C R Kahn, P De Meyts

J Clin Invest. 1976;58(5):1123-1135. https://doi.org/10.1172/JCI108565.

Research Article

The binding of 125I-insulin to insulin receptors on circulating monocytes of obese patients and normal volunteers has been determined under various dietary states. In the basal, fed state the monocytes of obese patients with clinical insulin resistance (n= 6) bound less insulin than normals (n =10) because of a decrease in insulin concentration (obese = 6,000-13,000 sites per monocyte versus normals 15,000-28,000 sites per monocyte). The single obese patient without evidence of clinical insulin resistance demonstrated normal binding of insulin with 16,000 sites per monocyte. In all patients, the total receptor concentration was inversely related to the circulating levels of insulin measured at rest after an overnight fast. For the obese patients with basally depressed insulin binding, a 48-72-h fast lowered circulating insulin and increased binding to normal levels but only at low concentrations; this limited normalization of 125I-insulin binding was associated with increased receptor affinity for insulin without change in receptor concentration. Refeeding after the acute fasting periods resulted in return to the elevated plasma insulin levels, the basal receptor affinity, and the depressed insulin binding observed in the basal, fed state. Chronic diet restored plasma insulin levels, insulin binding, and receptor concentration to normal without change in affinity. When the data from this study are coupled with previous in vivo and in vitro findings they suggest […]

Find the latest version: https://jci.me/108565/pdf Fluctuations in the Affinity and Concentration of Insulin Receptors on Circulating Monocytes of Obese Patients

EFFECTS OF STARVATION, REFEEDING, AND DIETING

ROBERT S. BAR, PHILLIP GORDEN, JESSE ROTH, C. RONALD KAHN, and PIERRE DE MEYTS

From the Branch, National Institute of Arthritis, , and Digestive Diseases, Nation(al Instituites of Healthi, Bethlesda, Maryland 20014

A B S T RA C T The binding of 125I-insulin to insulin the of human monocytes is more sensi- receptors on circulating monocytes of obese patients tive to regulation by ambient insulin than the recep- and normal volunteers has been determined under tors of obese mice and cultured human lymphocytes. various dietary states. In the basal, fed state the mono- The results further indicate that an insulin receptor cytes of obese patients with clinical insulin resistance undergoes in vivo modulation of its interaction with (n = 6) bound less insulin than normals (n = 10) be- insulin by changing receptor concentration and by cause of a decrease in insulin receptor concentration altering the affinity of existing receptors. (obese = 6,000-13,000 sites per monocyte versus nornals 15,000-28,000 sites per monocyte). The single INTRODUCTION obese patient without evidence of clinical insulin re- sistance demonstrated normal binding of insulin with Obesity is commonly associated with hyperinsu- 16,000 sites per monocyte. In all patients, the total re- linemia and resistance to the action of endogenous ceptor concentration was inversely related to the circu- and exogenous insulin (1, 2). In both genetic and lating levels of insulin measured at rest after an over- acquired forms of obesity in rodents, the increased night fast. concentrations of circulating insulin are associated For the obese patients with basally depressed in- with decreased insulin binding to liver (3-6), suilin binding, a 48-72-h fast lowered circtulating in- muscle (7), fat (8-10), and thymus lymphocytes (11,12). sulin and increased binding to normal levels but only at The decrease in insulin binding is due entirely to a low hormone concentrations; this limited normaliza- decreased concentration of insulin receptors; these tion of 125I-insulin binding was associated with in- residual receptors are normal receptors by multiple creased receptor affinity for insulin without change in criteria (6). With chronic caloric restriction of obese receptor concentration. Refeeding after the acute fast- mice, as well as with acute starvation of obese and ing periods resulted in return to the elevated plasma thin mice, the plasma insulin concentrations fall and insulin levels, the basal receptor affinity, and the de- receptor concentrations rise (3). In both the fed and pressed instulin binding observed in the basal, fed food-restricted states, the receptor concentration is state. Chronic diet restored plasma insulin levels, inversely related to the level of circulating insulin. insulin binding, and receptor concentration to normal The insulin receptors in obese humans are not as well without change in affinity. characterized. With circulating mononuclear cells, When the data from this study are coupled with we have previously shown that some obese patients prevous in vivo and in vitro findings they suggest that have impaired binding of 1251-insulin and that chronic caloric restriction improves this defect in hormone binding (13). The nature of the defect and the mecha- A portion of this work was presented at the National Meeting nism of this adaptation have not been clearly defined. of the American Federation For Clinical Research, 2 May, 1976. Clin. Res. 1976. 24: 269A. These initial studies measured insulin binding to Received for publication 5 January 1976 and in revised mixed mononuclear leukocyte preparations derived form 29 July 1976. from Ficoll-Hypaque gradients (13, 14). We have re- ThleJouirnal of Clinical Investigation Volume 58 Noveember 1976-1123-1135 1123 TABLE I Metabolic Characteristics of Obese Patients in Basal State and after Acute Fast, Refeeding, and Chronic Diet

Age, sex, and percent Subject ideal body weight Conditioni Weight Glticose Instuliu

kg mg/dl Ulml A 19 Basal 114 85 28 Female 72 h fast 111 54 8 207% B 54 Basal 132 95 60 Female 72 h fast 129 75 22 224% refeeding 132 93 50 chronic diet 113 84 12 C 23 Basal 163 86 50 Male 72 h fast 161 80 25 199% refeeding 162 91 55 chronic diet 145 74 12 D 37 Basal 130 96 50 Female 72 h fast 127 60 15 215% refeeding 130 85 35 chronic diet 117 85 15 E 33 Basal 149 470 60 Male 72 h fast 147 253 28 215% chronic diet 123 105 35 F 47 Basal 161 90 35 Female 288% G 58 Basal 108 160 42 Female 209% cently demonstrated that monocytes, which constitute hormone binding studies were done after hypokalemia and about 20%o of this mixed cell population, account for alkalosis had been corrected. Patients were initially maintained on a 3,500 calorie, 85-90% of the insulin binding (15). Therefore, weight-maintaining diet (=45% carbohydrate, 20% protein, changes in monocyte number could result in major and 35% fat) for periods of 3-7 days. Oral glucose tolerance alterations in insulin binding. In an attempt to clarify tests with 100 g of glucose and one to three insulin binding the mechanisms of insulin resistance in human obesity, studies were done in this basal, fed state. Patient A. was we have examined insulin binding to circulating further tested after a 72-h fast. The four patients demonstrat- ing lowest basal insulin binding (B.-E.) were selected for monocytes of obese patients under various dietary detailed study. After basal studies were completed, these conditions including the basal, fed state, after 24-72 four patients were fasted (<50 calories per day = "acute fast") h of starvation, after refeeding, and finally after chronic with insulin binding studies performed after 24, 48, and 72 h diet. of fast (the designated time intervals are in addition to the initial 10 h overnight fasting period). Upon completion of the 72-h fast the 3,500 calorie diet was reinstituted ("refeeding") METHODS with repeat binding studies performed after 2-10 days. Patients were then begun on a 600 calorie diet (45% carbo- Patients hydrate, 20% protein, 35% fat) for 6-12 wk ("chronic diet"). Plasma glucose and insulin levels were measured after over- A. Obese patients. Seven obese patients (A.-G.), selected night fasting on days that insulin binding studies were per- to represent the range of carbohydrate metabolic defects formed. seen in obesity, were studied while inpatients in the Clinical B. Normal volunteers. 10 normal volunteers, 7 males and Center of the National Institutes of Health (Fig. 1, Table I). 3 females, aged 18-32 yr were studied while inpatients. They were taking no medications and were free of disease All were within 10% of their ideal body weight, had fasting except for patient E. who had congenital alkalosis and has plasma insulin concentrations of 5-18 ,uU/ml, and showed previously been reported (16). In patient E. all metabolic and normal plasma glucose and insulin responses after ingesting 1124 R. S. Bar, P. Gorden, J. Roth, C. R. Kahn, and P. DeMeyts Glucose Insulin 700 - 400 _ l / F~~~~~~~~~~1 ~ 15 600

-~13 500

0 01 300 (I) E 2 I LLI D En C,, 0 2 F- D 200 i -j

100 D~~~~~~~C A 3

0 60 120 180 0 60 120 180 TIME (Minutes) FIGURE 1 Basal glucose tolerance tests in obese patients A.-G. Shaded areas represent thle normal range+±2 SD of plasma glucose and insulin responses in 39 normal stubjects.

100 g of glucose. Oral glucose tolerance tests, as well as insulin TABLE II binding studies, were performed in the basal state in all nor- Monocyte Percentages in Normal and Obese Patietits* mal volunteers. The normal volunteers were given a 3,500 calorie diet (=45% carbohydrate, 20% protein, and 35% fat) for at least 4 days before all basal testing. Four normal Cl.,roic volunteers also had insulin binding studies after periods of - BaIsaIlt 72 h fi st R.f tCdinii (liet acute fast, three for 48 h and the other for 72 h. '25I-insulin binding study. Whole blood was drawn into I. Normal acid-dextrose-citrate solution (Fenwal plasmapheresis volunteers triple blood pack Fenwal Inc., Walter Kidde and Co., Inc., Ashland, Maine) and centrifuged. The erythrocytes and 1 17 plasma were returned to the patient. The buffy coat was 2 29 diluted 1:1 with phosphate buffered saline (pH 7.4), layered 3 29,30,23 33 onto Ficoll-Hypaque gradients, and centrifuged according 4 26,36 19 29 to the method of Boyum (17). The mononuclear cell layer 5 24,27 was removed and diluted in 100 mM HEPES buffer (pH 8) 6 21,30 to a final concentration of 50 x 106 mononuclear cells per ml. Viability, as assessed by trypan blue exclusion, was al- 7 22,21 ways greater than 98%. The percentage of monocytes in 8 26,29 this final mononuclear preparation was determined by phago- 9 22 cytosis of latex beads (18) and verified by esterase staining 10 23 of monocytes (19). Good agreement was found between these two methods, with esterase staining giving results -10% II. Obese higher than found with latex bead ingestion.' There was no systematic difference in monocyte content in mononuclear A 25,28 37 cell preparations of the obese or normal subjects (Table II). B 24,26 20 26 20 125I-insulin was prepared at a specific activity of 150-200 C 30,36 31 33 21 ,uCi/,g insulin (=0.5 I per insulin) as previously described (20-22). 1251-insulin, 0.2 ng/ml, was incubated with 40 x 106 D 14,21 19 16 20 mononuclear cells per ml in the absence or presence or un- E 31,25 30 20 labeled insulin over a range of insulin concentrations from F 21 0.2 through 100,000 ng/ml with final incubation volume of 0.5 G 21 ml per assay tube. In this study the assay buffer was 100 mM * Values represented are determined by latex particle I The monocyte count actually used for calculation of bind- ingestion. ing data was determined by latex bead ingestion. t Patients were studied on one to three separate occasions. Fluctuations in the Affinity and Concentration of Insulin Receptors 1 125 z A-G = Obese, Basal 0Go Shaded Area= Normals,±1 S D z 4

C,) z 3

2 z U 1 w

0.1 1 10 102 103 104 105 [INSULIN (ng/ml)

FIGURE 2 '25I-insuliin binding to monocytes of obese patients stuidied in the basal state. Miono- cytes were prepared by Ficoll-Hypa(qtue gradient centrifugation of buffy coats. 1251-insutlin (0.2 ng/mI) was inctubated with 40 x 106 mononuclear cells/ml in the absence or presence of unlabeled insulin over a range of instulin concentrations from 0.2 to 100,000 ng/ml. After 180 min at 22°C, replicate 0.2-ml-aliquots of the assay mixture were transferred to microfuge ttubes, centrifluged, and the supernatant fluid was aspirated. The tips of the tubes containing the pellet were excised and counted. Nonspecific binding, defined as the radioactivity in the cell pellet in the presenice of 105 ng/ml insulin, was subtracted from the total binding at each instulin concentration and(l nor- malized to a monocyte concentration of 107 cells/ml. Each point is the meani of replicate samples. The shaded area represenits the mean+ 1 SD of 17 instulin binding sttudies performed in 10 nornilal vol uinteers.

HEPES, pH 8.0, and the assay tubes were incubated for malized the data to correct fb)r the vairiationis in monocvte con- 180 min at 22°C. These assay conditions produce a steady- tent (15). With the improved methodology, wve noow findl tlht state binding with less than 10% degradation of labeled the total bindin-g of '25I-insu1in to the cells of the niorml-sll hormone, no receptor degradation, and pH change of less than subjects is in ac narrower range aind slightly lhigle r than 0.15 pH U during the 180-min incubation. 1251-insulin binding previouisly reported (13, 14). The nonispecific binding aniid tle with these modified conditions was also consistently greater concentration of hormonie that reduced the binding of 1']- than previously found.2 After 180 min of incubation replicate insulin by half, both of which had beenl higlher with tle cells 0.2-ml-aliquots of the incubation mixture were transferred to of the obese (13, 14), were nlow the samiie for the obese andl microfuge tubes and centrifuged. The supemate was aspirated thin adults and lower than previously rel)orted. Miore im- and the cell pellet contained in the tip of the microfuge portantly, the improved precision of the datal as wvell ats a tube was excised and the radioactivity counted. Under these greater appreciationi of the complexities of tlhe initeractionl of conditions nonspecific binding, defined as the amount of 1251_ insulin with its receptor, halve permitte(d a iore dletaile(d insulin bound to the cell pellet in the presence of 105 ng/ml analysis of the insulin binding. insulin, was 0.3-0.7% of the total radioactivity for both normal Data analysis. The data on binding of '25j-in.stulinl to r-e- and obese patients and was subtracted from total binding to ceptor is presented in three ways whichl emplhasize differenlt give specific binding. 1251-insulin specifically bound was then quantitative aspects of the bindinig interaction. (1) The expressed as a function of monocyte concentration. percent of total radioactivity that is specifically bound to The methodology of this stuidy introdtuces modest clhanges receptors is plotted as a fuinction of log total] insulinl coni- in the preparation of the cells aind several changes in the centration; this most closely represenits the actual experi- condition of the 125I-insulin binding assay including pH, mental results. (2) Bound/free of '25I-instulin, (B/F)4 is temperattire, aind buffer composition.3 Wve halve also nor- plotted as a function of bound (B) hormiionie (Scatelhard plot) (23). For insulin biniding to its receptors this llot is curvilinieatlr. 2 Manuscript in preparation. 3 Previous reports from this laboratorv performed binding 4Abbreviatiotns used in this papeer: B/F, hound/free; K, studies with Tris buffer (25 mM, pH 7.6) and a 90-min incuba- affinity; R, limiting high affinity state; Kf, limiting low affinity tion period at 15°C. state; Ro, total concentration of receptor sites per cell.

1126 R. S. Bar, P. Gordenj,. Rothi, C. R. Kalahn, acnd P. DeMeyts 0.05 N- A- F-

h-4 A o-o B 0-0 C Obese Patients - D E LL a-l F G-o G 0--N=--.N Mean of Normals Shaded Area = Range of Normals

i4E C 4*D 1.00 4 1.5.a 2.0 2.5 33.0 E C DB G F A N INSULIN BOUND (ng/ml) FIGURE 3 Scatchlard analyses of l25I-insulin binding to monocytes of obese patients A.-G. and normal volunteers. The ordinate, B/F, is the ratio of bound to free lhormone and the abscissa represents the insulin bound (ng/ml), both expressed per 107 monocytes per ml. For each patient maximal B/F and the intercept at the abscissa are designated by arrowvs. The two shaded areas represent the range of normal for maximal B/F and abscissa-intercepts, respectively. The inset is an enlargement of the portion of the graph enclosed by the rectangle.

The total binding capacity or total concentration of receptor this paper, xve have designated K, as the value of K wlven sites (RO) is derived from the point '%'here the linear extrapola- the 125I5-insuliin concentration is 0.2 ng/ml.5 tion of the cutrve intersects the horizontal axis. (3) The third It should be noted that the validity of this analysis (and method of data analysis is the "average affinity profile". of the derived parameters) does not depend on assigning a Because the insulin receptor sites are not independent of particular model to the molecular mechanisms involved in one another, traditional methods for deriving the affinities the cooperativity (27). of the receptors from curvilinear Scatchard plots are not valid (24). Experimental data suggest that the insulin recep- tors are a single set of homogeneous binding sites that undergo RESULTS negatively cooperative site-site interactions such that the affinitv of the receptors for insulin is inversely related to the I. Basal 1251-insulin Binding in Normal and fractional occupancy (25, 26). The average affinity profile ex- Obese presses the relationship between the average affinity for insulin (k) and receptor occupancy (Y) (27). At anx' A. Normiial volutnteers. 17 instulin sttudies were per- i on the Scatchard curve, the average affinity = K1 point formed in 10 normal volunteers. At every insulin con- = (B/F)1 where B1 = the concentration of bound hormone insulin to fell within Ro- B, centration binding monocytes 2, When a indi- and (B/F)i = the bound/free hormone at that point (for details narrow limits (Fig. shaded area). given of this analysis see reference 27). When the affinity (K) is vidual was studied on separate occasions variability of graphed versus log of the fractional occupancv of the recep- data was also small. tor (Y' or B/RB) the plot displays the average affinity of the receptor at all levels of receptor occupancy and is referred to as the "average affinity profile" (27). In this analysis, the 3 If 125I-insulin binding to monocytes could be measured limiting high affinity state, obtained at lowv levels of receptor accurately under equilibrium conditions at insulin concen- occupancy, is designated k,; the limiting low affinity state, trations below 0.2 ng/ml, higher values of &, might be obtained at high levels of receptor occupancy, is Kf. In expected. Fltuctutations in the Affinity and Concentration of Instulin Receptors 1 127 (Fig. 3). This suiggested tlht receptor affinities and 2.2 A*- A niegative cooperativitv were largely uinialtere(d aniid tlait o-o B the ol)served reduictioni in '21I-instilin binding wats dute o-cJ C to a decrease in receptor concentration. The lack of 1.8 *-U D clhange in receptor affinity was conifirmiied by the aver- *-_ E age affinitv profiles (Fig. 4). These plots slhowed that both and as well as the entire affinity profile in 0 A-~~\ F ke kf 1.4 _ the obese were withiin the range of the normal suil)jects. x O-0 G Tlhuis the diminislhed basal binding of 1251-instlin to Range of mnonocyte receptors of obese patients was accouinted for 1.0 _ Normals by changes in receptor concentrationi withouit ainy sig- Ise nificant alteration in receptor affinity.

0.6 H II Effect of Acute Fast and Chronic Diet in Obese Patients The fouir obese patients with the most impaired basal 0.2 125I-instl in binding (B., C., D., and E.) were futrtlher I .I,...... I...... I1I.I...... 1 . Il sttudied dturing periods of actute fi:sting and after chronic 0.1 1.0 10 100 diet. Witlh actute fuisting, elevated basal plasmia insutlin conicentrations fell (Table I), body weiglht decreased l)v PERCENT SATURATION 2-3 kg, and turinary ketones became strongly positive (7 x 100) in all patients. Dturing the period of chronic diet restric- FIGURE 4 Average affinity profiles of 1251-insulin binding to tion obese patients lost 13-19 kg of body weight aind obese patients' monocvtes. The average K is eqnial to patients B., C., aind D. lowered their plasma insulin to the normal range, while patient E. lowered his B/F . The percentage of Ro that are occuipied is given by Ro - B plasma insulin from 60 to 35 /iU/ml. Both types of diet Y x 100, where Y' = B/R0,. The shaded area is defined by the (accute fast and chronic diet) resulted in normalization plots of the two normal voluinteers with highest and lowest of binding of tracer concentrations of '251-insulin in Ke. all four patients; however, the mechanisms whereby these changes were effected clearly differed. To illus- When these binding data were analyzed by the trate these phenomena a single patient, E., will first be mietlhod of Scatclhard, curvilinear plots were obtained. considered in detail. The instil in receptor conceintrationis on normiial cells A. Obese patieWt E. In the basal state, obese ranged from 15,000 to 28,000 sites per cell witlh a mean patient E. lhad the most profouind depression of' 1251_ of 22,000 sites per cell. The relationship between chang- insulin binding with monocytes binding -30% as muchl ing receptor affinity for lhormone and occtipanicy of re- insulin as normal (Fig. 5). This decreased 1251-insulin ceptor sites by the hormiionie is expressed by the binding was accompanied by a comparable decrease in average affinity profile. In miioniocytes from normal Ro, i.e. -6,000 sites per monocyte basally compared to volunteers, the highest or "empty sites" affinity, a normal mean of 22,000 sites per monocyte. When Ke, ranges from 0.10 to 0.23 nM-1 and begins acutely fasted, patient E. had no changes in instlin to decrease when only 0.3% of total receptor sites binding at 24 h but at 48 and 72 h of fast increases in are occutpied. With increasing occupancy of receptors '251-instulin binding became evident (Fig. 5). This by hormone, apparent K progressively decreases uintil increased hormone binding was present only at rela- a Kf, is reached. In normal voluinteers Kf ranges be- tively low, physiologic concentrations of instilin. Thus, tween 0.02 and 0.04 nM-1 and is reached when only at an insulin concentration of 0.2 ng/m] (5 ,uU/ml) 10-20% of available receptor sites are occupied. there was a fivefold increase in 1251-insulin binding B. Obese. Studies of monocytes from the seven while at insulin concentrations greater than 1.0 ng/ml obese patients demonstrated a wide range of 1251_ (25,uU/ml) no significant difference was seen between insulin binding curves, consistent with the wide range binding in the basal state and binding after a 72-h fast. of basal levels of circulating insulin seen in this obese The binding alterations with acute fasting were asso- group (Fig. 2). Three patients were within +2 SD of ciated with little or no change in Ro (Fig. 5) but with normal while in the others binding was decreased by a marked increase in k, the affinity of the "empty 40-70% as compared to normals. On Scatchard analysis site" or high affinity state. Basally Ke was 1.2 x 108 the plots were all cturvilinear and rouighly parallel to (M-') and with acute fasting increased to 6.0 x 108 one another and to the cturves from the normal stubjects (M-1), while Kf (0.04 nM-1) and Ro remained constant. 1128 R. S. Bar, P. Gorden, J. Roth, C. R. Kahn, and P. DeMeyts 72 H FAST CHRONIC DIET

a z 0 6 __ Basal Basal (B) Diet 0 24 H Chronic t 48 H Shaded Area = Normals z 5 * *- 72H ±1 SD

4 Shaded Area = Normals z ±1 SD 3 I-2 Z 1 L;

0.).1 1.u 1o 102 10i 10 10o 0.1 1.0 10 102 103 102 JINSULIN] (ng/mll SCATCHARD ANALYSES 0.07 Basal -Basal 0.05 - -4 72 H Fast Chronic Diet Shaded Area = Range of Shaded Area = Range of 0.04 Normals Normals

LL 0.03

0.02 '.

.., I 0.01 -.~~~~~~

Me11,1 9 0.2 0.4 0.6 0.8 1.0 1.2 1.4 2.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 2.0 INSULIN BOUND (ng/ml) AVERAGE AFFINITY PROFILES 6u0

-Basal Basal 2.2 5-4 72 H Fast - Chronic Diet Shaded Area = Range of __ Shaded Area = Range of ;tNormals Normnals 1.8 -0I t-0t."A', x I >-' 1.4 .:It

Ile 1.0 N-K 0.6- 4 02 0.1 1.0 1C 100 0.1 1.0 10 100 PERCEENT SATURATION Y x 100

FIGURE 5 125I-insulin binding during a 72-h fast and after chronic diet in obese patient E. Binding-competition curves are determined by the methods described in Fig. 2. Scatchard analyses and average affinity profiles are calculated as described in Analysis of Data. For Scatchard analyses, the shaded area is defined by the two normal volunteers with highest and lowest RO; for the average affinity profiles the shaded area is defined by plots of the two normal volunteers with highest and lowest Ke*

Chronic diet also produced increases in 1251-insulin after 72 h of fasting 1251-insulin binding was increased binding (Fig. 5). In this case, however, the binding to normal levels but the increase was only found at increase was apparent at all hormone concentrations insulin concentrations less than 5 ng/ml (100lU/ml). and Scatchard plots demonstrated that this was due to Scatchard plots and average affinity profiles of these an increase in Ro from 6,000 sites per cell basally data revealed that the increased binding was associated to 22,000 sites per cell after chronic diet. This increase with little change in R& but with markedly increased in Ro was not accompanied by any change in affinity Ke. At higher concentrations of hormone, the affinity (Fig. 5). Therefore, the restoration of insulin binding to differences between acute fast and basal study became normal by the chronic diet was solely caused by an less pronounced, so that when more than 7-10% of increase in receptor concentration. total receptor sites were filled, the affinity curve with B. Obese Patients B., C., and D. During acute acute fasting became indistinguishable from the af- fasting, patients B., C., and D. showed binding changes finity curve derived from basal data. After 2-10 days similar to those of patient E. (Fig. 6). In these patients of refeeding in patients B.-D., body weight, plasma Fluctuations in the Affinity and Concentration of Insulin Receptors 1129 72 H FAST CHRONIC DIET

(B) D OBESE PATIENT D Basal OBESE PATIENT -- Basal (B) 5 24 H,. Fast -- Chronic Diet 72 48 H Fast Shaded Area = 4 72V >-4 72 H. Fast \X:S Normals 48a,Refeeding (Re) ;'t +±1 SD 3 - Shaded Area = Normals- \0 \' ±1 SD 2 -B_ B' 24

4 a z 3 0 m z

z I- iw - 17 5 11\ 72 H Fast z 4 \ OBESE PATIENT B w cc 3 Re a. B 2

0.1 1.0 10 102 103 104 105

B, [INSULIN] (ng/ml) OBESE PATIENT A

3 -4\' 72 H_ _ 2 -Fast \,

0.1 1.0 10 102 103 104 105 [INSULIN] (ng/mI)

FIGURE 6 1251-insulin binding during a 72-h fast, with refeeding and after chronic diet in obese patients A.-D. The binding competition curves shown were determined by the method described in Fig. 2. The shaded area represents the mean±1 SD of 17 1251-insulin binding curves in 10 nornal volunteers. glucose and insulin concentrations, and insulin bind- parameters of affinity were unchanged and well within ing returned to basal, fed state levels (Table I, Fig. 6). the normal range. The binding changes induced by refeeding were due C. Patient A. Patient A. was an obese patient with entirely to a reduction of Ki to the level observed normal basal 125I-insulin binding to her circulating in the basal state. monocytes (Fig. 6). Although equally obese (207% of Chronic diet, which was associated with a fall in ideal body weight), her basal plasma insulin concen- plasma insulin concentrations to normal levels, also tration was the lowest of the obese group (Table I). resulted in normalization of 1251.insulin binding in Further, this patient had normal plasma glucose and patients B.-D. As with patient E., chronic diet was insulin responses to oral glucose challenge (Fig. 1). associated with an increase in Ro to normal while all After 72 h of fasting her insulin binding did not sig- 1130 R. S. Bar, P. Gorden, J. Roth, C. R. Kahn, and P. DeMet.ts nificantly change (Fig. 6) and no change in R0 or K.3 was detected during the acute fast. Similar results were ob- tained when four normal volunteers were fasted for 48 or 72 h (data not shown). III. Summary and relationship of Ro to circulating plasma insulin concentrations *-- ...... The effects of acute fast, refeeding, and chronic diet on insulin binding parameters in patients B.-E. are 04 summarized in Fig. 7. In the basal, fed state patients ::::::-:-: -:--:ws\,,. B.-E. have decreased 1251-insulin binding, reduced :::--:::::::--:-:, ,.:....:.:v>.>:...... z R, but normal kR suggesting the presence of decreased U, numbers of normal receptors accounting for the X. diminished insulin binding by these monocytes. With ° _.12 -...'. acute fasting insulin binding increased to normal (B., .. ... C., and D.) or supranormal (E.) levels with little change in H and 1.5 to 5-fold increase in k. Refeeding of E a in return all and JO f-R*. high calorie diet resulted of binding tf>. metabolic parameters to the basal state. With chronic diet, insulin binding, receptor number, and receptor affinity for hormone became indistinguishable from FIGURE240ummary of acute fast, refeeding, and chronic diet normal (Fig. 7). onwl25I-insulin binding to monocytes of obese patients. The In the basal state, when plasma insulin concentra- upperWpanel is tracer...... 2...... the percent of tion is plotted against receptor concentration (or initial 0120ifically boundEto 107 monocytes/ml, themiddlepanelis B/F), an inverse relationship is found (Fig. 8A), i.e., C-ttl) oeprsea...... prmno.ex... the higher the patient's plasma insulin concentration 6hel the lower the R. This inverse relationship between R0 and circulating insulin concentration was also tion0of>insulin expressed as found during the refeeding stage and with chronic diet * 36 .. *...... (Fig. 8B); however, after acute fasting R0 was essen- tially unchanged despite significant falls in plasma insulin concentrations (Fig. 8B).

DISCUSS ION insulincytes, havat physioogicalimmunologc10.concentraionsfunction that are(10--ensitiveMt Clinical states of hormone resistance can be due to an (28).Thes culturedmacrophage have inslin recep torshumanwhich are indistingishablevr fromim lrtthose ofnuith impairment in the delivery of the hormone to the target teceptorsmonocyte...... o liver anEloand ther tissue by numerou cell (for example, antihormone antibodies) or to defects at the target cell itself. At the target cell, the defect can be in the first step, the binding of the hormone to onofinsulin exp ressedlogicasconMentrations its receptor, or at any one of the multitude of steps Fhough 7h Summary mofnacutefast reedng, aefnedcronic diet between binding to receptor and the final effects of the guppserpanel ostheisper hvrcentorcrislin(.fong/mhat hormone. Of the many conditions characterized by specifically broun tohe0 monosyes/mleten middi el panltuis hormone resistance, the insulin resistance of obesity is thectotalh haexpesdafroemtomolesmiper monocytenxmono- the most common and best studied. In obese mice, ctheslwe panevste receptorgiafinityontat tare conenstra-t insulin resistance is associated with a decreased concentration of insulin receptors (3-6). This decrease vio(18).Bheecaustuedthrphgsaeinsulinreceptoso-uaie in insulin receptor concentration is reversible since receptor concentration can be restored toward normal thes winsli arecpoofnhecirutingusal frm tonoythe.l by both acute fasting and chronic dieting (3). thuhtehuman monocyte hasalsnor deineda roinuin To study the insulin receptors of obese humans under dietary conditions similar to those in the obese whichporboth havepreranotieisessimla tonumanrono- rodents, we should ideally study the insulin receptors of the liver, since the function of that organ is the overwhelming determinant of glucose homeostasis in Fluctuations in the Affinity and Concentration of Insulin Receptors 1131 sensitive aiiid precise physico-chemical criteria (28). [INSULIN] (ng/ml) Fturtlhermiore, in patients xvith the syndromes of' instulin 0.5 1.0 1.5 2.0 2.5 resistanice associated 'with acanitlhosis nigricanis, the degree of impairmilenit of bindinag of '25I-instulin to the circuilating monocytes of these paitienits correlated very 500 k 28,000 closelv \vith the severity of' tle inisuilin resistanice ob- A-E = Obese, Basal x a-e = Acute Fast served clinically (29, 30). Thuts, we consider that the 0 Obese, specific instulini binding sites of lhtumlani monocvtes are z B'-E' = Obese, Chronic Diet u 5O Shaded Area= Range of Normals representative of the state of inistuilin receptors in liver 400 B' cn 0 and other tisstues throuiglhotut the body. E E' 21,000 C In the presenit study, wve have confirmed the finding z that hvperinsulinemic obese patients have decreased 0z z- insuilini bindinig (13, 14) and we nowN find that the 300 0 decrease in instulin binding is duie entirel to a decrease Ea: G.) z cn in the concenitration of instulini receptors. In these w.S a 14,000 mq hvperinstulinielmiic obese patienits chronic diet lowered 0z circtulating inistulin levels anid retturnied insutlinl binding 0 n cc 200 k r- and receptor concenitrationis to normiial. In the basal 0 b (fed) and chronically dieted states, the total receptor HIL d a:Ul concentrationi oni the cells of' the obese patienits was 7,000 inversely related to the restinig levels of circtulating 100k e instulin. Thuts, obese people aind obese miiice have E quiite simiilar defects in insuilini binding in the fed state and respond identically to chlonic dietinig. 10 20 30 40 50 60 In addition to the previous repolts firomi ouir labora- [INSULIN] (R,U/ml) FIGURE 8B Effect of acute fast and chronic diet on plasma insulin concentration and insulin receptor concentration in [INSULIN] (ng/mi) obese patients. Basal values are designated by capital 0.5 1.0 1.5 2.0 2.5 letters without superscripts, those after acute fast by small letters, while values after chronic diet are designated by prime (') superscripts. 500K 28,000 A-G = Obese 0 N = Mean of 17 Normals tory (13, 14), tlhree other grouips haiive evaluiated the x Shaded Area = Range of Normals statuis of instulin receptors in lhtumani obesity. Malrinetti 0 et al, in a stuidy with fat cell membranes (A 400 K preliminilary 0 from obese people, was the first to stuggest that insutlini E binding was decreased in obesity (10). In at detailed sttudy of obese patients, Amatrtuda and his co-workers 0z 300 K reported that, when expressed on a peri cell basis, ft't Er cells from obese patients bouind inlsulin as well as did z cells from normal sulbjects (31). However, if these data are expressed per adipocvte suirfcfee aireat rather 0z 0 200 K than per whole cell, the obese patients in Amia- cr truda's study then show a receptor concentration decrease 0 comparable to that of' ouir patients.63 In cl recenit Bo sttudy (32), Olefsky fotund that the conicentration of cc insuilin receptors on f'at cells of obese patients is de- 100 1 creased whether expressed per cell or- per uinit of cell sturface althouigh clearly the defect is more marked 10 20 30 40 50 60 when expressed per uinit of cell stirf'ace. He also fouindl [INSULIN] (A,U/ml) 6 Fat cell size is known to increase in the obese state, con- FIGURE 8A Basal insulin receptor concentration as a func- sistent with the data from Amatruda's study in which the tion of plasma insulin concentration in obese patients. The diameter and surface area of the fat cells from the obese total insulin Ro is expressed both as femtomoles (fmoles) per patients were 1.6 and 2.5 times greater, respectively, than monocyte and as binding sites per monocyte. those in the normal subjects. 1132 R. S. Bar, P. Gordeni,J. Roth, C. R. Kahn, and P. DeMei ts excellent correlation in his patients between instulin circulating levels of insulin and the receptor concen- binding to monocytes and instulin binding to fat cells. tration. When obese mice are fasted for 24 h, plasma The present sttudies add stupport to the idea that one insulin levels fall and receptor concentrations increase of the major infltuences on the receptor concentration (3). Treatment ofthe mice during the fast with repeated is the chronic level of insulin to which the cells are doses of insulin prevents the restoration of the receptor being exposed (3, 33). We show that in obese patients concentration. Streptozotocin treatment of obese mice uinder basal conditions, the concentration of insulin also corrects the hyperinsulinemia, and elevates recep- receptors is inversely related to the resting level of tor concentrations (35). Additionally, diabetic Chinese circulating instulin, and in obese patients who are not hamsters, who have abnormally low levels of insulin in hyperinstulinemic or insulin resistant, receptor concen- both plasma and pancreas, have elevated concentra- trations are normal. Chronic dieting of these hyper- tions of insulin receptors in plasma membranes of instulinemic obese patients is accompanied by a fall liver (36). in the ambient insulin and a commensurate rise in the The effect of insulin on the concentration of its own receptor concentration. In thin hyperinstl inemic receptors is direct and can be reprodtuced in vitro by diabetics and in hyperinsulinemic obese patients, incubating cultured htuman lymphocytes (IM-9 line) in Olefsky and Olefsky and Reaven demonstrated that the the presence of insulin (33, 37). When exposed to concentration of insulin receptors on circulating insulin these cells show a decrease in the concentra- monocytes is decreased (32, 34). Correlations between tion of insulin receptors; the speed and magnitude of the ambient insulin concentration and the receptor the fall in the insulin receptor concentration is de- concentration are even more extensive in mice. In both pendent on the concentration of insulin in the medium obese and thin mice studied in the fed, fasted and (33, 37, Fig. 9). Recent sttudies employing insulin dieted states, there is an excellent correlation between analogtues and inhibitors of protein synthesis fuirther

MOLES/LITER 10-10 10-9 10-8 10-7 10-6

z 100 _ 0

Z'0cr 0z zo0 50 LU z 5 00 LUcrw

Obese Human Monocyte

- 1.0 10 100 1000 10,000 AMBIENT INSULIN CONCENTRATION (ng/ml) FIGURE 9 Effect of ambient insulin concentration on insulin Ro in human monocytes, mouse liver cell plasma membranes, and in cultured human lymphocytes. For the human monocytes and mouse liver membranes, the insulin concentration is the basal level of plasma insulin in vivo measured by radioimmunoassay with porcine insulin and mouse insulin standards, respec- tively; the receptor concentration is expressed as percent of normal volunteers (mean) for human studies and percent of thin littermates for the obese mice. The cultured human lympho- cytes were preincubated with the designated insulin concentrations for 18 h, then washed ex- tensively before binding studies were performed. For the cultured lymphocytes, 100% recep- tor concentration represents the R& when cells were preincubated with culture medium alone. Data for the obese mice and cultured lymphocytes are found in references 3, 33, and 37. Fluctuations in the Affinity and Concentration of Insulin Receptors 1133 indicate that the effect of instulini on the concentration effects of other , dietary composition, age, of its own receptors is at direct regulatory process metabolic alterations suich as acidosis or ketosis, as well effected at the target cell via the instlin receptor itself as stages of growth, development, and differentiation. (37). In addition, observations here and elsewhere of sig- The insulin receptors on hulmiani monocytes appear nificant changes in plasma insulin levels without con- more sensitive to the effects of chronic elevations comitant changes in receptor concentrations (as well as in ambient instulin than either mice in vivo or culttured the converse) should serve to emphasize that the hutman lymplhocytes in vitro (Fig. 9). For hulmians a ambient insulin concentration is buit one of the many 50% decrease in receptor conicentration was found influiences on the receptor (38-40). with circulating levels of :2 ng/mI (50 ,uU/ml), while In stummary, we have demonstrated that an insulin in mice, a comiiparable 50% decrease in receptor con- receptor undergoes changes in both its affinity and centration required ambient inisulin conicentrations of concentration in vivo. By coordinating changes in 30 ng/ml (Fig. 9). This is consistenit witlh the observa- receptor affinity and receptor concentration with other tions that, in general, rodents in vivo are less sensitive regtulatory events within the cell, the target cell is to insulin than are humans. The IM-9 lymphocytes are thereby capable of a broad range of responses to even more resistant; 400 ng/ml of insulin is needed hormonal stimtuli. to reduce receptor concentrations by 50%. Further studies will be required to determine if this re- ACKN OWLE DG ME NTS duced sensitivity is a general clharacteristic of lympho- We wish to thank Mrs. Carolyn Siebert, Ms. Maxine cvtes in ctultture or varies froim cell line to cell line. Lesniak, Mrs. Dorothy Beall, Mrs. Carol Shinn, and Dr. The receptor affinity chaniges associated with acuite Richard Eastman for their help in preparing this manuscript. fasting of hyperinsulinemic obese patients represent a We also wish to thank Mrs. Catherine Cushing, the Clinical uiniquie finding of our stuidy. Wlihen obese mice were Center Ward 8 staff, Mrs. Wanda Chappell, and the staff of the actutely fassted, receptor concenitrations increased while Blood Bank for their help with the patient studies. clhanges in receptor affinity were not observed (3). The affinity clhaniges seen in the hyvperinstl inemic obese REFERENCES hutmians were ratlher specific sinice thev were not 1. Rabinowitz, D. 1970. Some endocrine and metabolic observed when tlhin normals andcl obese patients with aspects of obesity. Atintn. Rev. Med. 21: 241-258. minimal hyperinstulinemia were similarly fasted. This 2. Porte, D., Jr., and J. D. Bagdade. 1970. Htuman insulin change in affinity would have the effect in vivo of secretion: An integrated approach. Annu. Rev. Med. 21: normalizing instlin binding at low resting levels of 219-240. 3. Soll, A. H., C. R. Kahn, D. M. Neville, Jr., and J. Roth. circulating inssulin while mainitaining the relatively 1975. Insulin receptor deficiency in genetic and acquired impaired binding at stimuclated levels of' hormone. obesity. J. Clin. Invest. 56: 769-780. The molectular mediators of these affinity changes are 4. Kahn, C. R., D. M. Neville, Jr., and J. Roth. 1973. Insulin- tinknown. It is uinlikely that the ambient instlin level receptor interaction in the obese-hyperglycemic mouse: A model of insulin resistance. J. Biol. 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It is im- brane in the obese hyperglycemic mouse. Excerpta Med. Int. Congr. Ser. 280: 75. (Abstr. 164). portant to emphasize that receptor concentration, as 10. Marinetti, G. V., L. Shlatz, and K. Reilly. 1972. Hormone- well as receptor affinity, are probably stubject to membrane interactions. In: Insulin Action. I. B. Fritz, nuimerouis other potential influiences, including the editor. Academic Press, Inc., New York. 207-276. 1134 R. S. Bar, P. Gorden, J. Roth, C. R. Kahn, and P. DeMeyts 11. Sol], A. H., I. D. Goldfine, J. Roth, C. R. Kalhn, and D. M. interactionis among instil in receptors. Chalracterization of Neville, Jr. 1974. Thymic lymnphocytes in obese (ob/ob) the negative cooperativity. J. Biol. C11eCin. 251: 1877- mice. A mirror of the instulin receptor defect in liver and 1888. fat.J. Biol. Chemii. 249: 4127-4131. 27. De Meyts, P., and J. Roth. 1975. Cooperativity inligand 12. Goldfine, I. D., J. D. Gardner, D. M. Neville, Jr. 1972. binding: A new graphic analysis. Biochletin. Biophyts. Res. Instulin action of isolated rat thymocytes. I. Binding of Communm1 66: 1118-1126. l251_insiil in and stimulation of a-aminoisobuityric acid 28. Bar, R. S., H. Koren, and J. Roth. 1976. Physiological transport.J. Biol. Cheem. 247: 6919-6926. instilin concentrations affect macrophage fulnction. 13. Archer, J. A., P. Gorden, and J. Roth. 1976. Defect in Diabetes. 25 (Suippl. 1): 348. (Abstr. 111). insuilin binding to receptors in obese man. Amelioration 29. Flier, J. S., C. R. Kahn, J. Roth, and R. S. Bar. 1975. Anti- with calorie restriction.J. Cliii. Incest. 55: 166-174. bodies that impair insuilini receptor- binding in an uinulstual 14. Archer, J. A., P. Gorden, J. R. Gavin, III, M. A. Lesniak, diabetic syndrome witlh severe instulini resistanice. Sci- and J. Roth. 1973. Instulin receptors in hluimani circtulating ence. (Wash. D.C.) 190: 63-65. lymphocytes: Application to the study of insuilin resis- 30. Kahn, C. R., J. S. Flier, R. S. Bar, J. A. Archler, P. Gordein, tasnce in mani.J Cliii. Endocriniol. Metab. 36: 627-633. M. M. Martin, and J. Roth. 1976. The syndromes of 15. Schwartz, R. H., A. R. Bianco, B. S. Handwerger, and instilini resistanlce aind acanitlhosis nligr-icanis. Inisullinl- C. R. Kahn. 1975. Demonstration that monocytes rather receptor disorders in miian. N. Engl.J. Aled. 294: 739-745. than lymphocytes are the insuilin-binding cells in 31. Amatrtuda, J. M., J. N. Livingston, and D. H. Lockwood. preparations of huimnan peripheral blood mononuiclear 1975. Instulin receptor: role in resistance of hmtiian leuikocytes: preparationis: Implications for stuidies of in- obesity to instulini. Scienice. (Wash. D.C.) 188: 264-266. stulin-resistance states in mani. Proc. Natl. Acad. Sci., 32. Olefskv, J. M. 1976. Decreased instlini binding to adipo- U.S.A. 72: 474-478. cytes and circtulatinig monocytes fromii obese patienits. J. 16. Gorden, P., and H. Levitin. 1973. Congeniital alkalosis Cliii. Itncest. 57: 1165-1172. witlh diarrhea. A sequial to Darrow's original description. 33. Gavin, J. R., III, J. Rotlh, D. MI. Neville, Jr., P. De Mevts, Ann. Initertn. Med. 78: 876-882. and D. N. Btiell. 1974. Instulin-depenidenit regtlation of 17. Bbyunm, A. 1968. Separation of leuikocytes from blood and instulin receptor conicentrationis. A direct demonistrationi in bone marrow. Scantd. J. Clin. Itnvest. 21 (Suppl. 97): cell ctultture. Proc. Natl. Acad. Sci. U.S.A. 71: 84-88. 77-89. 34. Olefsky, J. M., and G. M. Reaven. 1974. Decreased inistulin 18. Cline, M. J., and R. I. Lehrer. 1968. Phagocytosis by binding to lymplhocytes from diabetic subjects. J. Cliii. huiman monocytes. Blood. 32: 423-435. Inivest. 54: 1323-1328. 19. Li, C. Y., K.W. Lam, and L. T. Yaim. 1973. Esterases in 35. Freychet, P. 1976. Interactionis of polypeptide hormiionies hlumani leuikocytes.J. Histochleiii. Cytochlem. 21: 1-12. witlh cell memll)branie specific receptors: Stuidies witl 20. Freychet, P., C. R. Kalhnl, J. Rotlh, and D. M. Neville, Jr. instulini and gltucagoni. Diabetologia. 12: 83- 100. 1972. Insuilin interactions witlh liver plasma membranes. 36. Hepp, K. D., J. Lanigley, J. J. von Funcieke, R. Rennler, anid Independenice of binding of the lhormone and its degrada- W. Kemmlner. 1975. Incr-eased instilini binding capacity tion.J. Biol. Chietin. 247: 3953-3961. of liver membranes fromii diabetic Chiniese Hamiisters. 21. Huinter, W. M., and F. C. Greenwood. 1962. Preparation Natuire. (Lonid.) 258: 154. of iodine-131 labelled huiman growthi lhormone of high 37. Kosmakos, F. (;., anid J. Rotlh. 1976. Cellular basis of specific activity. Natuire (Loni.) 194: 495-496. instulini-inidtuced loss of instulin receptors. Enidocrinie 22. Yalow, R. S., and S. A. Bersoni. 1960. Immuinoassay of Society, 58th Ainutal Mleetinig, Juniie 23-25, Sani Franii- endogenouis plasmiia insuilin in mani. J. Clini. Inciest. 39: cisco, p. 69. (Abstr.) 1157- 1175. 38. Livingstoni, J. N., P. Ctiatrecasas, anid D. H. Lockwood. 23. Scatchard, G. 1949. The attractions of proteins for small 1972. Insuilin inisensitivity, of large flit cells. Scienice. molecuiles and ionls. Ann. N. Y. Acad. Sci. 51: 660-672. (Wash. D.C.) 177: 626-628. 24. Klotz, I. M., and D. L. Huinstoni. 1975. Protein interactions 39. Goldfinie, I. D. 1975. Biniding of iistiliin to thymnocvtes with smnall molecuiles. Relationslhips between stoichiio- friom suicklinig and hypophysectomnized rats: Evidence for metric binding constants, site binding constants, and two meclhaniismiis reguilatinig inistil in sensitivity. E udo- empirical binding parameters. J. Biol. Chleini. 250: crinologly 97: 948-954. 3001-3009. 40. Kahni, C. R., I. D. Goldfinie, D. NI. Neville, Jr., J. Rotlh, 25. De Meyts, P., J. Roth, D. M. Neville, Jr., J. R. Gavin, III, M. Garrison, and R. W. Bates. 1973. Inisuilini receptor de- and M. A. Lesniiak. 1973. Insuilini interactions with its fect: A major factor in the inisuilini resistaniee of glulco- receptors: Experimental evidence for negative coopera- corticoid excess. Enidocrinie Society, 55th Annual tivity. Biochzemii. Biophys. Res. Cotiin uni. 55: 154-161. Meetinig, April 28-30, Atlanitic City, New Jersey. 26. De Mevts, P., A. R. Bianco, and J. Roth. 1976. Site-site P. 168A. (Abstr.)

Flucitu(ations in thze Affinity and Concentration of Insulin Receptors 1135