Effects of Age and Obesity on Insulin Binding to Isolated Adipocytes 12 JERROLD M. OLEFSKY3 AND GERALD M. REAVEN4 Department of Medicine, Stanford University School of Medicine and Veterans Administration Hospital, Palo Alto, California appreciable effect on the ability of adipocytes to bind insulin. The influence of the obesity associated variables—hyperinsulinemia and increased fat cell size—on insulin binding was also examined. These latter studies are consistent with the concept that elevated in vivo plasma insulin levels lead to decreased insulin receptors, and further suggest a role for additional factors in regulating the adipocyte insulin receptor. In conclusion: 1. Decreased insulin binding to adipocytes is closely related to the obese state. 2. This decrease in insulin binding can be accounted for by decreased numbers of receptor sites per cell, and 3. The mechanism(s) underlying this decreased insulin binding are complex and probably represents an effect of more than one variable. (Endocrinology 96: 1486, 1975)
ABSTRACT. We have measured insulin binding to isolated adipocytes prepared from rats of varying ages and body weights. The ability of adipocytes to bind insulin progressively decreases as animals get older and fatter until about 70 days of age and 300 g body weight are reached. From this point on further decreases in insulin binding to adipocytes were not seen as rats got older and fatter. Analysis of the data indicated that this decrease in insulin binding could be accounted for by decreased numbers olf insulin receptor sites per cell. Further studies were conducted in which animals were allowed to age, but obesity was prevented or reversed by hypocaloric diets. In these experiments decreased insulin binding was either prevented or restored to normal by the negative caloric state, indicating that age had no
T
HE initial step of insulin's action is binding to specific receptors located on plasma membranes of target tissues (1), and methods are now available to directly study this binding reaction. Using these methods, several physiologic states, which are characterized by insulin resistance, have been found to be associated with decreased insulin binding to receptors (2-10). Of these insulin resistant states, obesity has been most thoroughly studied. Decreased insulin binding to receptors on circulating lymphocytes has been described in obese humans (7); and decreased insulin binding to liver (2,3) lymphoid (6), muscle (5), and adipose tissue (4) Received October 9, 1974. 1 This work was supported in part by a grant from the National Institute of Health, HL08506, and from the Veterans Administration. 2 Requests for reprints should be addressed to: Jerrold M. Olefsky, M.D. Veterans Administration Hospital, 3801 Miranda Avenue, Palo Alto, California 94304. 3 Dr. Olefsky is a Research and Education Associate, Veterans Administration (MRIS #6488). 4 Dr. Reaven is a Medical Investigator, Veterans Administration (MRIS #7363).
has been reported in a variety of genetically abnormal or artificially induced obese states in mice. However, naturally occurring, spontaneous obesity has not been extensively examined in animals, and, in the only studies of this type which have been reported (11-13), decreased insulin binding to adipocytes from spontaneously obese, genetically normal rats has not been observed. Furthermore, since obesity is associated with a number of additional abnormalities, it seemed important to investigate the relationship between these factors and insulin binding. Thus, in this current report we have carried out detailed studies of insulin binding to adipocytes from lean and spontaneously obese, genetically normal rats, and have attempted to correlate changes in insulin binding with other variables commonly associated with obesity such as age, caloric balance, adipocyte size, and hyperinsulinemia. Materials and Methods Materials. Porcine monocomponent insulin was generously supplied by Dr. Ronald Chance of the Eli Lilly Co. Na1Z5I was purchased from the
1486
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1487
INSULIN BINDING TO ADIPOCYTES New England Nuclear Co., bovine serum albumin (fraction V) from Armour and Co., collagenase from Worthington Biochemicals, and guinea pig anti-insulin antibody from Pentex Co.
phthlate has a specific gravity intermediate between buffer and cells, and 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 Iodination of insulin. [125I]iodoinsulin was pre- triplicate. The amount of buffer remaining in pared at specific activities of 100-150 //,Ci//i,g the cell layer was measured using [14C]inulin to according to Freychet et al.'s modification (14) determine the extracellular water space of the of the method of Hunter and Greenwood (15) as cell layer according to the method of Gliemann previously described (16). This preparation has (22). Adipocyte number and size were measured an average of 0.2-0.3 atoms of I25I per insulin according to a modification of method III of molecule. Freychet (14) and others (17,18) have Hirsch and Gallian (23), in which the cells were demonstrated that with this degree of iodination fixed in 2% osmium tetroxide in 0.05M collidine over 90% of the iodinated species is monoiodi- buffer (made isotonic with saline) for 72 h at nated insulin which retains full biological activ- 37 C and then taken up in a known volume of ity (14,18). 0.154M NaCl for counting. Counting was performed using a Celloscope Model 112H particle Preparation of isolated adipocytes. Male counter with a 400 /x aperture. The weight of Sprague-Dawley rats were used for all experi- triglyceride (/ag) in the sample was then divided ments. Animals were divided, by body weight, by the number of cells per sample to give the into three groups: group 1 (160-220 g), group 2 average adipocyte size. Cell size is expressed as (250-400 g), and group 3 (>400 g). Unless fjig of triglyceride (TG) per cell, and it should be otherwise stated all studies were performed in pointed out that this approach gives only the the morning on animals who had free access to average adipocyte size in the preparation, and standard rat chow. Animals were stunned by a does not indicate the distribution of cell sizes blow to the head, decapitated, and epididymal about the mean. fat pads removed. Isolated fat cells were prepared by shaking at 37 C for 60 min in Krebs- Insulin degradation. Degradation of insulin was Ringer bicarbonate buffer containing col- determined by examining the ability of the lagenase (3 mg/ml) and albumin (40 mg/ml), radioactive material remaining in the incubation according to the method of Rodbell (19). Cells media to precipitate with 10% TCA, adsorb to were then filtered through 250 /u.m nylon mesh, talc, or precipitate with excess anti-insulin anticentrifuged at 400 rpm for four minutes, and body (24,25). The percent of insulin which is washed three times in a buffer containing 35 intact according to these three methods is HIM Tris, 120 mM NaCl, 1.2 mM MgSO4, 2.5 DIM compared to that of [125I]iodoinsulin which has KC1, 10 mM glucose, 1 mM EDTA, and 1% BSA been incubated in the absence of adipocytes. (20). Percent [125I]iodoinsulin which remains intact is then determined by the following formula: Binding studies. Isolated fat cells were suspended in the Tris buffer containing 1% BSA pH 7.6, and incubated with [125I]iodoinsulin and unlabeled insulin in siliconized 10 ml Erlenmeyer flasks in a 24 C shaking water bath. Methodologic studies (data not shown) have indicated that optimal steady state binding conditions are achieved at 24 C following 45 min of incubation using cells from all three groups of animals. The incubations were terminated as described by Gammeltoft and Gliemann (21) by removing 200 fi\ aliquots from the cell suspension and rapidly centrifuging the cells in plastic micro tubes to which 100 /xl of dinonyl phthalate oil has been added. Dinonyl
% intact of incubated insulin % intact of unexposed insulin
x 100
Unexposed [l25I]iodoinsulin is at least 98% precipitable by 10% TCA, 95% adsorbable to talc, and 94% precipitable with excess antiinsulin antibody for up to 3 weeks after preparation. Calculations. Specific [125I]iodoinsulin binding was calculated by subtracting the amount of [125I]iodoinsulin nonspecifically bound from the total amount of [125I]iodoinsulin bound at each insulin concentration (2,6,10,25). Nonspecific
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binding is defined as the amount of [1S5I]iodoinsulin remaining "bound" in the presence of a large excess—200 /xg/ml (3 x 10~5M)—of unlabeled insulin (2,6,10,25). Apparent binding affinity was estimated by the use of Scatchard plots (26). As recently reviewed by Kahn et al. (27), this method of analysis relies upon a number of currently unproved assumptions, and the interpretation of the resulting curvilinear Scatchard plots is highly complex. Nonetheless, these plots are still useful for comparing average binding affinities and are used for this purpose. Binding capacity (number of insulin receptor sites per cell) can be calculated by determining the intercept on the abscissa of the Scatchard plot, and, again, this method remains useful for comparative purposes. However, because the terminal points of the Scatchard plot are obtained at very high insulin concentrations, and because non-specific binding is proportionately high and hard to determine at these concentrations, it is difficult to accurately define the precise point of intercept. Consequently, we have used as an operational definition of numbers of receptor sites per cell the number of 125
1
TABLE 1. Characteristics of [ I]iodoinsulin eluted from adipocytes
[12iI]iodoinsulin
Fresh
Eluted from cells
% precipitable by 10% TCA
99
90
% absorption to talc
96
87
% precipitable by excess antiinsulin antibody
95
88
Specific binding to liver plasma membranes2 (pmol x 10~2/200 fig membrane protein) 1
Endo • 1975 Vol 96 • No 6
OLEFSKY AND REAVEN
1488
l25
8.2 9
7.6
Adipocytes and [ I]iodoinsulin (10~ M) are incubated for 45 min at 24 C. Cells are then removed, washed twice with cold buffer (4 C) and then resuspended and incubated in fresh in&ulin-free buffer at 37 C for 60 min. The mixture is then centrifuged, and the infranate (containing 125 the eluted [l25I]iodoinsulin) is saved. The eluted [ I]iodoinsulin is then compared to equivalent amounts of fresh [125I]:iodoinsulin for its ability to precipitate with 10% TCA, adsorb to talc, precipitate with excess anti-insulin antibody, and specifically bind to liver plasma membranes. 2 Prepared according to the method of Neville et al. (31),
insulin molecules specifically bound at an insulin concentration of 100 ng/ml (2400 /u,U/ml or 1.7 x 10"8M). The rationale behind this approach is that if a receptor does not bind insulin at a concentration of 100 ng/ml (higher than can be achieved in the rat circulation) then it is probably not functioning physiologically as a receptor in vivo. Furthermore, our data show (25, and results section) that approximately 75% of all receptors are filled at this insulin concentration, and the number of molecules bound at this insulin concentration can be accurately measured with an intra-assay coefficient of variation of 5%. Thus, we believe that this measurement provides an accurate representation of receptor site concentration and is useful for comparative purposes. Nevertheless, we realize that 100 ng/ml is a somewhat arbitrary cutoff, and the same argument could be applied to lower insulin concentrations. Thus, this measurement is suggested only for comparative purposes, and should not be interpreted as having precise physico-chemical meaning.
Results
7. Nature of the bound insulin. Previous studies have indicated that essentially all of the insulin binding activity of adipocytes can be recovered in the particulate, nonnuclear cell fraction (28,29), and that treatment of intact fat cells with trypsin abolishes their insulin binding capacity (28-30). Thus, essentially all of the cell associated [125I]iodoinsulin is bound to the plasma membrane. To see if the bound [125I]iodoinsulin could be eluted from the cells, [125I]iodoinsulin and adipocytes were incubated for 45 min at which time an excess of unlabelled insulin (3 x 10~5M) was added. Dissociation of the bound [125I]iodoinsulin was measured over the subsequent 120 min. 50% of the previously bound [125I]iodoinsulin dissociates from the cells in 25 min, and greater than 90% is dissociated by 120 min. The nature of previously bound insulin was further characterized as described in Table 1. The data in Table 1 indicate that the insulin which had been bound to receptors retains most of its immunological activity, and that its ability to precipitate with TCA and
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INSULIN BINDING TO ADIPOCYTES
1489
pared to adipocytes from group 1 animals. On the other hand, increasing concentrations of unlabeled insulin appear equally capable of competing for binding with [125I]iodoinsulin (0.2 ng/ml) using cells from all three groups (IB). For example, 1 //. Insulin binding to adipocytes from rats ng/ml (24 /xU/ml) of unlabeled insulin 125 of different weights. Figure 1A sum- inhibits 16-17% of the [ I]iodoinsulin marizes the ability of adipocytes from 3 binding in all groups, and binding can be groups of rats to bind insulin. Animals from 50% inhibited at an insulin concentration ng/ml. At 100 ng/ml 77-80% of Group 1 (n = 16) weighed 160-220 g, of 10-11 125 group 2 (n = 19) weighed 250-400 g, and the [ I]iodoinsulin binding is inhibited. group 3 (n = 10) weighed > 400 g. It can Nonspecific binding averaged 5% of the be seen that cells from fatter rats (groups 2 total binding (corrected for "trapped and 3) bind only 50-60% as much insulin buffer," Table 2) and all the data in Fig. 1 at each insulin concentration when com- are corrected for this factor (see legend). adsorb to talc was comparable to fresh [125I]iodoinsulin. Furthermore, the eluted [125I]iodoinsulin specifically bound to liver plasma membrane receptors 93% as well as fresh [125I]iodoinsulin.
FIG. 1A (upper). Relationship between insulin concentration and the amount of insulin specifically bound per cell. Group 1 (•—•) consisted of 16 rats whose weights ranged from 160-220 g, group 2 (o—o) 19 rats whose weights ranged from 250-400 g and group 3 (•—•) 10 rats whose weights were >400 g. FIG. IB (lower). Inhibiting effect of unlabelled pork insulin on
[
l25
500-
300
200
I]iodoinsulin binding to
adipocytes. Incubations are carried out at 24 C for 45 min in the presence of 0.2 ng [125I]iodoinsulin. Brackets represent ±SE. Data represent the mean of 16 separate studies for group 1 (•—•), 19 for group 2 (o—o) and 10 for group 3 (•—•). All data are corrected for nonspecific binding by subtracting the amount of [125I]iodoinsulin remaining "bound" in the presence of 200 /xg/ml insulin from the amount of [l25I]iodoinsulin bound at all other insulin concentrations (2,6,10,25). Binding is a linear function of cell concentration up to 3.5 x 10* cells/ml, and all experiments were normalized to 2 x 10s cells/ml (average cell concentration used in these studies). B/F is the bound to free [l25I]iodoinsulin ratio.
2.0-
1000 Insulin concentration (ng/ml)
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OLEFSKY AND REAVEN
1490
TABLE 2. Total binding and nonspecific binding of [l25I]iodoinsulin to adipocytes from rats of varying weights (± SE)
% of total [lnI]iodoinsulin bound' % of total ['"Ijiodoinsulin nonspecifically bound* % of total [lt5r]iodoinsulin "trapped" in cell layer
Group I (160-220 g)
Group 2 (250-400 g)
Group 3 (>400 g)
2.20 ± 0.12
1.31 ± 0.10
1.28 ± 0.09
0.21 ± 0.01 (0.17-0.26)
0.20 ± 0.01 (0.16-0.27)
0.22 ± 0.01 (0.16-0.30)
0.11 ± 0.01 (0.09-0.12)
0.13 + 0.01 (0.10-0.14)
0.15 ±0.02 (0.12-0.16)
1 This represents the total per cent of the ['"Ijiodoinsulin bound at an insulin concentration of 0.2 ng/ml. The data are expressed per 2 x 105 cells/ml which is the average cell concentration used. "This represents the amount of ['"Ijiodoinsulin remaining in the cell layer in the presence of 200 /xg/ml of unlabeled insulin, divided by the total amount of ['"Ijiodoinsulin available. In these studies the amount of nonspecifically bound insulin accounted for by "trapped" buffer was 0.11% for group 1, 0.13% for group 2, and 0.15% for group 3. Thus the amount of nonspecifically bound [l25I]iodoinsulin net accounted for by trapped buffer was 0.10% for group 1, 0.07% for group 2, and 0.07% for group 3. Although the unaccounted for nonspec fie binding decreases as the rats get larger, since the total ['"Ijiodoinsulin binding also decreases as rats get larger, the proportion of the ['"Ijiodoinsulin bound which is nonspecifically bound remains the same: i.e., J>%. Specific ['"Ijiodoinsulin binding represents the difference between to:al and nonspecific binding.
The similarity of the shapes of the curves of Fig. IB indicates that the ability of unlabeled insulin to inhibit [125I]iod'.oinsulin binding is similar (10). Since the ability of unlabeled insulin to inhibit the
Endo • 1975 Vol 96 • No 6
[125I]iodoinsulin binding is a reflection of the binding affinity, the inhibition curves in Fig. IB suggest that the affinity of adipocytes for insulin is the same for all 3 groups. Thus, the decreased insulin binding to cells from rats of groups 2 and 3 is primarily due to decreased numbers of available receptor sites per cell. The idea is further supported by Fig. 2 which shows the Scatchard plots derived from the data in Fig. 1. When the bound/ free ratio is plotted as a function of bound hormone, similarly shaped curvilinear plots are obtained for all three groups. This type of curve can reflect a constantly decreasing overall affinity of the unoccupied insulin receptors as the per cent occupied receptors increases (negative cooperativity (32)), two heterogeneous insulin receptor populations of high and low affinity (27), or a combination of both of the above phenomenon. Regardless of which explanation holds, the slope of the plot over any given range represents the apparent affinity of the receptor population for that degree of occupancy. Fitting the data to two linear components over the same range of insulin concentrations and per cent receptor occupancies yields an average high affinity constant of 1.9 x 10~!)M and an average low affinity constant of 1 x 10~8M for all
FIG. 2. Scatchard plots of the data in Fig. IB. The K ^ of the low affinity site is obtained from the slope of the least squares straight line drawn to fit the data obtained at insulin concentrations >10 ng/ml. The contribution of this line is then subtracted from the points obtained at lower insulin concentrations and a second least squares straight line is drawn to fit the resultant points. The K ^ of the high affinity site is obtained from the slope of this latter line. .02 .06 .1
2
.3
.4
.5
Insulin bound per cell (p moles x
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INSULIN BINDING TO ADIPOCYTES
groups. These apparent affinity constants are calculated for comparative purposes, and are not to be interpreted as having precise physico-chemical meaning. The intercept on the abscissa of the Scatchard plot approximates the total receptor site concentration, and it is clear that the decrease in insulin binding to adipocytes from group 2 and 3 animals can be primarily accounted for by a decreased number of receptor sites per cell. Numbers of receptor sites per cell can be more accurately compared by following the approach outlined in the methods section. Thus, with this approach, the average number of available receptor sites per cell is 23 ± 1.5 x 104 sites per cell (mean ± SE) for group 1, 14 ± 1 x 104 sites per cell for group 2, and 12 ± 1 x 104 sites per cell for group 3. These differences in insulin binding are not due to differences in insulin degradation. Insulin degradation was studied (see Materials and Methods) and 91-93% of the insulin was still intact following 45 min of incubation at 24 C using an initial insulin concentration of 0.2 ng/ml. At higher insulin concentrations the percent insulin degradation is even less (21,24 and unpublished observations). No differences in the ability of adipocytes to degrade insulin were observed using cells from all three groups of rats. HI. Effect of age versus obesity on insulin binding. It should be noted that while striking differences in insulin binding were noted between group 1 versus groups 2 and 3, no differences were observed between groups 2 and 3 themselves. This is more clearly demonstrated in Fig. 3A which gives the relationship between the amount of insulin bound per cell and body weight, using the individual data obtained from each animal. As can be seen, the ability of adipocytes to bind [125I]iodoinsulin (0.2 ng/ml) steadily decreases as rats get larger from 160 g to about 300 g. Once 300 g of weight is reached it appears that no further decline in the ability of adipocytes to bind
1491
insulin occurs. Since these animals were allowed free access to food, weight and age increased together. Consequently, as seen in Fig. 3B, the relationship between age and insulin binding to adipocytes is essentially the same as in Fig. 3A. In an attempt to dissociate the influence of age from obesity on insulin binding to adipocytes we have carried out a number of caloric manipulations as described below. Fasting studies. In these studies a group of rats whose initial weights averaged 312 ± 5 g (range 300-336 g) were fasted in groups of 4 for 24, 48, and 72 h. These animals lost an average of 17 g of weight at 24 h of fasting, 32 g by 48 h, and 52 g by 72 h. Eight AM plasma insulin levels fell from 42 ± 3 /xU/ml to 5 ± 1 /LtU/ml by 24 h, 5 ± 1 /xU/ml by 48 h, and 3 ± 1 /u,U/ml by 72 h of fasting. The ability of adipocytes from these groups of animals to bind insulin is seen in Fig. 4A. The bars on the left side of the figure represent the amount of insulin bound at an insulin concentration of 0.2 ng/ml while the bars on the right represent the maximal amount of insulin bound (see Materials and Methods) i.e., the amount bound at an insulin concentration of 100 ng/ml. After 24 h of caloric deprivation the amount of insulin bound per adipocyte had almost doubled. Binding remained at this level through 48 h of fasting but declined significantly by 72 h. It should be pointed out that 72-h fasted rats are quite ill-appearing, and this may account for the decline in binding. Complete inhibition studies1 were performed in all of these experiments, and no differences in the ability of unlabeled insulin to inhibit the [125I]iodoinsulin binding were noted, and, thus, for the same reasons as discussed earlier binding affinity did not seem to be changed. Consequently, the increase in insulin binding noted following fasting could be predominately ac1
This means that parallel incubations were performed at all insulin concentrations on cells obtained from each rat as in Fig. 1.
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Endo • 1975 Vol96 • No 6
OLEFSKY AND REAVEN
1492 4.424.08 °o
3.74-
ABSTRACT. We have measured insulin binding to isolated adipocytes prepared from rats of varying ages and body weights. The ability of adipocytes to bind insulin progressively decreases as animals get older and fatter until about 70 days of age and 300 g body weight are reached. From this point on further decreases in insulin binding to adipocytes were not seen as rats got older and fatter. Analysis of the data indicated that this decrease in insulin binding could be accounted for by decreased numbers olf insulin receptor sites per cell. Further studies were conducted in which animals were allowed to age, but obesity was prevented or reversed by hypocaloric diets. In these experiments decreased insulin binding was either prevented or restored to normal by the negative caloric state, indicating that age had no
T
HE initial step of insulin's action is binding to specific receptors located on plasma membranes of target tissues (1), and methods are now available to directly study this binding reaction. Using these methods, several physiologic states, which are characterized by insulin resistance, have been found to be associated with decreased insulin binding to receptors (2-10). Of these insulin resistant states, obesity has been most thoroughly studied. Decreased insulin binding to receptors on circulating lymphocytes has been described in obese humans (7); and decreased insulin binding to liver (2,3) lymphoid (6), muscle (5), and adipose tissue (4) Received October 9, 1974. 1 This work was supported in part by a grant from the National Institute of Health, HL08506, and from the Veterans Administration. 2 Requests for reprints should be addressed to: Jerrold M. Olefsky, M.D. Veterans Administration Hospital, 3801 Miranda Avenue, Palo Alto, California 94304. 3 Dr. Olefsky is a Research and Education Associate, Veterans Administration (MRIS #6488). 4 Dr. Reaven is a Medical Investigator, Veterans Administration (MRIS #7363).
has been reported in a variety of genetically abnormal or artificially induced obese states in mice. However, naturally occurring, spontaneous obesity has not been extensively examined in animals, and, in the only studies of this type which have been reported (11-13), decreased insulin binding to adipocytes from spontaneously obese, genetically normal rats has not been observed. Furthermore, since obesity is associated with a number of additional abnormalities, it seemed important to investigate the relationship between these factors and insulin binding. Thus, in this current report we have carried out detailed studies of insulin binding to adipocytes from lean and spontaneously obese, genetically normal rats, and have attempted to correlate changes in insulin binding with other variables commonly associated with obesity such as age, caloric balance, adipocyte size, and hyperinsulinemia. Materials and Methods Materials. Porcine monocomponent insulin was generously supplied by Dr. Ronald Chance of the Eli Lilly Co. Na1Z5I was purchased from the
1486
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1487
INSULIN BINDING TO ADIPOCYTES New England Nuclear Co., bovine serum albumin (fraction V) from Armour and Co., collagenase from Worthington Biochemicals, and guinea pig anti-insulin antibody from Pentex Co.
phthlate has a specific gravity intermediate between buffer and cells, and 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 Iodination of insulin. [125I]iodoinsulin was pre- triplicate. The amount of buffer remaining in pared at specific activities of 100-150 //,Ci//i,g the cell layer was measured using [14C]inulin to according to Freychet et al.'s modification (14) determine the extracellular water space of the of the method of Hunter and Greenwood (15) as cell layer according to the method of Gliemann previously described (16). This preparation has (22). Adipocyte number and size were measured an average of 0.2-0.3 atoms of I25I per insulin according to a modification of method III of molecule. Freychet (14) and others (17,18) have Hirsch and Gallian (23), in which the cells were demonstrated that with this degree of iodination fixed in 2% osmium tetroxide in 0.05M collidine over 90% of the iodinated species is monoiodi- buffer (made isotonic with saline) for 72 h at nated insulin which retains full biological activ- 37 C and then taken up in a known volume of ity (14,18). 0.154M NaCl for counting. Counting was performed using a Celloscope Model 112H particle Preparation of isolated adipocytes. Male counter with a 400 /x aperture. The weight of Sprague-Dawley rats were used for all experi- triglyceride (/ag) in the sample was then divided ments. Animals were divided, by body weight, by the number of cells per sample to give the into three groups: group 1 (160-220 g), group 2 average adipocyte size. Cell size is expressed as (250-400 g), and group 3 (>400 g). Unless fjig of triglyceride (TG) per cell, and it should be otherwise stated all studies were performed in pointed out that this approach gives only the the morning on animals who had free access to average adipocyte size in the preparation, and standard rat chow. Animals were stunned by a does not indicate the distribution of cell sizes blow to the head, decapitated, and epididymal about the mean. fat pads removed. Isolated fat cells were prepared by shaking at 37 C for 60 min in Krebs- Insulin degradation. Degradation of insulin was Ringer bicarbonate buffer containing col- determined by examining the ability of the lagenase (3 mg/ml) and albumin (40 mg/ml), radioactive material remaining in the incubation according to the method of Rodbell (19). Cells media to precipitate with 10% TCA, adsorb to were then filtered through 250 /u.m nylon mesh, talc, or precipitate with excess anti-insulin anticentrifuged at 400 rpm for four minutes, and body (24,25). The percent of insulin which is washed three times in a buffer containing 35 intact according to these three methods is HIM Tris, 120 mM NaCl, 1.2 mM MgSO4, 2.5 DIM compared to that of [125I]iodoinsulin which has KC1, 10 mM glucose, 1 mM EDTA, and 1% BSA been incubated in the absence of adipocytes. (20). Percent [125I]iodoinsulin which remains intact is then determined by the following formula: Binding studies. Isolated fat cells were suspended in the Tris buffer containing 1% BSA pH 7.6, and incubated with [125I]iodoinsulin and unlabeled insulin in siliconized 10 ml Erlenmeyer flasks in a 24 C shaking water bath. Methodologic studies (data not shown) have indicated that optimal steady state binding conditions are achieved at 24 C following 45 min of incubation using cells from all three groups of animals. The incubations were terminated as described by Gammeltoft and Gliemann (21) by removing 200 fi\ aliquots from the cell suspension and rapidly centrifuging the cells in plastic micro tubes to which 100 /xl of dinonyl phthalate oil has been added. Dinonyl
% intact of incubated insulin % intact of unexposed insulin
x 100
Unexposed [l25I]iodoinsulin is at least 98% precipitable by 10% TCA, 95% adsorbable to talc, and 94% precipitable with excess antiinsulin antibody for up to 3 weeks after preparation. Calculations. Specific [125I]iodoinsulin binding was calculated by subtracting the amount of [125I]iodoinsulin nonspecifically bound from the total amount of [125I]iodoinsulin bound at each insulin concentration (2,6,10,25). Nonspecific
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binding is defined as the amount of [1S5I]iodoinsulin remaining "bound" in the presence of a large excess—200 /xg/ml (3 x 10~5M)—of unlabeled insulin (2,6,10,25). Apparent binding affinity was estimated by the use of Scatchard plots (26). As recently reviewed by Kahn et al. (27), this method of analysis relies upon a number of currently unproved assumptions, and the interpretation of the resulting curvilinear Scatchard plots is highly complex. Nonetheless, these plots are still useful for comparing average binding affinities and are used for this purpose. Binding capacity (number of insulin receptor sites per cell) can be calculated by determining the intercept on the abscissa of the Scatchard plot, and, again, this method remains useful for comparative purposes. However, because the terminal points of the Scatchard plot are obtained at very high insulin concentrations, and because non-specific binding is proportionately high and hard to determine at these concentrations, it is difficult to accurately define the precise point of intercept. Consequently, we have used as an operational definition of numbers of receptor sites per cell the number of 125
1
TABLE 1. Characteristics of [ I]iodoinsulin eluted from adipocytes
[12iI]iodoinsulin
Fresh
Eluted from cells
% precipitable by 10% TCA
99
90
% absorption to talc
96
87
% precipitable by excess antiinsulin antibody
95
88
Specific binding to liver plasma membranes2 (pmol x 10~2/200 fig membrane protein) 1
Endo • 1975 Vol 96 • No 6
OLEFSKY AND REAVEN
1488
l25
8.2 9
7.6
Adipocytes and [ I]iodoinsulin (10~ M) are incubated for 45 min at 24 C. Cells are then removed, washed twice with cold buffer (4 C) and then resuspended and incubated in fresh in&ulin-free buffer at 37 C for 60 min. The mixture is then centrifuged, and the infranate (containing 125 the eluted [l25I]iodoinsulin) is saved. The eluted [ I]iodoinsulin is then compared to equivalent amounts of fresh [125I]:iodoinsulin for its ability to precipitate with 10% TCA, adsorb to talc, precipitate with excess anti-insulin antibody, and specifically bind to liver plasma membranes. 2 Prepared according to the method of Neville et al. (31),
insulin molecules specifically bound at an insulin concentration of 100 ng/ml (2400 /u,U/ml or 1.7 x 10"8M). The rationale behind this approach is that if a receptor does not bind insulin at a concentration of 100 ng/ml (higher than can be achieved in the rat circulation) then it is probably not functioning physiologically as a receptor in vivo. Furthermore, our data show (25, and results section) that approximately 75% of all receptors are filled at this insulin concentration, and the number of molecules bound at this insulin concentration can be accurately measured with an intra-assay coefficient of variation of 5%. Thus, we believe that this measurement provides an accurate representation of receptor site concentration and is useful for comparative purposes. Nevertheless, we realize that 100 ng/ml is a somewhat arbitrary cutoff, and the same argument could be applied to lower insulin concentrations. Thus, this measurement is suggested only for comparative purposes, and should not be interpreted as having precise physico-chemical meaning.
Results
7. Nature of the bound insulin. Previous studies have indicated that essentially all of the insulin binding activity of adipocytes can be recovered in the particulate, nonnuclear cell fraction (28,29), and that treatment of intact fat cells with trypsin abolishes their insulin binding capacity (28-30). Thus, essentially all of the cell associated [125I]iodoinsulin is bound to the plasma membrane. To see if the bound [125I]iodoinsulin could be eluted from the cells, [125I]iodoinsulin and adipocytes were incubated for 45 min at which time an excess of unlabelled insulin (3 x 10~5M) was added. Dissociation of the bound [125I]iodoinsulin was measured over the subsequent 120 min. 50% of the previously bound [125I]iodoinsulin dissociates from the cells in 25 min, and greater than 90% is dissociated by 120 min. The nature of previously bound insulin was further characterized as described in Table 1. The data in Table 1 indicate that the insulin which had been bound to receptors retains most of its immunological activity, and that its ability to precipitate with TCA and
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INSULIN BINDING TO ADIPOCYTES
1489
pared to adipocytes from group 1 animals. On the other hand, increasing concentrations of unlabeled insulin appear equally capable of competing for binding with [125I]iodoinsulin (0.2 ng/ml) using cells from all three groups (IB). For example, 1 //. Insulin binding to adipocytes from rats ng/ml (24 /xU/ml) of unlabeled insulin 125 of different weights. Figure 1A sum- inhibits 16-17% of the [ I]iodoinsulin marizes the ability of adipocytes from 3 binding in all groups, and binding can be groups of rats to bind insulin. Animals from 50% inhibited at an insulin concentration ng/ml. At 100 ng/ml 77-80% of Group 1 (n = 16) weighed 160-220 g, of 10-11 125 group 2 (n = 19) weighed 250-400 g, and the [ I]iodoinsulin binding is inhibited. group 3 (n = 10) weighed > 400 g. It can Nonspecific binding averaged 5% of the be seen that cells from fatter rats (groups 2 total binding (corrected for "trapped and 3) bind only 50-60% as much insulin buffer," Table 2) and all the data in Fig. 1 at each insulin concentration when com- are corrected for this factor (see legend). adsorb to talc was comparable to fresh [125I]iodoinsulin. Furthermore, the eluted [125I]iodoinsulin specifically bound to liver plasma membrane receptors 93% as well as fresh [125I]iodoinsulin.
FIG. 1A (upper). Relationship between insulin concentration and the amount of insulin specifically bound per cell. Group 1 (•—•) consisted of 16 rats whose weights ranged from 160-220 g, group 2 (o—o) 19 rats whose weights ranged from 250-400 g and group 3 (•—•) 10 rats whose weights were >400 g. FIG. IB (lower). Inhibiting effect of unlabelled pork insulin on
[
l25
500-
300
200
I]iodoinsulin binding to
adipocytes. Incubations are carried out at 24 C for 45 min in the presence of 0.2 ng [125I]iodoinsulin. Brackets represent ±SE. Data represent the mean of 16 separate studies for group 1 (•—•), 19 for group 2 (o—o) and 10 for group 3 (•—•). All data are corrected for nonspecific binding by subtracting the amount of [125I]iodoinsulin remaining "bound" in the presence of 200 /xg/ml insulin from the amount of [l25I]iodoinsulin bound at all other insulin concentrations (2,6,10,25). Binding is a linear function of cell concentration up to 3.5 x 10* cells/ml, and all experiments were normalized to 2 x 10s cells/ml (average cell concentration used in these studies). B/F is the bound to free [l25I]iodoinsulin ratio.
2.0-
1000 Insulin concentration (ng/ml)
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OLEFSKY AND REAVEN
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TABLE 2. Total binding and nonspecific binding of [l25I]iodoinsulin to adipocytes from rats of varying weights (± SE)
% of total [lnI]iodoinsulin bound' % of total ['"Ijiodoinsulin nonspecifically bound* % of total [lt5r]iodoinsulin "trapped" in cell layer
Group I (160-220 g)
Group 2 (250-400 g)
Group 3 (>400 g)
2.20 ± 0.12
1.31 ± 0.10
1.28 ± 0.09
0.21 ± 0.01 (0.17-0.26)
0.20 ± 0.01 (0.16-0.27)
0.22 ± 0.01 (0.16-0.30)
0.11 ± 0.01 (0.09-0.12)
0.13 + 0.01 (0.10-0.14)
0.15 ±0.02 (0.12-0.16)
1 This represents the total per cent of the ['"Ijiodoinsulin bound at an insulin concentration of 0.2 ng/ml. The data are expressed per 2 x 105 cells/ml which is the average cell concentration used. "This represents the amount of ['"Ijiodoinsulin remaining in the cell layer in the presence of 200 /xg/ml of unlabeled insulin, divided by the total amount of ['"Ijiodoinsulin available. In these studies the amount of nonspecifically bound insulin accounted for by "trapped" buffer was 0.11% for group 1, 0.13% for group 2, and 0.15% for group 3. Thus the amount of nonspecifically bound [l25I]iodoinsulin net accounted for by trapped buffer was 0.10% for group 1, 0.07% for group 2, and 0.07% for group 3. Although the unaccounted for nonspec fie binding decreases as the rats get larger, since the total ['"Ijiodoinsulin binding also decreases as rats get larger, the proportion of the ['"Ijiodoinsulin bound which is nonspecifically bound remains the same: i.e., J>%. Specific ['"Ijiodoinsulin binding represents the difference between to:al and nonspecific binding.
The similarity of the shapes of the curves of Fig. IB indicates that the ability of unlabeled insulin to inhibit [125I]iod'.oinsulin binding is similar (10). Since the ability of unlabeled insulin to inhibit the
Endo • 1975 Vol 96 • No 6
[125I]iodoinsulin binding is a reflection of the binding affinity, the inhibition curves in Fig. IB suggest that the affinity of adipocytes for insulin is the same for all 3 groups. Thus, the decreased insulin binding to cells from rats of groups 2 and 3 is primarily due to decreased numbers of available receptor sites per cell. The idea is further supported by Fig. 2 which shows the Scatchard plots derived from the data in Fig. 1. When the bound/ free ratio is plotted as a function of bound hormone, similarly shaped curvilinear plots are obtained for all three groups. This type of curve can reflect a constantly decreasing overall affinity of the unoccupied insulin receptors as the per cent occupied receptors increases (negative cooperativity (32)), two heterogeneous insulin receptor populations of high and low affinity (27), or a combination of both of the above phenomenon. Regardless of which explanation holds, the slope of the plot over any given range represents the apparent affinity of the receptor population for that degree of occupancy. Fitting the data to two linear components over the same range of insulin concentrations and per cent receptor occupancies yields an average high affinity constant of 1.9 x 10~!)M and an average low affinity constant of 1 x 10~8M for all
FIG. 2. Scatchard plots of the data in Fig. IB. The K ^ of the low affinity site is obtained from the slope of the least squares straight line drawn to fit the data obtained at insulin concentrations >10 ng/ml. The contribution of this line is then subtracted from the points obtained at lower insulin concentrations and a second least squares straight line is drawn to fit the resultant points. The K ^ of the high affinity site is obtained from the slope of this latter line. .02 .06 .1
2
.3
.4
.5
Insulin bound per cell (p moles x
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INSULIN BINDING TO ADIPOCYTES
groups. These apparent affinity constants are calculated for comparative purposes, and are not to be interpreted as having precise physico-chemical meaning. The intercept on the abscissa of the Scatchard plot approximates the total receptor site concentration, and it is clear that the decrease in insulin binding to adipocytes from group 2 and 3 animals can be primarily accounted for by a decreased number of receptor sites per cell. Numbers of receptor sites per cell can be more accurately compared by following the approach outlined in the methods section. Thus, with this approach, the average number of available receptor sites per cell is 23 ± 1.5 x 104 sites per cell (mean ± SE) for group 1, 14 ± 1 x 104 sites per cell for group 2, and 12 ± 1 x 104 sites per cell for group 3. These differences in insulin binding are not due to differences in insulin degradation. Insulin degradation was studied (see Materials and Methods) and 91-93% of the insulin was still intact following 45 min of incubation at 24 C using an initial insulin concentration of 0.2 ng/ml. At higher insulin concentrations the percent insulin degradation is even less (21,24 and unpublished observations). No differences in the ability of adipocytes to degrade insulin were observed using cells from all three groups of rats. HI. Effect of age versus obesity on insulin binding. It should be noted that while striking differences in insulin binding were noted between group 1 versus groups 2 and 3, no differences were observed between groups 2 and 3 themselves. This is more clearly demonstrated in Fig. 3A which gives the relationship between the amount of insulin bound per cell and body weight, using the individual data obtained from each animal. As can be seen, the ability of adipocytes to bind [125I]iodoinsulin (0.2 ng/ml) steadily decreases as rats get larger from 160 g to about 300 g. Once 300 g of weight is reached it appears that no further decline in the ability of adipocytes to bind
1491
insulin occurs. Since these animals were allowed free access to food, weight and age increased together. Consequently, as seen in Fig. 3B, the relationship between age and insulin binding to adipocytes is essentially the same as in Fig. 3A. In an attempt to dissociate the influence of age from obesity on insulin binding to adipocytes we have carried out a number of caloric manipulations as described below. Fasting studies. In these studies a group of rats whose initial weights averaged 312 ± 5 g (range 300-336 g) were fasted in groups of 4 for 24, 48, and 72 h. These animals lost an average of 17 g of weight at 24 h of fasting, 32 g by 48 h, and 52 g by 72 h. Eight AM plasma insulin levels fell from 42 ± 3 /xU/ml to 5 ± 1 /LtU/ml by 24 h, 5 ± 1 /xU/ml by 48 h, and 3 ± 1 /u,U/ml by 72 h of fasting. The ability of adipocytes from these groups of animals to bind insulin is seen in Fig. 4A. The bars on the left side of the figure represent the amount of insulin bound at an insulin concentration of 0.2 ng/ml while the bars on the right represent the maximal amount of insulin bound (see Materials and Methods) i.e., the amount bound at an insulin concentration of 100 ng/ml. After 24 h of caloric deprivation the amount of insulin bound per adipocyte had almost doubled. Binding remained at this level through 48 h of fasting but declined significantly by 72 h. It should be pointed out that 72-h fasted rats are quite ill-appearing, and this may account for the decline in binding. Complete inhibition studies1 were performed in all of these experiments, and no differences in the ability of unlabeled insulin to inhibit the [125I]iodoinsulin binding were noted, and, thus, for the same reasons as discussed earlier binding affinity did not seem to be changed. Consequently, the increase in insulin binding noted following fasting could be predominately ac1
This means that parallel incubations were performed at all insulin concentrations on cells obtained from each rat as in Fig. 1.
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Endo • 1975 Vol96 • No 6
OLEFSKY AND REAVEN
1492 4.424.08 °o
3.74-
