The Relationship between the Insulin-binding Capacity of Fat Cells and the Cellular Response to Insulin

SUMMARY The effects of trypsin on the insulin-binding capacity of fat cells were studied with [12jI]iodoinsulin, which was shown to be a valid tracer of native insulin. The binding of insulin to isolated fat cells was approximately 5 microunits/lOO mg when the concentration of the hormone in the incubation medium was 100 microunits per ml. The initial step of the insulin receptor interaction followed the law of mass action. When the cells were exposed to trypsin (1 mg per ml) for 15 set and for 15 min, the binding capacity was reduced by more than 80 and 98%, respectively. Upon subsequent incubation of trypsin-treated cells for 2 hours after inactivation of the enzyme, the binding capacity was partly restored. How-ever, the maximum binding capacity of “recovered” cells was only 9 and 4 microunits/lOO mg (depending upon the length of the initial trypsin treatment) while that of untreated cells was 62 microunits/ mg. The apparent dissociation constant of the insulin receptor system (approximately I milliunit per ml or 7 XIM) was not significantly altered by the above treatment. in recovered cells was less sensitive to insulin as compared with that of untreated cells; however, the metabolism in all cell preparations responded normally to higher concentrations of insulin when more than 1.5 microunits


SUMMARY
The effects of trypsin on the insulin-binding capacity of fat cells were studied with [12jI]iodoinsulin, which was shown to be a valid tracer of native insulin.
The binding of insulin to isolated fat cells was approximately 5 microunits/lOO mg when the concentration of the hormone in the incubation medium was 100 microunits per ml. The initial step of the insulin receptor interaction followed the law of mass action. When the cells were exposed to trypsin (1 mg per ml) for 15 set and for 15 min, the binding capacity was reduced by more than 80 and 98%, respectively.
Upon subsequent incubation of trypsin-treated cells for 2 hours after inactivation of the enzyme, the binding capacity was partly restored.
However, the maximum binding capacity of "recovered" cells was only 9 and 4 microunits/lOO mg (depending upon the length of the initial trypsin treatment) while that of untreated cells was 62 microunits/ mg. The apparent dissociation constant of the insulin receptor system (approximately I milliunit per ml or 7 XIM) was not significantly altered by the above treatment. Glucose metabolism in recovered cells was less sensitive to insulin as compared with that of untreated cells; however, the metabolism in all cell preparations responded normally to higher concentrations of insulin when more than 1.5 microunits of insulin were bound to 100 mg of fat cells.
This indicates that glucose metabolism in intact fat cells is stimulated maximally when only approximately 2.4% of the total insulin receptors on the cells are occupied by insulin.
Since the binding follows the law of mass action, it appears that the presence of "excess" receptors on fat cells renders the cells highly sensitive to insulin.
Upon incubat.ion with fat cells, trypsin initially mimics the effects of insulin on cellular metabolism but subsequently renders the cells unresponsive to both insulin and trypsin (1,2). Our previous data suggested that these effects of trypsin were induced upon interaction of the enzyme with a certain cellular element, * This work was supported by United State Public Health Service Grants It01 AR1[ 06725 and 074WARIP.
which was possibly the insulin receptor (1,2) t.hat is generally considered to be located on t>he cell surface (3). Accordingly, the present work was initiated to examine whether trypsin modifies the insulin-binding capacity of fat cells. Previously, the binding of insulin to muscle or adipose tissue cells has been studied with either modified insulin labeled with %, 1311, or lz51 (4-ll), or native unlabeled insulin (12,13). In the latter case, the uptake of insulin was determined by measuring the changes in t'he horrnone concentration in the incubation medium.
Since the ch anges in hormone concentration were relatively small as compared wit,h the background, the method was not very sensitive.
Nevertheless, it had a definite advantage over the "tracer" method since the biological activities of insulins modified by labeling were not known with certainty (cf. Reference 13). Consequently, prior to the work presented in this paper, attempts were made to study the effects of trypsin on the binding of native insulin to fat cells. The results were compatible with the assumption that l-rypsin lowered the insulin-binding capacity of fat cells (14,15). In addition, it initially appeared t,hat upon furt,her incubation of trypsin-treated cells for recovery, the cells rapidly regained their insulin-binding capacity (14, 15). However, this observation was not substantiated in subsequent experiments,l although the physiological response to t,he hormone (at 1 milliunit per ml) was restored on all occasions.
In the meantime, separate tests in this laboratory indicated that an iodoinsulin preparation labeled with I261 was a valid tracer of insulin for studyin g the insulin-binding capacity of isolated fat cells. Accordingly, the effects of trypsin on the insulin-binding capacity were re-eyxmined with the [12jI]iodoinsulin preparation as the tracer.
Results of this work are presented in this paper along with cert,ain observations that show the validity of the iodoinsulin prep:rrat,ion as the tracer. A preliminary account of the present work has been published (16).

MATERIALS AND RZETHODS
Iodinated pork insulin labeled with lz51 (approximately 100 PCi per pg) was purchased frorn Cambridge Nuclear Radiopharmaceutical.
According to the manufacturer, most of the iodoinsulin in the preparation was a monoiodo derivative. The biological activity of the preparation in the fat cell system (determined in this laboratory) was approximately 807, of that of native insulin on a weight basis, and approximately 80% of the  a certain amount of radioactivity was found on the filter membrane; the amount was proportional to the dry weight of fat cells (Fig. 1). The amount of radioactivity (per cell) was not significantly lowered w-hen the cells were washed by repeated centrifugation at O-2" (Table I, Experiment A), or when the cells were washed in the cold, kept at a 10~ temperature for 30 to 60 min, and washed again in the cold (Table I, Experiment  U). However, a significant loss of activity was observed when the cells were washed at higher temperatures (  (Table  II). This suggested that almost all radioactive material bound to fat cells was iodoinsulin.
If it was assumed that all the radioactivity was coming from iodoinsulin, the binding of the compound was approximately 5 microunits (as insulin) per 100 mg (dry cells) when the concentration in the incubation medium was 100 microunits per ml (Table II).
The rate of binding of iodoinsulin to fat cells was temperature dependent (Fig. 2). At 37", the reaction proceeded rapidly, but the amouut of binding was decreased after 15 min. This was probably caused by a partial destruction of iodoinsulin by either a fat cell enzyme (19)  In the plotting, however, a few data obtained at extremely high insulin concentrations were rejected since it was observed that a small amount of radioactivity would always bind to fat cells even in the presence of a large dose of cold insulin (see also Figs. 4 and 5).
The amount of this nonspecific binding was 0.05 to 0.07% of the total radioactivity added to the incubation mixture.
Each point in this figure represents the mean value of two observations. ZR, bound insulin (microunits per 100 mg in I ml) ; I, free insulin (milliunits per ml).
at 25" (or under standard conditions for the incubation; see "Materials and Methods") \vas less than 1O7o of the total (the data are not shown), as it might be expected from previous data (12, 13). The rate of reaction was very slow at 0".
EJects of Insulin and Trypsin on Binding-The binding of iodoinsulin to fat cells was greatly inhibited by the addition of cold native insulin (Table III, Experiments 1 and 2), or by preliminary exposure of cells to trypsin for either 15 set or 15 min (Table III, Experiments 3 through 6). The degree of inhibition was greater when the cells were exposed to the enzyme for a longer period (i.e. 15 min).
In contrast, no inhibition was observed when the cells were mised with trypsin and soybean trypsin inhibitor in the reverse order (Table III, Experiment 7). These data were consistent with an interpretation that both iodoinsulin and trypsin interact with the insulin-binding sites of fat cells. As it has been reported previously, trypsin induces insulin-like effects in fat cells during a 15.set treatment (2) and abolishes the responsiveness of the cells to the hormone during n 15.min treatment (1).
When the cells exposed t'o insulin were washed and incubated for 2 hours in an insulin-free medium, the capacity to bind iodoinsulin was restored almost in full (Table III, Experiments 1 and 2). This observation was consist'ent with the data of our physiological experiments (2). In contrast, when the cells treated with trypsin for either 15 set or 15 min were incubated  The experiment was carried out as described in Fig. 4 except t,hat cells were exposed to trypsin for 15 min.
for recovery, only a fraction (a few per cent) of the original binding capacity was restored in 2 hours (Table III, Experiments 3 through 6). These results were unexpected since, according to our physiological experiments, fat cells were able to regain a near normal metabolic activity within a few hours of trypsin treatment (1,2), although the "recovered" cells were not as sensitive to insulin as untreated fat cells (2).
The results presented in Figs. 3 through 5 indicated that the apparent maximal insulin-binding capacity (B,,,) of untreated fat cells was approximately 62 microunits/lOO mg of fat cells (Fig. 3), but those of cells recovered from a 15.set treatment and a 15.min treatment were only approximately 9 and 4 microunits, respectively, per 100 mg (Figs. 4 and 5). In contrast, the apparent dissociation constant (KJ of the insulin receptor   system was not increased at all by the trypsin treatments: the K, values estimated in Figs. 3, 4, and 5 were 1.5, 0.9, and 1.0 milliunits per ml. As a measure of the validity of these estimated figures, amounts of insulin that, would be bound to fat cells at a given hormone concentration were calculated with the B,,, and K, values estimated above. As it call be seen in Table IV (Project A), the calculated values were not. greatly different from the corresponding data that were obtained in separate experiments.
The mean value of the Zi, values estimated above was approsimately 1 milliunit per ml. However, t'he apparent I(, value for the glucose metabolism was only approximately 7 microunits per ml (Fig. 6). The latter observation was in agreement with the results obtained by Crofford (13). It was significant that no break was detected in the "binding" curve in Fig. 6 at a range where the metabolic activity was saturated.

CorreZation between Binding and Physiological
Effects of Insdin -The data shown in Fig. 7 indicated t,hat glucose metabolism in fat cells was stimulated maximally (see arrows in Panel ;1) when the cells had bound a fixed amount of insulin (1.5 microunits/100 mg; see arrows in Panel B) regardless of the type of cell preparation: I, ZZ, or ZZZ (see legend).
In addition, the data in the figure indicated that the necessary amount of insulin (1.5 microunits/lOO mg) was bound to these cell preparations when the hormone concentrations were 26, 170, and 680 microunits per ml. As it can be seen in Table IV (Project B), these values were comparable to those that werr calculated from the B,,, and K, values estimated earlier. Issue of O(Slober 25, 1971 'I'. Kono and F. W. Barham 6215 Pui tiul protection by insulin of iodoinsztlin-bintlinll rapacity of jut The untreated, treated, and "rccovrred" cells were prepared as r~sual, cxccpt (a) that the indicated amounts of inslllilr were added to the reaction mixture 10 min before trypsin treatment and (b) that all t.he cell preparations lvcre extensively washed to remo~ e insulin from the "pills-insulin" preparations prior to the recovery period.
The time for recovery was 2 hours. The bindilrg of iodoinsulin was determined lmder standard conditions. Pnrtinl Protection of Hornlone-binding Sile with Insulin jrom cflect oj Trypsin-When trypsin treatment for 15 set was carried out in the presence of insulin (100 millimGts per ml), the decrease in the binding capacity (as observed in recovered cells) was significantly smaller than the control (Table V). In colltrast, no such protective effect of the hormone was observed when the cells (and insulin) were exposed to the enzyme for 15 min (Table  T'), llresumably because insulin was digested by the enzyme under these conditions (2). DISCUSSION =\lthough the exact physiological ac+ivitics of iodoinsulin have not been established, it has been suggested by Izzo et al. (al), by Garratt (22), and by Freychet, Roth, and Seville (11) that a11 iodoinsulin preparation is a valid tracer of native insulin provided th7.t the compound is not too heavily iodinntcd.
In agreement with this suggestion, it was observed in the present n-ork that a monoiodoinsulin preparation was a valid tracer of insulin in studying the binding of t,he hormone to fat cells. Thus, the binding of the compound was almost entirely blocked upon 1,reliminary treatment of the iodoinsulin preparation with antiinsulin serum, or upon addition of native insulin to the incubation mixture.
Furthermore, it was noted that the binding could be reversed (as are the physiological effects of native insulin) upon washing the loaded cells at room temperature.
These results differ from some of the earlier observatiolls which indicated that the binding of iodoinsulin to fat cells or adipose tissue was only partially blocked by either anti-insulin serum (9) or cold native insulin (11).
The amount of iodoinsulin bound to fat cells was estimated to be approximately 5 microunits (a~ insulin) per 100 mg when it,s concentration was 100 microunits per ml (Tables II and III). Previously, it was reported by Crofford (13) that the amount of native insulin taken up by fat cells \yas approximately 10 microunits/100 mg when the insulin concentration was 100 microunits per ml. However, a smaller value, 4.2 f 0.5 microunits/lOO mg (n = 9), was obtained when the uptake experiment was repeated prior to the work reported in this paper (unpublished,' but a part of the data is in Rcfcrencc 15). Therefore, as a first approximation, it was postulated in the present work that the affinities of fat cells for native and iodinated insulins are equal (Figs. 3, 4, 5, and 7). Although certain errors may have been introduced by this postulate, the effects of the approximation are probably insignificant to the present discussioll. The binding of insulin to fat cells was saturable (Fig. 3). Nevertheless, the physiological effect of 11-1~ hormone on glucose oxidation was saturated when only approximately one-fortieth (1.5 microunits/lOO mg) of the apparent total binding sites (62 microunits/lOO mg) were occupied by the hormone (Figs. 6 and  7). The difference in the apparent R, values for the binding (1 milliunit per ml; Figs. 3 through 5) and for the glucose oxidation (approximately 7 microunits per ml; Fig. 6) mits more than 100-fold, which was too great to be accounted for by a possible difference in the affinities of fat cells for native and iodinated insulins.
In addition, similar results have previously been obtained in studies on the uptake of iodoinsulin by frog muscle (8), rat diaphragm (6, 7), and rat adipose tissue (lo), and of native insulin by isolated fat cells (13).
Because of these observations mentioned above, it was suggested in the past that either (n) there is little correlation between the observed binding and the physiological effects of insulin (6, 9) or (b) only a fraction of the observed binding is "specific" or "active," while the rest is not (7, 8, 10). However, it was noted in the present work that both the binding and metabolic effects of insulin at low physiological (see below) concentrations were greatly affected by the apparent maximal insulin-binding capacity (B,,,) of the cell preparation ( Fig. 7 and Table IV) and that glucose oxidation in fat cells was stimulated in full when a fixed amount of insulin was bound to the cell preparation (Figs. 6 and 7). It appears that these and other observations described in t,his paper are consistent wiiith the interpretation (a) that normal fat cells can generate, or accumulate, a certain intracellular hormonal signal strong enough to stimulate the cellular metabolism when only a fraction of the total insulin receptors are occupied by hormone, and (b) t,hat the cells equipped with a large number of receptors are highly sensitive to insulin since, according to the law of mass action, these cells are able to bind the necessary amount of insulin even when the hormone concent,ration is low. With this interpretation, one can easily explain how the metabolism in recovered cells, which are equipped with a small number of receptors, can be stimulated to a near normal level when the hormone concentration is increased (cj. Fig. 7).
The above argument does not' imply, however, that the individual insulin receptor is inefficient.
The estimated K, value for the binding was approximately 7 IIM (or 1 milliunit per ml), which is very good when compared with the R, values of many enzymes and is roughly comparable to the K, value of (a) the receptor systems for estradiol (0.7 nM) and cstriol (2 nM) of uterus (23, 24) and (b) the glucagon receptor system (approximately 4 nu) of liver (25). Nevertheless, the normal insulin concentration in the blood of rats is probably less than 1 nM (150 microunits per ml; e.g. see Reference 26) and the estimated K, value for glucose utilization in fat cells was only approximately 50 PM (or 7 microunits per ml; Fig. 6). Besides, it has been known that in both fat cells and adipose tissue insulin regulates glucose utilization (13, 27; Fig. 6), lipolysis (2,27,28), and protein synthesis (29, 30) at approximately the same concentration range (roughly 7 to 300 phx or 1 to 50 microunits per ml), although it is also known that isolated cells are somewhat more Al., J. Biol. _ that protein synthesis in fat cells was greatly stimulated when the insulin conceiitration was increased from 50 to 800 microunits per ml (29), but the physiological significance of the observation is questionable.
If the total insulin-binding capacity of fat cells is assumed to be 62 microunit,s/lOO mg as estimated in Fig. 3, one can calculate that this value is roughly equivalent to (a) 4 pmoles of insulin per g of fat cells, or (0) 160,000 receptor sites in a single cell when the mean cell diameter is 50 pm, or (c) 21 receptors per pm2 of the cell surface if t'he receptors are evenly distributed on the surface. Likewise, one can calculate from the present data that glucose metabolism in fat cells is stimulated to one-half of the maximum and t,o the maximum when approximately 1,200 and 4,000 insulin molecules, respectively, are bound to each cell. The latter value is not greably different from that of Crofford and Mincmura, who estimated that both glucose and amino acid metabolism in fat cells arc stimulated maximally when approximately 3,000 insulin molecules are taken up by each cell (15).
Finally, the present data are consistent with our previous hypothesis that trypsin induces insulin-like effects upon interaction (presumably binding) with the insulin receptor site, which is modified by the enzyme upon prolonged incubation (2). The data are also compatible with our theory that fat cells have a capacity to restore, or regenerate, the insulin receptors after trypsin treatment (I, 14, 15) ; however, it is now suggested that the rate of restoration is only a few per cent' per hour (cf. Table  III).
Ac/cnowZedg?,ze1Lts~--We are indebted to Doctors C. R. Park, S. I'. Colowick, and ,J. H. Eston for valuable discussions and help in the preparation of this paper.