Effect of aging on the kinetic characteristics of the insulin receptor autophosphorylation in rat...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 296, No. 1, July, pp. 231-238,1992

Effect of Aging on the Kinetic Characteristics of the Insulin Receptor Autophosphorylation in Rat Adipocytes

Pilar Ruiz, Juan A. Pulido,’ Carmen Martinez, Jo& M. Carrascosa, Jorgina Satrtistegui, and Antonio And&s2 Departamento de Biologiix Molecular, Centro de Biologia Molecular, (U. A. M. -C. S. I. C.), Facultad de Ciencias, Ukversidad Auto’noma-de Madrid, 28049 Madrid, Swain

Received November 1,1991, and in revised form March 4,1992

The effect of aging on the insulin binding parameters and on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipose tissue has been in- vestigated. Using solubilized receptors from adipocyte plasma membranes, no significant differences were identified in either affinity or receptor number in adult vs old rats. Time courses for in vitro receptor phosphorylation revealed that both the initial rate of autophosphorylation and the maximal “P incorporation were decreased by 40% in old (24-month) animals as compared to adult (3- month) control rats. The tyrosine phosphatase activity associated with the adipocyte plasma membranes does not account for the decreased kinase activity found in old rats. Insulin sensitivity (measured as the dose of insulin required for 50% maximal stimulation of kinase activity) was similar in both groups of rats. However, the kinase activity showed a decreased responsiveness to the hormone in the old rats. Double reciprocal plot analysis of receptor phosphorylation revealed that the Km for ATP was not modified. In contrast, the insulin- stimulated V,,,, value was decreased by two-fold in 24- month-old rats. The decrease in V,,,, does not appear to be related to an increased basal phosphorylation level on Ser/Thr residues of the C terminus of the receptor &subunit. Thus, we conclude that the reduced insulin receptor kinase activity in adipose tissue from old rats is due, at least in part, to a defect of the intrinsic kinase activity of the insulin receptor. 0 1992 Academic PRESS, IIW.

The decrease in glucose tolerance that occurs with aging has been well documented (1, 2). In both humans and

r Present address: Departamento de Bioquimica y Biologia Molecular, Universidad de Alcall de Henares, 28871 Madrid, Spain.

’ To whom correspondence should be addressed. Fax: l-3974799.

ooo3-9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

animals, aging is associated with insulin resistance at the cellular level. The binding of insulin to its cell surface receptor is the first event in insulin action. In human and rat aging, insulin binding has been reported to be decreased (3-5) or unchanged (6-9). However, even though a decreased insulin receptor number may contribute to alterations in receptor function, it has been proposed that a postbinding defect is the major contribution to the insulin resistance of aging (10).

The binding of insulin to its receptor results in autophosphorylation of the receptor through activation of an intrinsic tyrosine kinase activity (11). In addition, con- siderable evidence suggests that this kinase activity plays an essential role in at least some of the intracellular ac- tions of insulin (12). Activation of the receptor kinase is one of the earliest postbinding events in insulin action so far recognized. Moreover, alterations in the tyrosine kinase activity have been associated with various insulin resistant states (13).

We have previously reported that in 24-month-old rats, both autophosphorylation of the insulin receptor (1R)3 from adipocyte plasma membranes and phosphorylation of an exogenous substrate were significantly reduced as compared to adult (3-month) rats (14). In the present work, we attempt to further investigate the properties of the decreased kinase activity found in old rats. To this end, we have searched for alterations in (a) the insulin sensitivity and/or responsiveness of IR kinase activity, (b) the structure of IR covalently crosslinked to lz51-insulin, and (c) the kinetic properties of IR autophosphorylation in rats of 3 and 24 months of age. Moreover, since protein tyrosine phosphatases are likely to play a role in

a Abbreviations used: IR, insulin receptor; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TPCK, L-l-p-tosylamino-2- phenylethyl chloromethyl ketone.

231

232 RUiZ ET AL.

the control of signal transduction by insulin (15), the contribution of a tyrosine phosphatase activity associated with plasma membranes, influencing the kinase activity in old rats, has also been measured. Finally, since an increase in phosphorylation on serine and/or threonine residues in the P-subunit of the IR by CAMP-dependent protein kinase (PK-A) and/or protein kinase C (PK-C) leads to a reduced tyrosine kinase activity (12, 13), the role of an increased basal Ser/Thr phosphorylation of the C terminus of the receptor P-subunit, as a possible cause of the decreased kinase activity found in old rats, has also been studied. Our results point toward an alteration at insulin receptor autophosphorylation level as a possible cause of the decreased kinase activity in old rats.

MATERIALS AND METHODS

Materials. Porcine insulin (Velosulin) was purchased from Nordisk. Collagenase was from Boehringer Corp. Bovine serum albumin fraction V was from Armour Pharmaceutical Co. Reagents for electrophoresis and phenylmethylsulfonyl fluoride (PMSF) were from Serva. [r-??]ATP (3000 Ci/mmol) and monoiodinated [Al41 “?-insulin (1775 Ci/mmol) were obtained from Amersham. Disuccinimidyl suberate was purchased from Pierce Chemical Co. A synthetic peptide bearing the sequence of the insulin receptor at residues 1314-1324 as numbered by Ullrich et al. (16) was purchased from Peninsula Laboratories. Polyclonal antibodies against this specific sequence were produced as described in (17). All other reagents used were of the highest grade commercially available.

Isolation of fat cells and plasma membrane preparation. Male adult (3 months) and 24-month-old Wistar rats fed ad libitum on a standard laboratory diet and water were used throughout this study. The metabolic characteristics and the mean size of the fat cell of both groups of rats have been previously reported (14). Adipocytes were prepared according to Rodbell (18) in Krebs-Ringer-bicarbonate buffer, pH 7.4, containing 3% bovine serum albumin. Adipocyte plasma membranes were purified in the presence of 1 mM PMSF as previously described by Massague and Czech (19). These membranes were stored as a pellet at -7O’C until they were used. Preparations from adult and old rats, and assays performed in this study, were always conducted in parallel.

Preparation of detergent-solubilized plasma membranes, insulin binding assay, and cross-linking of ‘251-insulin to solubilized insulin receptors. Adipocyte plasma membrane proteins were solubilized in 50 mM Tris-HCI buffer, pH 7.4, containing 1% Triton X-100 and 0.5 mM PMSF for 60 min at 4°C. Insoluble material was removed by centrifugation at 100,OOOg for 60 min. The resulting supernatant was used in receptor binding and phosphorylation experiments. Solubilized insulin receptor (40 pg) was incubated at 4°C for 16 h with ‘l-insulin (20,000 cpm, 64 PM) and varying concentrations of unlabeled insulin (O-160 nM), in a final volume of 0.5 ml of a medium containing 25 mM Tris-HCl, pH 7.4,0.2% BSA, 0.05% Triton X-100. Nonspecific binding was assayed by incubation in the presence of 5 pM unlabeled insulin. Separation of the free and receptor bound insulin was achieved by precipitation with 10% polyethylene glycol/O.5 mg/ml y-globulin and subsequent filtration in GF/C filters. Binding data were analyzed by the Scatchard method and the use of a computer program based on the two binding sites model (20). [A141 ‘261-insulin was cross-linked to solubilized receptors in 50 mM Hepes, pH 7.4, with 0.2 mM disuccinimidyl suberate in dimethyl sulfoxide as described (21). ‘x51-insulin cross-linking to the receptors was prevented in the presence of 1.6 /.cM unlabeled insulin, demonstrating the specificity of the insulin binding to its receptor. Insulin binding was measured prior to cross-linking, and equal insulin binding activities from adult and old rats were used. After addition of electrophoresis sample buffer, the mixture was boiled for 3 min, and samples were sub-

jetted to 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions according to the method of Laemmli (22).

Measurement of ATPase andphsphutuse activities. ATPase activity was measured as described by Freidenberg et al. (23). Protein phosphatase activity was assessed after autophosphorylating insulin receptors with 40 nM ATP/2 &i [y-32P]ATP for 5 min at room temperature (see below). The dephosphorylation reaction was started after addition of unlabeled ATP at a final concentration of 10 mM. At different time intervals al- iquots were taken and the reaction was stopped as described above for the cross-linking experiments. Samples were subjected to 7.5% SDS- PAGE under reducing conditions. Radioactive proteins were identified by autoradiography of the stained and dried gel. Incorporation of 32P into the receptor P-subunit was quantified by densitometric analysis of the film.

Pkospkorylation ussuys. In all experiments equal numbers of receptors from 3- and 24-month-old rats, determined by binding assays, were used. Unless otherwise indicated autophosphorylation reactions were done as follows. Solubilized insulin receptors were preincubated for 10 min at room temperature in the absence or presence of 10m7 M insulin. The phosphorylation reaction was carried out in the presence of 12 mM MgC12, 4 mM MnC&, 25 mM Tris-HCl, pH 7.4, 40 PM ATP/2 PCi [y- 32P]ATP. After 2 min incubation the reaction was stopped by the addition of electrophoresis sample buffer and heating to 95°C for 3 min, and samples were subjected to 7.5% SDS-PAGE under reducing conditions. Radioactive proteins were identified by autoradiography and incorporation of 3zP into the receptor o-subunit was quantified by densitometric analysis of the film or by scintillation counting. Background was esti- mated by counting an equivalent size area of the gels determined to be free of discrete phosphoproteins. In some experiments, the labeled insulin receptor was immunoprecipitated with anti-receptor antibodies generated toward the C terminus of the P-subunit (residues 1314-1324) or with anti-phosphotyrosine antibodies as previously described (24).

Mild trypsin digestion experiments were performed as described by Goren et al. (25). The solubilized insulin receptors were incubated, prior to the phosphorylation reaction, with 10 pg/ml TPCK-treated trypsin at 22°C for 1 min. The digestion was stopped by adding aprotinin (250 pg/ml final concentration), and the receptors were used for autophosphorylation assays as described above.

Other methods. Protein concentration was determined by a modification of the Lowry procedure (26) using BSA as standard. The reported values are the mean + SE. Statistical comparisons were carried out using unpaired Student’s t test.

RESULTS

Figure 1 illustrates the insulin binding to solubilized receptors from adipocyte plasma membranes from 3- and 24-month-old rats. The Kd and B,, values derived from the Scatchard analysis revealed no significant differences in either affinity or receptor number in adult vs old rats. These results demonstrate that, the insulin binding characteristics of solubilized receptors from adipocyte plasma membranes are not modified with aging, indicating that insulin signal transduction is probably impaired at a postbinding level (7, 8).

Insulin receptor autophosphorylation time courses were performed with solubilized receptors from adult and old rats. The incorporation of 32P into a 95-kDa band, identified as the insulin receptor P-subunit by virtue of its immunoprecipitability with rabbit polyclonal anti-insulin receptor antibodies generated toward the C terminus of

KINETIC ANALYSIS OF THE INSULIN RECEPTOR KINASE IN AGING 233

F 1 E

t m

0.0 - ti \ A O6 k 0.4 \

0.2 - +\

I -==2=-&

I I 1000 2000 3000

I3 (fmol/mgl

FIG. 1. Scatchard analysis of insulin binding to solubilized receptors from adipocyte plasma membranes. Insulin receptors from adult (3 months) (0) and old (24 months) (A) rats were incubated for 16 h at 4’C with ‘*‘I-insulin (20,000 cpm, 64 PM) and varying concentrations of unlabeled insulin as described under Materials and Methods. The data shown are from a representative experiment, reproduced three times. Each point is the mean of triplicate determinations.

the P-subunit (residues 1314-1324) (data not shown), was quantified by scanning densitometry of the resulting autoradiographs and the results are shown in Fig. 2. Both initial rate of autophosphorylation and maximal 32P incorporation into the insulin receptor P-subunit from old rats were decreased. Moreover, the 40% decrease in phosphorylation at all time points assayed in the old rats (Fig. 2) did not appear to be due to contaminating ATPase activity since the ATP hydrolysis under the assay conditions was minimal and values from adult and old rats were comparable, with 11 and 8% hydrolysis at 10 min,

2 5 10 15

Time (min)

FIG. 2. Time course of autophosphorylation of solubilized insulin receptors from adipocyte plasma membrane. Equal binding activities derived from adult (0) and old (A) rats were preincubated with lo-’ M

insulin for 10 min at room temperature and phosphorylated for various periods of time under the conditions described under Materials and Methods. After electrophoresis and autoradiography, the intensity of the band corresponding to the b-subunit was measured by scanning densitometry. Values are expressed as percentages of the maximal a2P incorporation and represent the means +_ SE of four separate experiments, each assayed in duplicate.

” ,Ot.----l b 10 20 30

Time tmin)

FIG. 3. Protein tyrosine phosphatase activity in solubilized plasma membrane receptor preparations. Phosphatase activity in receptor preparations from adult (0) and old (A) rats was assayed as described under Materials and Methods. The level of phosphorylation of the p- subunit band, measured by scanning densitometry, before the addition of 10 mM unlabeled ATP was taken as 100% and the values found after addition of unlabeled ATP are expressed as a percentage of this value. The data represent the mean + SE of four separate experiments.

respectively. Similar findings have been reported in receptor preparations from other tissues (27).

The decreased kinase activity found in old rats (Fig. 2) could be explained by the presence in these preparations of a protein tyrosine phosphatase with higher activity than in control adult rats. To investigate this possibility, the rate of dephosphorylation of the insulin receptor was assessed as indicated under Materials and Methods. As can be seen (Fig. 3) the rate of loss of 32P from the insulin receptor P-subunits was identical in adult and old rats. The extent of dephosphorylation is similar to previously published results in wheat germ agglutinin-purified receptors from rat liver (28) and Fao hepatoma cells (29).

1 2 --- ---- - . rbdr xwi3 I ‘.” .. ‘-

-205

-116 - 97

- 66

- 45

- 29

insulin ml 0 .I6 1.6 16 160 0 -16 1.6 16 160

FIG. 4. Autoradiograph showing insulin dose response of autophosphorylation of solubilized plasma membrane proteins of fat cells from adult (1) and old (2) rats. Equal binding activities from both receptor preparations were used. Insulin receptors were preincubated in the presence and absence of increasing concentrations of insulin for 10 min at room temperature. The kinase assay was performed as described under Materials and Methods. The figure shows a representative experiment, reproduced three times.

234 RUiZ ET AL.

.16 1.6 16 160

Insulin Concentration ( nM 1

FIG. 5. Dose-response curves of insulin action on receptor autophosphorylation in adult (0) and old (A) rats. The method is as described in the legend to in Fig. 4. Dose-response curves for stimulation of the p-subunit band were determined by scanning densitometry. A, results were expressed as percentage of maximal insulin effect in each case. B, values were represented as percentage of maximal 32P incorporation observed in receptors from adult rats. The data represent the mean + SE of three separate experiments.

Insulin receptor autophosphorylation was measured over a range of insulin concentrations (Fig. 4). The incorporation of 32P into the 95-kDa band was quantified by scanning densitometry of the resulting autoradiographs. As shown in Fig. 5A, when we express the results as percentage of maximal insulin effect in each group of rats, dose-response curves for receptors derived from adult and old rats appear to be superimposable, with half-maximal autophosphorylation occurring at 2 X lo-’ M insulin in receptors from both groups of rats (Fig. 5A). This value is in agreement with previously published data from liver and muscle rat tissues (24,30). However, when values are represented as percentage of maximal 32P incorporation, there was approximately 32% less 32P incorporated into the insulin receptor @-subunit derived from old (24- month) as compared to adult (3-month) rats (the range observed in numerous experiments was from 19 to 59%) (Fig. 5B). Similar results have been obtained when the insulin dose-response experiments were done in the presence of 1 mM orthovanadate (data not shown). These data demonstrate that the decrease in insulin-induced receptor autophosphorylation in old rats is due to a decrease in maximal insulin responsiveness and not to a change in insulin sensitivity.

Since insulin receptor kinase activity was always measured using the same amount of solubilized insulin receptors determined by insulin binding activity, we tested whether a structural modification of the insulin receptor from old rats could explain the results of autophosphorylation. To this end, ‘251-insulin was covalently cross- linked to the solubilized insulin receptors from both groups of rats, and the labeled receptors were subjected to sodium dodecyl sulfate electrophoresis in the absence or presence of a reducing agent. The results, shown in Fig. 6, demonstrate the presence of a band specifically labeled under nonreducing conditions corresponding to the molecular weight of the intact insulin receptor (~r2/32) heterodimer. No bands corresponding to the molecular weights of smaller receptor oligomers (19) or free a-subunit were detected. Free a-subunits were seen (Fig. 6) only upon addition of a reducing agent. Quantitation by scanning densitometry of the intensity of the labeled receptor band from three different preparations revealed no significant differences between receptors derived from adult and old rats (data not shown). Furthermore, the absence of smaller receptor oligomers in old rats may suggest that the decreased kinase activity found in these animals was not due to receptor degradation that could lead to the formation of a low-molecular-mass fragment with lower or no kinase activity.

To study if the cause for the decreased autophosphorylation of the receptor from 24- as compared to 3-month- old rats might reflect changes in the kinetic constants of the receptor kinase, we analyzed the effects of different ATP concentrations on the rate of autophosphorylation; 2-min phosphorylation intervals were used to measure

Insulin -+ -+ +- P-ME ,: - - -- ++

FIG. 6. Cross-linking of ‘=I-insulin to solubilized insulin receptors. Solubilized plasma membranes of fat cells from adult (1) and old (2) rats were incubated with ‘*‘I-insulin (150,000 cpm, 0.48 nM) for 16 h at 4°C in the absence (-) and presence (+) of 1.6 pM unlabeled insulin. After addition of dissuccinimidyl suberate for 15 min at 4”C, the reaction was stopped’ as described under Materials and Methods. Samples were subjected to 5% SDS-PAGE under reducing (f/3-mercaptoethanol) or nonreducing (-fi-mercaptoethanol) conditions. The autoradiograph shows a representative experiment, reproduced three times.


initial rates. Double reciprocal plots of the tyrosine kinase activity in the absence and presence of insulin are shown in Fig. 7. The K,,, for ATP and V,,, values derived from the double reciprocal plots are presented in Table I. As can be seen, insulin caused an increase in the V,,,,, of both receptor preparations, whereas the K,,, of the receptor kinase for ATP was not modified by the hormone and did not change with aging. The insulin stimulation of the receptor autophosphorylation was twofold lower in receptors derived from old (24-month) as compared to adult (3-month) rats, and the V,,, in old rats was also 40% lower. Moreover, after basal activity was subtracted from the activity present with insulin to get the “insulin-stimulated” component, double reciprocal analysis of this activity indicated a 55% reduction in the V,,, of the receptors from old rats (Table I). We have obtained similar results when using higher ATP concentrations (up to 160 PM ATP) for the kinetic experiments showing that the defect found in the insulin receptor kinase activity in aged rats is not due to a change in its affinity for ATP (data not shown). Thus, these results demonstrate that the decrease in the insulin receptor kinase activity in old rats is entirely attributed to a decrease in the V,,,,, of the enzyme.

To determine if the decrease in V,,, for receptor autophosphorylation in the 24-month-old rats could be explained by an increase in the basal phosphorylation level on serine and/or threonine residues of the C terminus of the receptor P-subunit, mild trypsin digestion experiments were performed (25). Solubilized insulin receptors were

TABLE I

Kinetic Characteristics of Insulin Receptor Autophosphorylation from Rat Adipocytes

Age (months)

V mar (fmol 32P/fmol binding. 2’) K, ATP (PM)

Ins- -Ins +Ins stimulated Ins +Ins

3 0.5 k 0.12 2.5 IL 0.19 1.81 t 0.16 1: +- 4 28 i 5 24 0.7 + 0.03 1.6 t 0.04 0.79 I? 0.07 14 It 2 23 + 6

NS P < 0.05 P < 0.0125 NS NS

Note. V,,,, and Km values for insulin receptor autophosphorylation were obtained after double reciprocal plot analysis as described in the legend to Fig. 7. The difference between the activity found in the presence of 1O-7 M insulin and basal phosphorylation, at each ATP concentration, was plotted to obtain the insulin-stimulated component. The results are the mean + SE of seven to nine different experiments.

preincubated with trypsin prior to the autophosphorylation reaction in order to remove a lo-kDa fragment of the C terminus of the insulin receptor ,&subunit. This fragment contains threonine-1336, recently identified as a phosphorylation site by protein kinase C, and some of the serine sites of phosphorylation (31). As shown in Fig. 8, trypsin-digested P-subunits had a molecular weight of 85 kDa on SDS gels. After removal of the lo-kDa fragment, autophosphorylation of the insulin receptor from 24-month-old rats was found to be reduced by 44 t 5% (n = 3) as compared to that found in 3-month-old rats.

LATH CAM 40 30 20 10 95k icl -

I 8

/I /I I I / I/--- l I I

0.05 0.10 0.05 010

I/ [ATP] (oM)-’

FIG. ‘7. Double reciprocal plot analysis of the insulin receptor autophosphorylation. Insulin receptors from adult (A) and old (B) rats were preincubated in the absence (open symbols) or presence of lo-? M insulin (dark symbols), and the kinase activity was assayed as indicated under Materials and Methods. Following the addition of [y-32P]ATP, the specific activity of [~J-~‘P]ATP was kept constant at all ATP concentrations (range from 10 to 40 PM). After electrophoresis and autoradiography, bands corresponding to the S-subunit were excised from the dried gels and counted. The results are the mean +_ SE of seven to nine separate experiments. Inset: autoradiographs of a representative experiment of insulin- stimulated receptor phosphorylation at different concentrations of ATP in adult (A) and old (B) rats.

236 RUiZ ET AL.

TRYPSIN l-=-l&

c95K +95K

a b a b

FIG. 8. Autoradiograph showing insulin-stimulated receptor phosphorylation from adult (a) and old (b) rats before and after mild trypsin digestion. Prior to the autophosphorylation reaction, equal amounts of solubilized insulin receptors from both groups of rats were digested (+) or not (-) with TPCK-treated trypsin (10 ag/ml) for 1 min at 22’C and trypsinisation was stopped by addition of aprotinin. Then, after prein- cubation with 1O-7 M insulin, the kinase activity was assayed as indicated under Materials and Methods. The insulin receptors were immunoprecipitated with anti-phosphotyrosine antibodies. The figure shows a representative experiment, reproduced three times.

Before trypsinization, the decrease in autophosphorylation in receptors derived from old rats was 37 f 2% (n = 3) with respect to adults. Thus, since the difference in the insulin receptor autophosphorylation remains essentially the same before or after trypsin treatment, these results suggest that basal Ser/Thr phosphorylation of the C terminus of the insulin receptor P-subunit could not be the cause of the decreased insulin receptor kinase activity found in old rats.

DISCUSSION

The findings reported here indicate that there is no age-related change in the insulin binding characteristics in solubilized receptors from adipocyte plasma membranes. Moreover, the structural studies using insulin cross-linked receptors have shown no appreciable differences in the electrophoretic migration of the affinity-labeled receptors between adult and old rats (Fig. 6) and indicate that the molecular structure of the insulin receptor is not grossly modified during the process of aging. Taken together, these results indicate that the insulin resistance of rat aging seems to be the consequence of an alteration of the hormone signaling mechanism at a postbinding level (10).

The reduced insulin receptor autophosphorylation that we found in old rats at all time points assayed (Fig. 2) could be due to the action of a protein tyrosine phosphatase with higher activity in these animals than in adult rats. However, our results indicate that the alterations in insulin receptor 32P incorporation in the old rats are not due to the tyrosine phosphatase activity associated with adipocyte plasma membranes since the rate of dephos- phosphorylation of the insulin receptor P-subunit from

3- and 24-month-ok&s rats was identical (Fig. 3), and under the conditions used to measure autophosphorylation, 2- min intervals, the phosphate released was less than 5% in all insulin receptor preparations. Thus, the activity of this enzyme does not account for the decreased autophosphorylation of the insulin receptor found in old rats.

Analysis of the insulin dose-response curves and the half-maximal stimulating insulin concentration suggest that the diminished kinase activity in receptor preparations from adipocyte plasma membranes from old rats is not due to its decreased insulin sensitivity, but rather to a lesser responsiveness to the hormone action. In contrast, Bryer-Ash and Freidenberg (32) found no differences in either insulin sensitivity or insulin responsiveness on insulin receptor kinase activity from rat liver membranes in old rats. However, others have presented evidence for a reduced kinase activity in insulin receptors isolated from old rats in animals previously injected with the hormone, showing an impaired responsiveness in the hepatic insulin receptor kinase activity at maximal stimulating concentrations of insulin as compared to hormone-treated adult rats (33). In addition, a decrease in kinase activity in solubilized receptors from rat liver in old rats has been also reported, although it is not clear if these results are found after an hyperglycaemic stimulus to the old rats (9). Taken together, these results suggest that the intrinsic kinase activity of hepatic insulin receptor from old rats is reduced after an in uivo insulin activation, and normal as compared to adult in untreated old rats. However, our findings show a reduced insulin receptor kinase activity in adipocyte plasma membranes from untreated old rats, and similar results have been recently reported in insulin receptors from skeletal muscle derived from 20-month-old rats (34). This discrepancy may suggest the existence of tissue-specific differences for insulin responsiveness of the insulin receptor kinase activity as has been previously reported (30, 35).

The kinetic studies provide some insight into the mechanism responsible for the inhibition of kinase activity in receptors from old rats. As shown in Table I, the apparent Km for ATP was not modified during the process of aging. However, we have found a twofold decrease in V,, for receptor autophosphorylation in the old rats. Since the receptor from old rats shows no change in the insulin binding characteristics and the molecular structure of the receptor is not grossly modified during the process of aging, the decrease in V,,,,, for receptor autophosphorylation may reflect a defect in the coupling between insulin binding and receptor autophosphorylation. Therefore if, as discussed in (36, 37), the P-subunit undergo conformational changes during autophosphorylation, it might be speculated that in receptor derived from old rats this conformational change is somehow altered, and as a result (a) tyrosine phosphorylation site(s) is/are impeded from incorporating phosphate. In fact, we have previously re-


ported that the phosphotyrosine content of the insulin receptor P-subunit is markedly lower in 24- than in 3- month-old rats (14).

Finally, in several experimental models of insulin resistance has been suggested that serine phosphorylation of the insulin receptor P-subunit appears to be responsible for the alteration in the kinetic parameters of receptor autophosphorylation (38-40). In addition, Karasik et al. (41) have shown that in liver of rats undergoing starva- tion, an insulin resistant state, an increase in basal phosphorylation on Ser/Thr residues of the C terminus of the insulin receptor P-subunit, was associated to a decreased autophosphorylation and function of the insulin receptor kinase. In that report (41), removal of a lo-kDa fragment of the C terminus of the P-subunit receptor from starved animals led to a normalization or equalization of insulin- stimulated autophosphorylation of the receptor as compared to fed control rats. In contrast, the experiments in the present study show that removal of the lo-kDa fragment of the C terminus of the receptor ,&subunit from old rats did not lead to an equalization of insulin-stimulated autophosphorylation as compared to adult trypsin- treated receptors (Fig. 8). Therefore, our data might indicate that the type of alterations of the insulin receptor kinase may be different in distinct models of insulin resistance.

Although the experiment in Fig. 8 does not exclude the possibility that other inhibitory serine phosphorylation sites, unidentified to date (42), might be still present in the 85kDa fragment of the insulin receptor P-subunit, our results suggest that the decreased autophosphorylation of the insulin receptor from 24-month-old rats is un- likely to be due to increased basal phosphorylation on Ser/Thr residues of the C terminus of the receptor p- subunit. This suggestion is further supported by data recently reported by Anderson and Olefsky (43) showing that, in phorbol ester-treated Rat-l fibroblasts expressing human insulin receptors, phosphorylation on Ser/Thr residues of the C terminus of the receptor P-subunit is not necessary for the TPA/PKC-mediated inhibition of insulin receptor autophosphorylation.

In conclusion, we have demonstrated that the reduced insulin receptor kinase activity in old rats resulted from a twofold decrease in V,,, for receptor autophosphorylation. Moreover, we present evidence suggesting that this inhibition does not seem to be related either to a higher dephosphorylation rate caused by a protein tyrosine phosphatase activity or to an increased basal Ser/Thr phosphorylation of the C terminus of the insulin receptor P-subunit. Taken together, our results suggest that a defect may exist in the intrinsic kinase activity in solubilized receptors derived from adipocyte plasma membranes from old rats. Further studies will need to be performed to identify the mechanism(s) leading to the decrease in the

intrinsic kinase activity of the insulin receptor in adipocytes from old rats.

ACKNOWLEDGMENTS

We are grateful to Drs. A. Martinez-Serrano and J. Vazquez for helping in the computer analysis of the binding data. This work was supported by Grant PM88-0008 from the Direction General de Investigacibn Cientifica y Tecnica (DGICYT), Spain. The Centro de Biologia Molec- ular is recipient of an institutional grant from the Ramon Areces Foun- dation. P. Ruiz and C. Martinez are recipients of predoctoral fellowships from Ministerio de Education y Ciencia, Spain.

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