Insulin-induced increases in the activity of the spontaneously active and ATP Mg-dependent forms of phosphatase-1 in alloxan-diabetic rat liver

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1 Eur. J. Biochem. 146, (1985) 0 FEBS 1985 Insulin-induced increases in the activity of the spontaneously active and ATP Mg-dependent forms of phosphatase-1 in alloxan-diabetic rat liver Carol J. DRAGLAND-MESERVE, Donald K. WEBSTER and Lynne H. PARKER BOTELHO Sandoz Research Institute, Sandoz, Inc., East Hanover, New Jersey (Received July 9/0ctober 16, 1984) - EJB Liver supernatant from normal and alloxan-diabetic rats was fractionated by DEAE-cellulose chromatography and the separated phosphoprotein phosphatase fractions were assayed with [32P]histone fzb, [32P]phosphorylase a and [32P]phosphorylase kinase as substrates. In diabetic rat liver, one of the phosphatase fractions found in the normal liver was significantly reduced. This fraction was identified as a mixture of the spontaneously active form and the ATP. Mg-dependent form of phosphoprotein phosphatase-i (F,) based on sensitivity to inhibitor-2, substrate specificity, and the fact that it could be activated 42-70% by glycogen synthase kinase-3 in the presence of ATP. Mg. Further analysis of this fraction showed that liver cytosol from diabetic rats contained 62-79% lower spontaneously active phosphatase-i activity and % lower combined spontaneously active and ATP. Mg-dependent protein phosphatase-1 (F,) activity. Insulin administration increased the spontaneously active and the ATP. Mg-dependent protein phosphatase-i activities approximately 45% and 36%, respectively, in alloxan-diabetic rats. These data imply that the lower levels of spontaneously active phosphatase-1 activity in diabetic rat liver cannot be explained by presuming phosphatase-1 to have been present as Fc, the inactive form. Moreover, insulin restored the total activity of the spontaneously active and activatable forms of phosphatase-i to those present in normal liver implying that both forms of phosphatase-i activity are under hormonal control. Reversible phosphorylation of rate-limiting enzymes in carbohydrate metabolism is recognized as a hormonally controlled mechanism by which enzymes involved in interconnected pathways are regulated simultaneously in response to one hormonal stimulus [I -51. Since the ratio of protein kinase to phosphoprotein phosphatase activity ultimately determines the phosphorylation state of the rate-limiting enzymes, and thus their physiological activity, studying the mechanisms by which these enzymes are controlled may lead to a better understanding of diabetes and other diseases where carbohydrate metabolism is not properly regulated in response to hormonal stimulation. Diabetic rat liver shows a greatly diminished rate of glycogenesis [6-91 and an enhanced rate of gluconeogenesis, both effects reflecting changes in the activity and presumably the phosphorylation state of the rate-limiting enzymes in these pathways. Insulin has been shown to influence the phosphorylation state of several enzymes through direct activation of specific phosphatases [I Supporting these findings are several reports showing a decrease in spontaneously active phosphatase activity in diabetic animals [6-19, 421 and restoration following insulin administration [I There have been no studies, however, of the ATP. Mg-dependent phosphatase (F,) activity in diabetic animals or of the effects of insulin on this enzyme. The present Abbreviation. U, international unit of insulin. Enzymes. Phosphoprotein phosphatase (EC ); a-amylase (EC ); glycogen phosphorylase (EC ); alkalinephosphatase (EC ); CAMP-dependent protein kinase (EC ); phosphorylase kinase (EC ); glycogen synthase kinase-3 (EC ); pyruvate kinase (EC ); acetyl-coa carboxylase (EC ); ATP-citrate (pro-3s)-lyase (EC ). paper compares DEAE-column chromatography elution peaks for the various phosphatases from normal versus diabetic rat liver, identifies the phosphatases as type l or type 2 phosphoprotein phosphatase [3] based on substrate specificity and sensitivity to inhibitor-2, and quantifies the spontaneously active and the ATP. Mg-dependent [I91 phosphatases present in normal and diabetic rat liver cytosol by rapid fractionation and small-scale DEAE-cellulose batch-elution. The purpose of these measurements was to determine whether the lower activity of phosphatases reported in diabetic rat liver was due to reversible inactivation of phosphatase-i to the Fc form or to irreversible inactivation of both of the major phosphatases - types 1 and 2A - involved in glycogen metabolism and glucose homeostasis. Finally, the effects of insulin on the various phosphatase activities were measured. MATERIALS AND METHODS DEAE-cellulose DE-52 was purchased from Whatman. Histone f2b was purchased from Worthington. Phosphorylase b, crystalline insulin, alkaline phosphatase type 111, were obtained from Sigma Chemical Company. [Y~~PIATP, 2-10 Ci/mmol, was purchased from New England Nuclear (Boston, MA, USA). Phosphorylase kinase, glycogen synthase kinase-3 (FA), protein phosphatase-i, ATP. Mg-dependent phosphatase (F,), and inhibitor-2 were prepared in this laboratory. Treatment of animals Male Sprague-Dawley rats ( g) were made diabetic by the administration of alloxan (40 mg/kg) intravenous-

2 700 ly and were used 5-7 days after treatment. For insulin studies, a mg piece of liver was ligated, excised and immediately frozen in liquid nitrogen; 5 min later insulin (4 U/kg) was administered intraportally and a second piece of liver was excised. Separation and identification of phosphoprotein phosphatases from rat liver - Fresh or frozen livers (40mg/ml) obtained from male Sprague-Dawley rats fasted for h were homogenized in 5 mm Tris (ph 7 3, 2 mm EDTA, and 0.5 mm dithiothreitol in the absence or presence of 0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm tosyllysylchloromethane, 0.1 mm tosylphenylalanylchloromethane and 0.1 mm p-aminobenzamidine and centrifuged at x g for 20 min. All steps were performed at 4 C. Aliquots of supernatant were applied to DEAE-cellulose columns and eluted as explained in the legend to Fig. 1. This procedure resulted in consistent phosphatase elution patterns and protein elution profiles for thirty animals, normal and diabetic. Elution patterns were found to be the same for fresh liver and frozen liver in the absence or presence of proteolytic enzyme inhibitors. The specific activity of each sample was measured before addition to the column, and recovery of phosphatase activity and protein was greater than 95% in all cases. The protein elution patterns for the two substrates were not significantly different. In experiments designed to quantify the changes in phosphatase-1 activity in the livers of fasting normal and fasting diabetic rats following intravenous insulin administration, the livers were homogenized as described above, but the supernatants were applied to analytical-size DEAE-cellulose columns and the phosphatases were rapidly separated by batch elution as described in the legend to Fig. 4. The column fractions were assayed immediately for protein content and for phosphoprotein phosphatase activity with [32P]histone fzb or [32P]phosphorylase a as substrates. Fractions were stored at -20 C at a final concentration of 50 mm Tris (ph 7.0), then adjusted to 1 mm EDTA, 50 mm 2-mercaptoethanol and 40% ethylene glycol. Preparation of enzymes and 32P-labelled phosphoprotein substrates 32P-labelled enzymes: [32P]Phosphorylase a [20,21], [3zP]- phosphorylase kinase [22, 231 and [32P]histone f2b [24], were prepared as previously described. Inhibitor-2 was isolated from rabbit skeletal muscle and purified to homogeneity according to Yang et al. [25]. The purified preparation (0.4 mg/ml) (MI = 30000) inhibited protein phosphatase-1 from rabbit skeletal muscle with an apparent Ki of 22 nm and was stored in 20 mm Tris (ph 7.0) containing 1 mm 2-mercaptoethanol and 40% ethylene glycol at -2O"C. Glycogen synthase kinase-3, the activating factor (FA) of the ATP. Mg-dependent protein phosphatase (F,), was isolated from rabbit skeletal muscle according to Vandenheede et al. [26] and Hemmings et al. [27]. FA was not purified to homogeneity, but gave a major band of M, % on sodium dodecyl sulfate slab-gel electrophoresis [28], catalyzed the activation of the ATP. Mg-dependent phosphatase (F,) and did not dephosphorylate [32P]phosphorylase a or inhibit the dephosphorylation of [32P]phosphorylase a by phosphatase-1. Protein phosphatase-1 [22] and the ATP. Mg-dependent protein phosphatase (F,) [28] were prepared as described previously. Phosphoprotein phosphatase assays The [32P]histone phosphatase assay [29] was carried out in a total volume of 200 pl containing a 50-pl aliquot of a DEAE-cellulose column fraction, [ 32P]histone f2b (30 pg), 50 mm Tris (ph 7.9, 1 mm dithiothreitol and 2.5 mm MgClz at 30 C. 32Pi radioactivity was counted following extraction with ammonium molybdate and isobutanol [30]. Activity is expressed as pmol 32Pi released/lo min. The [32P]phosphorylase a phosphatase assay [31] was conducted in the absence or presence of inhibitor-2 (0.8 pg) in a total volume of 75 p1 containing a 40-p1 aliquot of a DEAEcellulose column fraction, 50 mm Tris (ph 7.0), 2 mm EDTA, 50 mm 2-mercaptoethanol, and 1 mg/ml bovine serum albumin. When column fractions were assayed immediately following elution, MnC12 did not affect the activity and therefore it was not present in the assay buffer. The titration studies of the peak DEAE-cellulose column fractions with inhibitor-2 were similarly performed but the concentration of inhibitor-2 in each assay was varied. Phosphatase activity is expressed as pmol 32Pi released/lo min. The [32P]phosphorylase kinase phosphatase assay [32] was conducted in the absence and presence of inhibitor-2 (2.4 pg) in a total volume of 180 pl containing a 1204 aliquot of a sixfold concentrated DEAE-cellulose column fraction, 50 mm Tris (ph 7.0), 0.5 mm dithiothreitol, 2 mm MnClZ, and 0.3 mg/ml bovine serum albumin. Two 2 04 aliquots of the reaction mixture were removed after 0, 10, and 20 min of incubation. One aliquot was precipitated in 25% trichloroacetic acid and the radioactivity was counted directly. The other 204 aliquot was applied to a 5% sodium dodecyl sulfate/polyacrylamide disc gel and the separated a + a', p, and y subunits were sectioned and the radioactivity was counted separately. The results are expressed as pmol 32Pi released in the absence or presence of inhibitor-2 and as pmol 32Pi released by the selective dephosphorylation of the p or a subunit of phosphorylase kinase. The ATP. Mg-dependent protein phosphatase (F,) assay [ 191 of the analytical-size DEAE-cellulose column fractions was carried out in the absence or presence of 0.3 nmol of the activating factor (FA) and in the absence or presence of 1 pg of inhibitor-2 in a total volume of 37.5 p1 containing 12.5 p1 of column fraction, 0.2 mm ATP, 2.0 mm MgCl2, 0.3 mg/ ml bovine serum albumin, 50 mm Tris (ph 7.0), and 0.1% 2-mercaptoethanol. The increase in phosphatase activity resulting from the conversion of inactive Fc to an active protein phosphatase is shown as the percentage increase in the release of 32Pi from [32P]phosphorylase a. Protein phosphatase-2c was assayed [33, with [32P]phosphorylase kinase as the substrate following irreversible inactivation of protein phosphatase-2a with 50 mm NaF followed by exhaustive dialysis to remove the NaF. The assay mixture contained inhibitor-2 (0.8 pg), 50 mm Tris (ph 7.0), 10 mm MgC12, 0.5 mm EGTA, 0.3 mg/ml bovine serum albumin and 0.1% 2-mercaptoethanol in a total volume of 60 p1. 32Pi was determined after extraction with ammonium molybdate and isobutanol. Phosphatase 1,2A and 2C [33, account for virtually all of the dephosphorylation of enzymes involved in liver glucose metabolism, and the choice of [32P]phosphorylase and [32P]phosphorylase kinase as the main substrates for the

3 701 Fig. 1. Column elution profiles of liver phosphorylase phosphatase and liver histone phosphatase from x g supernatants prepared from normal24-h fastedrats (A, C) or alloxanized24-h fasted rats (B, D). Aliquots containing 3.6 mg of supernatant protein from rat liver in 0.7 mi were applied to columns of DEAE-cellulose (1 x 19 cm). Following a 50-ml wash, the samples were eluted at 30 ml/h with a 60-ml (A, B) or an 80-ml (C, D) linear gradient from M NaCl. 1-ml fractions were collected and analyzed for phosphatase activity and protein concentration. A and B show the phosphorylase phosphatase profiles expressed as pmoi 32Pi released/lo min for 40 pl of column fraction and C and D show the histone phosphatase profiles expressed as pmol 32P, released/lo min for 50 pl of column fraction. Recovery of protein was 95% and recovery of phosphatase activity was %. These profiles are representative of more than 30 experiments present study is consistent with this. The remaining phosphatase found in liver, protein phosphatase-2b [3, 21, 23, , was not measured and did not interfere with the quantification of other phosphatases since this phosphatase does not dephosphorylate either the enzymes involved in hepatic metabolism or the three substrates used in this study. RESULTS Fig. 1 shows representative DEAE-cellulose column phosphatase fractionation patterns for liver supernatants from a normal fasted rat (Fig. 1 A, C) and an alloxan-diabetic fasted rat (Fig. lb, D). The elution pattern obtained for the liver supernatant from a normal rat contained three distinct phosphatase peaks, observable with both substrates, whereas identical treatment of the sample prepared from diabetic rat liver produced a phosphatase elution pattern in which the first peak of phosphatase activity was greatly reduced or absent. The last elution peak had a very high specific activity which decreased significantly within hours following column elution even when stored on ice. Full activity could be restored by a 10-min preincubation with 2 mm MnClz and there were no significant differences between the activity of the last peak for liver samples from normal rats and samples from diabetic rats when assayed in the presence of 2 mm MnClZ. The activity in the first two peaks was stable for at least six hours when stored on ice and there were no changes in activity in the presence or absence of MnC12. The 48000xg pellet was assayed for glycogen-bound phosphatase activity with or without a ph 6.1 precipitation step [22] and no precipitable activity was found in the samples from h fasted animals. The peaks of phosphdtase activity eluted from the DEAEcellulose column were identified as phosphatase-1 or phosphatase-2 based on their sensitivity to inhibitor-2 in the presence of the three 32Pi-labeled substrates. Assay of the DEAEcellulose column fractions from normal and diabetic liver supernatant (Fig. 1) revealed the first two peaks of the normal to be missing or reduced in the diabetic. In the presence of 2 p1 (0.8 pg) of inhibitor-2, an amount shown to inhibit partially-purified phosphatase-1 by 90 - loo%, normal and diabetic elution patterns resembled each other closely with the first peak of phosphatase activity virtually missing and the second peak of activity reduced. This is illustrated in Fig. 2 which shows the titration data with inhibitor-2 for the normal and diabetic peak fractions from DEAE-cellulose column elutions. The titration results with inhibitor-2 obtained for both the normal and the diabetic samples, using either histone fzb or phosphorylase as substrates, showed that the first chromatography peak contained virtually 100% phosphatase- 1, the second peak approximately 20-30% phosphatase-1, and the last peak virtually 100% phosphatase-2. The differences in protein content of the column fractions for the normal sample and those for the diabetic rat sample were not statistically significant. The [32P]phosphoryla~e kinase assay data in Fig. 3 confirm the inhibitor-2 data. The maximal-activity fraction from

4 702 Fig. 2. Inhibitor-2 titration of the phosphorylase phosphatase activity contained in the D EA E-cellulose column fractions from a normal 24-hfasted rat and an alloxanized 24-h-fasted rat. 40-pl aliquots of the column fractions from the experiment described in Fig. 1 were assayed in the presence of 0, 1, 3 and 5 p1 of inhibitor-2 as described in Materials and Methods. The titration curves with inhibitor-2 are shown for peak fractions from elution peaks 1 (0), 2 ( x ) and 3 (M) from a normal rat (A) and from an alloxanized rat (B) age increase in the phosphorylase phosphatase activity was measured in the presence of ATP. Mg and the activating factor, FA. Each peak fraction was assayed individually for the presence of the inactive (F,) form of phosphatase-1 using the specific assay for phosphatase-i [31] plus ATP. Mg and FA or the specific assay for Fc [19]. The phosphatase activity of the DEAE-cellulose eluate of a normal fasted rat - specifically, the activity in the peak fraction from peak 1 (Fig. 2A) - increased 55% in the presence of FA, ATP and Mg; activation of the other phosphatase peak fractions was negligible (data not shown). The phosphatase activity of the DEAE-cellulose eluate of a diabetic rat - specifically, the peak fraction from peak 1 (Fig. 2 B) - increased only 11 % in the presence of FA, ATP and Mg; activation of the other phosphatase peak fractions was negligible (data not shown). After establishing that liver supernatant from diabetic rats contains less spontaneously active phosphatase and less ATP. Mg-dependent phosphatase eluting from DEAEcellulose between 0 and 0.2 M NaC1, small-scale rapidfractionation DEAE-cellulose batch-elution columns were used to quantify changes in the phosphatase activities. Prior to insulin administration, diabetic animals were found to have 62-79% lower spontaneously active phosphatase levels (P < 0.01) and % lower combined spontaneously active and ATP. Mg-dependent phosphatase levels (P < 0.025) than normal animals. Insulin (4 U/kg) increased the spontaneously active phosphatase-1 by an average of 45% and the ATP. Mg-dependent protein phosphatase-1 by an average of 36% in diabetic animals. The effect of insulin in normal animals was negligible. Results from a representative experiment with three normal and three diabetic animals are displayed in Fig. 4. Data from a total of six diabetic and seven normal rats showed the same pattern. After insulin treatment, phosphatase levels in normal rats were not significantly different from those in diabetic rats. There were no statistically significant differences in the levels of phosphatase-2a with or without insulin, nor were there any statistically significant differences between levels of phosphatase-2a in the livers of normal versus diabetic rats. Levels of phosphatase-2c were negligible in all samples. peak 1 had almost 100% specificity for dephosphorylation of the b-subunit of phosphorylase kinase indicative of phosphatase-1 ; the maximal-activity fraction from peak 2 had mixed a and 1 subunit activity; and the maximal-activity fraction from peak 3 had almost exclusively a-activity indicative of phosphatase-2. Endogenous inhibitor proteins in liver extracts assayed at low concentration and for short periods, such as 10 min, should not significantly inhibit the endogenous phosphatase-i [33], and this was indeed the case when boiled aliquots (40 PI) of each column fraction were assayed with 20 pl of a 1 :40 dilution of purified phosphatase-1 and [ 32P]phosphorylase a ~91. Peaks 2 and 3 were mainly phosphatase-2a. Phosphatase- 2C activity was found to be less than 4% of the total phosphatase activity, in agreement with the value found by Ingebritsen et al. [33]. No phosphatase activity towards p-nitrophenyl phosphate [34] was observed in any of the column fractions using the ph 7.0 assay mixture specific for detection of phosphatases 1 and 2. Phosphatase-2B was not measured. To determine whether the decrease in activity of phosphatase-i was due to reversible inactivation to Fc, the percent- DISCUSSION Data presented in this paper lead to the conclusion that the major decrease in phosphatase activity in rat liver as a result of alloxan treatment is due to a decrease in activity of phosphatase-i rather than of phosphatase-2. This is in agreement with a recent report by Foulkes and Jefferson [42] in which the only significant effect of alloxan diabetes in rat liver was found to be a 50% decrease in the specific activity of protein phosphatase-1. That the deficient phosphatase in liver supernatant from alloxan-diabetic rats turns out to be spontaneously active phosphatase-i is not surprising considering the fact that this enzyme accounts for a large percentage of the dephosphorylation of enzymes involved in glucose metabolism [33, 35-37]. Previous reports that the activities of both glycogen phosphorylase and glycogen synthase are defective in alloxandiabetic rat liver, presumably because of the decreased activities of both glycogen phosphorylase phosphatase [14, 161 and glycogen synthase phosphatase [4-9, 15, IS] are thus confirmed. The data presented in this paper also show that insulin administration specifically increases the activity of the

5 703 Fig. 3. Phosphorylase kinase phosphatase activity of eluted fractions fiom DEA E-cellulose column chromatography of liver supernatant,from 24-h-fasted rats. The data represent the rate of selective dephosphorylation of either the b-subunit (solid line) or the rx-subunit (broken line) of [3'P]phosphorylase kinase as determined by electrophoresis and described in detail in Materials and Methods. Samples are the peak fractions for peaks 1, 2 and 3 from the experiment shown in Fig. 1 for samples prepared from a normal rat (A) or an alloxanized rat (B) spontaneously active phosphatase-1 with no significant effect on the activity of phosphatase-2. This is expected since the activity of the potent inhibitor of phosphatase-1 activity, inhibitor-i, has been shown to be under insulin control [43, 441 and no such insulin control has been postulated for phosphatase-2 activity. These results are consistent with the observations of many investigators that insulin administration increases the activity of both glycogen synthase phosphatase [4-6, 9, 15, 181 and phosphorylase phosphatase [7, 8, 14, 161. The observed decrease in phosphatase-i activity in insulindeficient animals could logically be due either to a decrease in total synthesis of enzyme or, as Foulkes and Jefferson have recently demonstrated [42], to a decrease in specific activity. Since the insulin-induced increase in phosphatase-i activity occurred in 5 min, a mechanism involving changes in protein synthesis is highly unlikely. Assuming that insulin-deficiency results in a decrease in enzyme specific activity, the following possibilities must be considered: (a) changes in the activity of inhibitor-i, which has been shown to be under insulin control in vivo [43,44]; (b) changes in the activity of inhibitor-2, which has been shown to be under the control of glycogen synthase kinase-3 [45-471; (c) reversible inactivation of phosphatase-1 to the inactive Fc form or changes in any of the complex Fig. 4. E'fect of insulin on phosphatase-1 activity in liver supernatants,from normal versus alloxanized rats. Liver samples were from the same animal before insulin treatment and 5 min following a 4 Ujkg hepatic portal vein injection of insulin. Aliquots of x g liver supernatants containing 0.5 mg of protein in 100 pl were applied to DEAEcellulose columns (1 x 2 cm) and batch-eluted with four 2.0-ml volumes of equilibration buffer containing 0 M, M, M and 0.30 M NaC1, respectively. Recovery of protein and total phosphatase activity was 95% or higher in all experiments. An aliquot of each sample was assayed I: FA and inhibitor-2 as described in Materials and Methods. There were no significant differences in protein content between the normal and diabetic samples being compared. Results are expressed as pmol of 3zPi released/l5 min from [32P]phosphorylase a in the absence of inhibitor-2 minus pmol of 32Pi released from [32P]phosphorylase a in the presence of inhibitor-2. The results represent the phosphatase-i activity in the M NaCl and M NaCl fractions since no activity sensitive to inhibitor-2 was found in the M NaCl fraction. The data presented show the values & SEM for three separate experiments with three diabetic animals (shaded bars) and three normal animals (cross-hatched bars). The figure shows activities in the absence or presence of F4 at 0 min (**, P < 0.01 and *, P < 0.025, respectively) and at 5 min (differences not statistically significant). Data from a total of six diabetic and seven normal animals showed the same pattern control mechanisms involved in the regulation of this reversible inactivation mechanism [48, 491. With regard to this third possibility, the present study concludes that the decrease in the specific activity of phosphatase-1 is not simply a reversible inactivation to the ATP. Mg-dependent form of protein phosphatase-i, since even when the inactive form was activated the combined activity of the ATP. Mg-dependent and spontaneously active forms was still 46% lower in liver from diabetic rats than in liver from normal rats. The decrease in specific activity seems to result from deficient hormonal control of some regulatory factor(s) affecting both forms of protein phosphatase-1 activity, since administration of insulin to diabetic animals not only restores the spontaneously active protein phosphatase- 1 activity to normal levels but also restores the ATP. Mgdependent phosphatase activity. Elucidation of the regulatory mechanism(s) awaits studies designed to quantify changes in the liver concentrations of inhibitor-i, inhibitor-2, and glycogen synthase kinase-3 under different physiological conditions and in normal versus dia-

6 704 betic states and studies delineating the control mechanism(s) involved in hormonal regulation of the reversible inactivation of the Mg. ATP-dependent phosphatase. We would like to thank Dr Thomas Ingrebritsen, Dr Brian Hemmings and Dr Jackie Vandenheede for the invaluable instruction given in phosphoprotein phosphatase laboratory techniques including preparation of FA, Fc, inhibitor-2 and the 32Pi-labelled substrates. REFERENCES 1. Krebs, E. G. (1972) Curr. Top. Cell. Regul. 5, Nimmo, H. G. & Cohen, P. (1977) Adv. Cyclic Nucleotide Res. 8, Cohen, P. (1978) Curr. Top. Cell. Regul. 14, Krebs, E. G. & Beavo, J. A. (1979) Annu. Rev. Biochem. 48, Cohen, P. (1980) in Molecular Aspects of Cellular Regulation (Cohen, P. ed.) vol. 1, pp. 1 and 255, Elsevier/North Holland, Amsterdam. Miller, T. B., Jr & Larner, J. (1973) J. Biol. Chem. 248, Miller, T. B., Jr (1978) Am. J. Physiol. 234, E13-El9. Miller, T. B., Jr (1979) Biochim. Biophys. Acta 583, Langdon, D. R. & Curnow, R. T. (1983) Diabetes 32, Bishop, J. S. & Larner, J. (1967) J. Biol. Chem. 242, Gold, A. H. (1970) J. Biol. Chem. 245, Nuttall, F. Q., Gannon, M. C., Corbett, V. A. &Wheeler, M. P. (1976) J. Biol. Chem. 251, Bishop, J. S. (1970) Biochim. Biophys. Acta208, Tan, A. W. H. & Nuttal, F. Q. (1976) Biochim. Biophys. Acta 445, Bollen, M. & Stalmans, W. (1984) Biochem. J. 217, Khandelwal, R. J., Zinman, S. M. & Zebrowski, E. J. (1977) Biochem J. 168, Gold, A. H., Dickemper, D. & Haverstick, D. M. (1979) Mol. Cell. Biochem. 25, Shahed, A. R., Mehta, P. P., Chalker, D., Allman, D. W., Gibson, D. M. & Harper, E. T. (1980) Biochem. Znt. I, Yang, S. D., Vandenheede, J. R., Goris, J. & Merlevede, W. (1980) J. Biol. Chem. 255, Fischer, E. H. & Krebs, E. G. (1958) J. Biol. Chem. 231, Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P. (1977) Biochem. J. 162, Cohen, P. (1973) Eur. J. Biochem. 34, Antoniw, J. F. & Cohen, P. (1976) Eur. J. Biochem. 68, Meisler, M. H. & Langan, J. A. (1969) J. Biol. Chem. 244, Yang, S. D., Vandenheede, J. R. & Merlevede, W. (1981) FEBS Lett. 132, Vandenheede, J. R., Yang, S. D., Goris, J. & Merlevede, W. (1980) J. Biol. Chem. 255, Hemmings, B. A., Yellowlees, D., Kernohan, J. C. & Cohen, P. (1981) Eur. J. Biochem. 119, Hemmings, B. A., Resink, T. J. & Cohen, P. (1982) FEBS Leu. 150, Tamura, S., Kikuchi, K., Hiragia, A., Kikuchi, H., Hosokawa, M. & Tsuiki, S. (1978) Biochim. Biophys. Actu 524, Huprikar, S., Lang, M., Friedman, Y. & Burke, G. (1979) FEBS Lert. 99, Cohen, P. (1977) Biochem. J. 162, Stewart, A. A., Hemmings, B. A. & Cohen, P. (1981) Eur. J. Biochem. 115, Ingebritsen, T. S., Stewart, A. A. & Cohen, P. (1983) Eur. J. Biochem. 132, Garen, A. & Levinthal, C. (1960) Biochim. Biophys. Acta 38, Ingebritsen, T. S. & Cohen, P. (1983) Eur. J. Biochem. 132, Ingebritsen, T. S., Foulkes, J. G. & Cohen, P. (1983) Eicr. J. Biochem. 132, Ingebritsen, T. S. & Cohen, P. (1983) Science (Wash. DC) 221, Pato, M. F., Adelstein, R. S., Crouch, D. B., Safer, B., Ingebritsen, T. S. & Cohen, P. (1983) Eur. J. Biochem. 132, Stewart, A. A., Ingebritsen, T. S. & Cohen, P. (1983) Eur. J. Biochem. 132, Stewart, A. A., Ingebritsen, T. S., Manalan, A,, Klee, C. B. & Cohen, P. (1982) FEBS Lett. 137,SO Yang, S. D., Tallant, E. A. & Cheung, W. Y. (1982) Biochem. Biophys. Res. Commun. 106, Foulkes, J. G. & Jefferson, L. S. (1984) Diabetes 33, Foulkes, J. G., Cohen, P. & Jefferson, L. S. (1980) FEBS Lett. II2, Foulkes, J. G., Cohen, P., Strada, S., Everson, W. V. &Jefferson, L. S. (1982) J. Biol. Chem. 257, Vandenheede, J. R., Young, S. D., Goris, J. & Merlevede, W. (1980) J. Biof. Chem. 255, Hemmings, B. A., Resink, T. & Cohen, P. (1982) FEBSLert. 150, Resink, T., Hemmings, B. A,, Lintung, H. Y. & Cohen, P. (1983) Eur. J. Biochem. 133, Yang, S.-D., Vandenheede, J. R. & Merlevede, W. (1981) FEBS Lett. 126, Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S.-D., Vandenheede, J. R. & Merlevede, W. (1984) J. Biol. Cheni. 259, C. J. Dragland-Meserve, D. K. Webster, and L. H. Parker Botelho, Sandoz Research Institute, Sandoz, Inc. East Hanover, New Jersey, USA 07936