©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of Protein Phosphatase-1 Activity by Insulin in Rat Adipocytes
EVALUATION OF THE ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE PATHWAY (*)

(Received for publication, July 5, 1994; and in revised form, September 19, 1994)

Najma Begum (§)

From the Diabetes Research Laboratory, Winthrop University Hospital, Mineola, New York 11501 and the School of Medicine, State University of New York, Stony Brook, New York 11790

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

In this study, we examined the distribution of protein serine/threonine phosphatase-1 (PP-1) and analyzed the effect of insulin on PP-1 and its mechanism of activation in freshly isolated rat adipocytes. The adipocyte particulate fraction (PF) constituted approx80% of cellular PP-1 activity, while PP-2A was entirely cytosolic. Insulin rapidly stimulated PF PP-1 in a time- and dose-dependent manner (maximum stimulation at 5 min with 4 nM insulin). Immunoprecipitation of PF with an antibody against the site-1 sequence of rabbit skeletal muscle glycogen-associated PP-1 (PP-1G) subunit indicated that approx40% of adipocyte PP-1 activity was due to PP-1G form of the enzyme. Insulin stimulated PP-1G (120% over basal levels) without affecting the other forms of PP-1 in the PF. Insulin activation of PP-1 was accompanied by >2-fold increase in the phosphorylation state of the 160-kDa regulatory subunit of PP-1. Stimulation of p21/mitogen-activated protein kinase pathway (MAP) with GTP analogues also resulted in stimulation of PP-1 similar to insulin. The insulin effect on MAP kinase and PP-1 activation was blocked by a GTP antagonist, guanyl-5`-yl thiophosphate. The inhibitors of MAP kinase activation (viz. cAMP agonists, SpcAMP and ML-9) also blocked PP-1 stimulation by insulin. The time course of MAP kinase activation preceded the phosphorylation of PP-1 regulatory subunit and PP-1 activation. We conclude that insulin rapidly activates a membrane-associated PP-1 in adipocytes, which may be similar to rabbit skeletal muscle PP-1G, and the activation is mediated by p21/MAP kinase pathway.


INTRODUCTION

The major physiological role of insulin is to control body fuel metabolism by promoting the uptake of glucose and the other nutrients and their conversion into glycogen, proteins, and lipids. Insulin stimulates these pathways by controlling the activation state of certain key enzymes of glycogen and lipid metabolism via phosphorylation and dephosphorylation mechanisms(1, 2, 3) .

Protein phosphatase-1 (PP-1) (^1)is one of the four major types of serine hreonine phosphatases in mammalian cells, with a widespread tissue distribution and capability to dephosphorylate a variety of regulatory proteins in vitro(4) . The active forms of PP-1 are believed to be largely particulate, and their association with subcellular structures is mediated by ``targeting subunits'' distinct from the catalytic subunit(5) . Thus, PP-1 exists as a complex with other proteins that target it to particular subcellular locations, modify its substrate specificity, and appear to regulate the enzyme activity(5) . In the rabbit skeletal muscle, PP-1 is found associated with glycogen particles (PP-1G), the sarcoplasmic reticulum, the myofibrils, and the cytosolic inhibitor 2 protein(5, 6) . Insulin has been shown to promote site-specific phosphorylation (site-1) of the 160-kDa regulatory subunit (G subunit) of rabbit skeletal muscle PP-1(7) , an event that is believed to activate PP-1 with subsequent dephosphorylation (and activation) of glycogen synthase and phosphorylase kinase (inactivation). Phosphorylation of site-1 is catalyzed by an insulin-stimulated protein kinase, which is a mammalian homologue of ribosomal S6 kinase II(8) . In contrast to insulin, adrenalin via cAMP/protein kinase A inhibits the mitogen-activated protein (MAP) kinase pathway and also stimulates phosphorylation at site-2 on PP-1G subunit, which in turn causes dissociation of PP-1C subunit from the G subunit and its release from glycogen(9, 10) .

A number of recent studies with transfected cell lines indicate that p21/MAP kinase pathway plays a pivotal role in insulin's effect on mitogenesis(11, 12, 13, 14, 15) . However, the intracellular targets of MAP kinase have not been completely defined. MAP kinase phosphorylates and activates the transcription factors c-Myc, c-Jun, phospholipase A2, and the 90-kDa protein serine/threonine kinase, Rsk-2 (16, 17) , resulting in the phosphorylation of the ribosomal protein S6. In the skeletal muscle, PP-1G is phosphorylated and activated in response to insulin by an insulin-stimulated protein kinase, which is a mammalian homologue of Rsk-2(8) , thus providing a potential link between tyrosine kinase-mediated MAP kinase activation and stimulation of glycogen synthesis. In contrast to skeletal muscle, which has been widely studied with respect to glycogen metabolism and PP-1 regulation, no data exists in the literature on the regulation of PP-1 in freshly isolated rat adipocytes.

In view of the recent reports demonstrating the formation of p21 GTP and rapid activation of MAP kinase cascade in response to insulin and other growth factors in cultured 3T3 L1 adipocytes, in the present study, we have examined the regulation of PP-1 by insulin in freshly isolated rat adipocytes, identified the regulatory subunit of the enzyme that controls the enzyme activity, and studied the contribution of PI3 kinase and p21/MAP kinase pathway in insulin's effect on PP-1. To evaluate the contribution of PI3 kinase pathway, the recently described specific inhibitor of PI3 kinase, wortmannin (18) was used. The involvement of Ras or other GTP binding proteins in insulin response was investigated by a) treatment of permeabilized rat adipocytes with non-hydrolyzable GTP analogs, b) studying the effect of GTP antagonist, GDPbetaS on insulin response, and c) blocking MAP kinase and 90-kDa S6 kinase activation with SpcAMP, a cAMP agonist, and ML-9, a myosin light chain kinase inhibitor.


EXPERIMENTAL PROCEDURES

Materials

Porcine insulin was a kind gift from Eli Lilly Co. (Indianapolis, IN). [-P]ATP (specific activity, >3000 Ci/mmol), [P] orthophosphoric acid, and I-protein A were purchased from DuPont NEN. Phosphorylase a, phosphorylase kinase, and other reagents for PP-1 assay, myelin basic protein, and protein kinase inhibitor peptide were from Life Technologies, Inc. Okadaic acid (OA) was from Moana Bioproducts (Honolulu, Hawaii). Collagenase was obtained from Worthington. Reagents for SDS-polyacrylamide gel electrophoresis and immunoblotting were purchased from Bio-Rad, adenosine triphosphate, dithiothreitol, phenylmethylsulfonyl fluoride (PMSF), leupeptin, benzamidine, pepstatin A, aprotinin, antipain, TPCK-treated trypsin, soya trypsin inhibitor (STI), sodium fluoride, sodium pyrophosphate, sucrose, Triton X-100, SDS, guanosine triphosphate S (GTPS), and GDPbetaS were from Sigma. Wortmannin and rapamycin were purchased from Biomol Research (Plymouth Meeting, PA). alpha toxin was from List Biologicals (Campbell, CA). Antibody to the catalytic subunit of PP-1 (anti-C) was from UBI (Lake Placid, NY), while anti-regulatory subunit antibodies (anti-site-1) were generated by immunizing rabbits against a synthetic peptide corresponding to the sequence surrounding the site-1 of rabbit skeletal muscle PP-1G subunit (SPQPSRRGSESSEE). Affinity purification and characterization of the antibody was done according to the protocol described by Hiraga et al.(19) .

Preparation of Isolated Adipocytes

Isolated adipocytes were prepared from the epididymal fat pads of male Sprague-Dawley rats (body weight, 140-160 g) by collagenase digestion as described earlier(20) . Aliquots (0.5-3 ml) of adipocytes were resuspended in 2-3 ml of Krebs-Hepes buffer containing 3% bovine serum albumin and 30 mg/dl glucose and incubated with insulin (0-100 nM) for 0-30 min at 37 °C before or after treatment with various other agents as detailed in the figure legends. At the end of the incubation period, adipocytes were processed for a) extraction and the assay of PP-1 activity in the particulate (PF) and cytosolic (CF) fractions, b) P labeling, immunoprecipitation, and Western blot analysis of the PP-1 regulatory and catalytic subunits in the particulate and cytosolic fractions, and c) assay of MAP kinase activity in the cytosolic fractions.

Extraction of Protein Phosphatase-1

Control, insulin, or other agonist-treated adipocytes were rinsed twice with warm phosphate-buffered saline and resuspended in 1-2 ml of PP-1 extraction buffer containing 40 mM imidazole HCl buffer (pH 7.2), 2 mM EDTA, 0.2% 2-mercaptoethanol, 2 mg/ml glycogen, 10 µg/ml each of aprotinin, leupeptin, antipain, STI, pepstatin A, 1 mM benzamidine, and 1 mM PMSF. The cells were homogenized using a polytron for 5-10 s and centrifuged at 1000 times g for 5 min. Supernatants were centrifuged at 100,000 times g for 30 min at 4 °C to separate the PF from CF. PP-1 activity was assayed in the PF and CF.

Assay of PP-1 Activity

Adipocyte spontaneous PP-1 activity was measured according to the method of Cohen et al.(21) using purified P-labeled phosphorylase a as a substrate. Briefly, extracts (1-5 µg of protein) were pre-incubated with and without okadaic acid (3 nM) in 40 µl of assay buffer (same as the extraction buffer above) for 2 min at 30 °C. The reaction was initiated by the addition of 20 µl of P-labeled phosphorylase a. After 10 min, the reaction was stopped by adding 180 µl of 20% trichloroacetic acid and 10 µl of 3% bovine serum albumin as a carrier protein. The tubes were left on ice for 10 min and then centrifuged at 12,000 times g for 3 min at 4 °C. Aliquots of the clear supernatant were counted to determine the amount of radioactivity (P(i)) released. PP-1 activity was determined by including 3 nM OA in the assay. At this concentration, under the conditions of the assay, OA inhibits only PP-2A, and the remaining activity represents the contribution of PP-1(21) . This was further verified by the assay of enzyme activity in the presence of inhibitor 2. Pre-incubation of the enzyme extracts with 2 µg of inhibitor 2 for 2 min resulted in >80% inhibition in the phosphatase activity. The amount of activity inhibited by inhibitor 2 was equal to the activity assayed in the presence of 3 nM OA.

Because PP-1 activity can be present in its latent form(22) , the total PP-1 activity was estimated by incubation of extracts with TPCK-treated trypsin (40 µg/ml times 5 min at 37 °C). The reaction was stopped by the addition of 3 volumes of STI (100 µg/ml) followed by centrifugation and assay of enzyme activity in the supernatants.

P-Labeled phosphorylase a was prepared by reacting [-P]ATP with purified phosphorylase kinase and phosphorylase b as described earlier(23) .

In Vivo Phosphorylation and Immunoprecipitation of PP-1

1-2-ml adipocytes were incubated with [P]orthophosphate (0.3 mCi/ml) for 2 h followed by insulin (4.2 nM) for 5 min. The cells were rinsed 4 times with 1 ml of ice-cold phosphate-buffered saline containing phosphatase and protease inhibitors and homogenized in buffer containing 20 mM Hepes (pH 7.4), 1 mM EDTA, 2 mM sodium vanadate, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 40 mM beta-glycerophosphate, 1 mM benzamidine, 0.1 mM PMSF, 10 µg/ml leupeptin, aprotinin, antipain, STI, and pepstatin A, and 100 mM NaCl followed by subcellular fractionation to separate PF and CF. PP-1 was immunoprecipitated with an antibody raised against the site-1 sequence of rabbit skeletal muscle PP-1G subunit as described below.

A second experiment was run simultaneously in which cells were incubated with cold P(i) for the indicated time; PF and CF were prepared in a buffer (same as above except that sodium fluoride, beta-glycerophosphate, and sodium pyrophosphate were omitted), followed by immunoprecipitation with site-1 antibody and the assay of enzyme activity in the immunoprecipitates. Briefly, the cell lysates (100 µg of protein) from above were precleared by incubation with rat IgG (5 µg/ml, coupled to protein A-Sepharose) at 4 °C for 1 h. The supernatants were immunoprecipitated with PP-1G subunit antibody (10 µg/ml) at room temperature for 1 h, followed by treatment with 50 µl of protein A-Sepharose CL6B (50% v/v) for 1 h. In some experiments, the antibody was pre-incubated with the competing peptide before adding to the cell lysates. The pellets were washed four times with lysis buffer (see above) and resuspended in 40 µl of 2 times SDS-sample buffer. The samples were incubated at 37 °C for 10 min, followed by centrifugation (10,000 times g for 30 s) to pellet down the Sepharose beads. Electrophoresis of the immunoprecipitates was performed in 7.5% SDS-polyacrylamide gels followed by autoradiography (24) . The protein contents of PP-1 catalytic and regulatory subunits were determined by immunoprecipitating unlabeled cell lysates with G subunit antibody, followed by separation of immunoprecipitated proteins on SDS-PAGE and Western blot analysis with PP-1C or PP-1G subunit antibody. After the transfer of proteins to nitrocellulose membranes, the membranes were probed with PP-1C or PP-1G subunit antibody. The catalytic and the regulatory subunits of PP-1 were identified by incubating with I-protein A (0.2 µCi/ml) followed by autoradiography. The intensity of the signal was quantitated by densitometric analysis of the autoradiograms as well as by the ``cut and count'' technique.

For the measurement of enzyme activity in the immunoprecipitates of control and insulin-exposed cells, the immunoprecipitations were performed in the cold room at 4 °C as described above, except that the lysis buffer did not contain phosphatase inhibitors. The absence of phosphatase inhibitors might result in considerable dephosphorylation of PP-1G subunit, thereby resulting in an underestimation of insulin effect. Because all the steps were carried out in the cold room and the treatment groups were always compared with the controls, the magnitude of error is lesser than anticipated. The immune complexes were washed four times with wash buffer and resuspended in the same buffer containing 15 µg/ml site-1 peptide (against which the antibody was raised) and incubated at 4 °C for 1 h to release the bound enzyme from the immune complex. An aliquot of the supernatant was assayed for PP-1 activity as described above.

Assay of MAP Kinase Activity

MAP kinase activity was measured using myelin basic protein as substrate. Control and agonist-treated cells were washed thrice with cold phosphate-buffered saline containing phosphatase and protease inhibitors and extracted in 100 mM beta-glycerophosphate (pH 7.2), 10 mM EGTA, 10 mM magnesium chloride, 100 µM orthovanadate, 1 mM dithiothreitol, 10 µg/ml each of aprotinin, leupeptin, pepstatin, antipain, and STI, and 1 mM PMSF. The cells were sonicated at 70% output for 10 s and centrifuged at 2000 times g for 5 min to remove cellular debris. The supernatants were spun at 100,000 times g for 15 min. 1-5 µg of cytosolic protein was used for the assay, which was carried out for 10 min at 30 °C in a final volume of 50 µl containing 20 mM Hepes, pH 7.4, 48 mM beta-glycerophosphate, 60 µM orthovanadate, 0.3 µM okadaic acid, and 0.2 mg/ml myelin basic protein(25) . The reaction was stopped by transferring 25-µl aliquots onto phosphocellulose discs (Life Technologies, Inc.), which were dropped into 180 mM phosphoric acid. The discs were washed five times for 5 min each time in 180 mM phosphoric acid, rinsed with 95% ethanol, dried, and counted in scintillation mixture.

Protein Assay

The protein content in the cell extracts was determined by bicinchoninic acid(26) .

Statistics

The Student's t test or analysis of variance was used to evaluate the significance of the effect of insulin on PP-1 stimulation, PP-1 content, and phosphorylation status of PP-1 in control and insulin-treated cells. Results are presented as mean ± S.E. of 4-6 individual experiments performed in duplicate.


RESULTS

In the initial studies, the cellular distribution of protein phosphatase-1 and -2A activities was examined in adipocyte particulate and cytosolic fractions. Particulate fraction comprised of 75% of cellular PP-1 activity, while 25% of PP-1 was present in the soluble fraction. In contrast to PP-1, the contribution of PP-2A to the membranous phosphatase activity was very small, and most of the PP-2A activity was found in the cytosol (Fig. 1). Mild trypsin treatment resulted in approx40% increase in particulate PP-1 activity toward phosphorylase a, while the cytosolic PP-1 and PP-2A activities were not affected. Insulin treatment resulted in a rapid time-dependent increase in the spontaneous particulate PP-1 activity (50% increase over basal values within 5 min, with a return to basal levels within 20-30 min) (Fig. 2) without affecting the total (trypsin-released) PP-1. Insulin had no effect on cytosolic PP-1 or PP-2A activities (data not shown). Earlier studies by Villa-Moruzzi (27) had shown that insulin treatment of differentiated 3T3 L1 adipocytes in culture resulted in the stimulation of cytosolic PP-1 activity. More importantly, trypsin treatment of the cytosolic extracts was required to detect significant insulin effect. The insulin effect was attributed to stimulation of the FA/GSK-3. The present studies do not confirm the above observations. While trypsin treatment of the particulate fractions resulted in considerable increase in basal PP-1 activity toward phosphorylase a, the cytosolic PP-1 activity was not increased in control or insulin-stimulated cells. The reason for this discrepancy is not clear at present. It could be due to cultured cells as opposed to the freshly isolated adipose cells used in the present study.


Figure 1: Distribution of spontaneous and total (trypsinreleased) PP-1 and PP-2A activities in the adipocyte subcellular fractions. Protein phosphatases were extracted as detailed in the text, and subcellular fractions were prepared and assayed for the phosphatase activities using P-labeled phosphorylase as a substrate. Results are the mean ± S.E. of four independent experiments performed in duplicates. 1, spontaneous activities; 2, trypsin-released total activities.




Figure 2: Time course of PP-1 activation by insulin. Adipocytes were treated with 4 nM insulin for 0-30 min. PP-1 activity was measured in the particulate fractions. Results are the mean of four independent experiments ± S.E. performed in duplicate.



The stimulation of PP-1 activity by insulin was dose-dependent, with a maximal effect observed at 4 nM (Fig. 3). Higher concentrations of insulin were inhibitory. Although the molecular basis for the biphasic effect of insulin on PP-1 activity is not understood, these results are similar to the findings of Chan et al.(28) in 3T3-D1 fibroblasts.


Figure 3: Dose response of PP-1 activation by insulin. Adipocytes were treated with various doses of insulin for 5 min, followed by the assay of enzyme activity in the PF. Results are the mean of two independent experiments.



The activation of PP-1 by insulin could theoretically occur at the level of protein synthesis or by post-translational modification of the enzyme. Western blot analysis of particulate fractions with PP-1 catalytic subunit antibody revealed similar amounts of a 37-kDa catalytic domain in both control and insulin-treated adipocytes. The identity of 37-kDa protein as PP-1C subunit was verified by competition studies with a peptide corresponding to the 14-amino acid sequence at the COOH-terminal region of rabbit skeletal muscle PP-1C subunit (data not shown).

Because PP-1 is known to exist as a complex bound to different targeting subunits, the nature of the regulatory subunit of adipocyte PP-1 was examined. Adipocyte membranes were solubilized in 0.5% Triton X-100, and the proteins were subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted with an antibody raised against a peptide surrounding the site-1 sequence of rabbit skeletal muscle PP-1G. Earlier studies by Cohen et al.(5, 6) have suggested that the targeting subunit of PP-1 in the sarcoplasmic reticulum is similar to glycogen-bound PP-1G subunit, thereby raising the possibility that one targeting subunit might associate with different cell organelles. The results of immunoblot analysis indicate that adipocyte membranes contain a protein of molecular mass of 160 kDa that binds to PP-1G subunit antibody (Fig. 4, lane3). This protein was not detected in the cytosolic fraction (Fig. 4, lane4), and the binding of the antibody to this protein was blocked by the presence of peptide against which the antibody was made (Fig. 4, lane5). Further confirmation that the adipocytes contain a protein that might be the regulatory subunit of PP-1 came from experiments in which the solubilized membrane proteins were immunoprecipitated with PP-1G subunit antibody (Table 1). In control preparations, 40% of membranous PP-1 activity was immunoprecipitated by the antibody. Insulin treatment resulted in 120% increase in immunoprecipitable PP-1 activity while the supernatants did not show an insulin effect, suggesting that insulin activates PP-1G in the adipocytes without affecting the other forms of PP-1.


Figure 4: Detection of PP-1 regulatory subunit in the adipocyte particulate fractions by Western blot analysis with PP-1G subunit antibody. Lane1, molecular weight marker; lane2, rabbit skeletal muscle extract (10 µg of protein); lane3, adipocyte particulate fraction (50 µg of protein); lane4, cytosolic fraction; lane5, particulate fraction probed with PP-1G subunit antibody in the presence of antigen.





Since the activation of PP-1 by insulin in the skeletal muscle involves phosphorylation of site-1 on the regulatory subunit of the enzyme, experiments were performed comparing the phosphorylation status of the putative regulatory subunit of adipocyte PP-1 in the control and insulin-stimulated cells. In these experiments, adipocytes were metabolically labeled with [P]orthophosphate for 2 h followed by treatment with insulin. The labeled proteins present in the PF and CF were immunoprecipitated with PP-1G subunit antibody, and the immunoprecipitates were subjected to SDS-PAGE followed by autoradiography. Parallel experiments were performed on cells that were treated with cold phosphate followed by immunoprecipitation and Western blot analysis to quantitate the amount of the regulatory subunit. The amount of P incorporated into the regulatory subunit was determined by densitometric analysis of the autoradiograms of phosphorylated proteins. The extent of phosphorylation was quantitated after normalizing for variations in the contents of proteins in the immunoprecipitates. Insulin treatment resulted in a 2-fold increase in the P incorporation into 160-kDa protein (Fig. 5, lane2). This protein was absent in the cytosolic fractions (Fig. 5, lanes3 and 4) and it was not immunoprecipitated by the preimmune serum (Fig. 5, lanes5 and 6); the immunoprecipitation was completely blocked by the competing peptide (Fig. 5, lanes7 and 8).


Figure 5: Phosphorylation of adipocyte PP-1 regulatory subunit by insulin. P-Labeled adipocytes were exposed to insulin (4 nM) for 5 min. Triton-solubilized particulate or cytosolic fractions (100 µg of protein) were precleared by treatment rat IgG (10 µg) precoupled to protein A-Sepharose. The supernatants were incubated with PP-1G subunit antibody (20 µg) precoupled to protein A-Sepharose for 1 h at room temperature with constant mixing. The immunoprecipitates were washed four times in lysis buffer, eluted with 2 times LSB, followed by separation on a 7.5% SDS-polyacrylamide gel and autoradiography. A representative autoradiogram is shown. Lane1, control PF; lane2, insulin-treated adipocyte PF; lanes3 and 4, CF from control and insulin-treated adipocytes; lanes5 and 6, adipocyte PF from control and insulin-treated cells immunoprecipitated with preimmune serum; lanes7 and 8, immunoprecipitates from control and insulin-treated adipocyte PFs in the presence of excess antigen.



From the above results, it appears that in the intact adipose tissue, insulin stimulates PP-1 activity by increasing the phosphorylation on the regulatory subunit. Further studies are needed on a highly purified enzyme preparation to examine the sites and the amino acid residues that are phosphorylated in response to insulin and whether the activation state of PP-1 could be modulated by another phosphatase.

To understand the mechanism of PP-1 activation by insulin and to determine the upstream activators of this enzyme, the contribution of PI-3 kinase and Ras/MAP kinase signaling pathways were examined. To investigate the role of PI3 kinase, cells were incubated with wortmannin, a specific inhibitor of PI3 kinase. Pretreatment of cells with maximally effective concentrations of wortmannin did not block insulin's effect on PP-1 (Fig. 6). Studies by Klarlund et al.(29) have shown that incubation of permeabilized 3T3 L1 adipocytes in culture with the non-hydrolyzable GTP analogues results in a rapid activation of MAP kinase and S6 kinases. To examine the role of Ras/MAP kinase pathway, two complimentary approaches were used. In the first approach, alpha toxin-permeabilized adipocytes were exposed to a non-hydrolyzable analogue of GTP, GTPS, or a GTP antagonist, GDPbetaS, in the presence or absence of insulin, and PP-1 activation was monitored. In the second approach, MAP kinase activation was blocked by treatment of cells with a cAMP agonist, SpcAMP, and ML-9, a MAP kinase/S6 kinase inhibitor previously reported to inhibit insulin effect on glucose transport(30) . If MAP kinase pathway is indeed involved in insulin's effect on adipocyte PP-1, then treatment with GTPS should result in PP-1 stimulation. Treatment of permeabilized cells with GTPS for 5-10 min resulted in 30-40% increase in PP-1 activity (Fig. 7). The extent of stimulation of the enzyme was comparable with insulin. Inclusion of insulin with the GTP analogue did not further increase the stimulation of PP-1. In contrast, presence of a GTP antagonist, GDPbetaS, during insulin exposure completely blocked the effect of insulin on PP-1 activation (Fig. 7). Basal levels of the enzyme were not affected by GTP antagonist or by the alpha toxin that was used to permeabilize the cells to GTP analogues.


Figure 6: Effect of wortmannin, a PI3-kinase inhibitor on PP-1 activation. Adipocytes were exposed to wortmannin (1 µM) for 15 min prior to insulin treatment. PP-1 activity was measured in the PF. Results are the mean ± S.E. of three independent experiments.




Figure 7: GTPS activates PP-1 in permeabilized cells. Adipocytes were permeabilized by treatment with alpha toxin (15 µg/ml) for 10 min, the medium was removed, and adipocytes were resuspended in fresh KRP buffer containing GTPS (1 mM) with and without insulin and incubated for 10 min. A second batch of cells was incubated with GDPbetaS for 10 min followed by insulin exposure for 5 min. Results are the mean ± S.E. of four independent experiments performed in duplicate.



To further confirm that MAP kinase activation indeed results in the activation of PP-1, the kinetics of stimulation of MAP kinase by insulin was studied using myelin basic protein as a substrate. Within 2 min of exposure to insulin, there was a 2-fold increase in MAP kinase activity, with a peak at 5 min and a return to basal levels in 10 min. The effect of insulin was dose-dependent, with a maximum response at 4 nM in 5 min. Treatment of cells with ML-9, a myosin light chain kinase inhibitor, not only blocked the insulin effect on MAP kinase (Fig. 8), it also inhibited PP-1 activation (Fig. 9). In contrast to ML-9, rapamycin, an immunosuppressant drug known to inhibit the 70-kDa S6 kinase I, had no effect on PP-1 stimulation by insulin (results not shown).


Figure 8: Inhibition of MAP kinase activation by ML-9, a myosin light chain kinase inhibitor. Adipocytes were incubated with ML-9 (100 µM) for 10 min followed by insulin (4 nM) treatment for 5 min. MAP kinase activity was assayed as described in the text. Results are the mean of four independent experiments ± S.E.




Figure 9: ML-9 also blocks insulin activation of PP-1. Results are the mean of four independent experiments ± S.E. performed in duplicate.




DISCUSSION

The results presented in this report indicate that approx40% of the membrane-associated PP-1 activity in rat adipocytes is complexed to a protein that may be similar to the G subunit, which targets the rabbit skeletal muscle PP-1 to glycogen protein particles. This conclusion is based on immunoblotting and immunoprecipitation studies with an antibody raised against site-1 sequence of rabbit skeletal muscle PP-1G. The contribution of the other forms of PP-1 could not be assessed in the present study due to lack of specific antibodies. Studies by Hubbard et al.(31) have shown that sarcovesicular PP-1 in rabbit skeletal muscle is similar to PP-1G, suggesting that the G subunit may play a dual role in targeting PP-1 to two different subcellular locations. A dual function for the G subunit is further supported by the predicted amino acid sequence, which contains a hydrophobic region near the COOH terminus that could serve as a membrane-spanning or -anchoring domain(32) . The demonstration of PP-1G/sarcovesicular PP-1-like subunit in adipocyte membranes further reinforces the possibility that reversible targeting by organelle-specific subunits may be a general but important regulatory mechanism for controlling the location, substrate specificity, and activity of PP-1. Insulin rapidly activates the membrane-associated PP-1 in rat adipocytes in a time- and dose-dependent manner. The insulin effect on PP-1 (40-50% above basal values) was consistent and significant (p < 0.05). Insulin did not influence the abundance of particulate PP-1 catalytic subunit (data not shown), thereby suggesting that the observed increase in the enzyme activity is due to activation of the enzyme rather than an increase in the content of PP-1C subunit. Immunoprecipitation studies indicated that insulin activates the PP-1G/sarcovesicular PP-1 form >2-fold without altering the other forms of the enzyme (Table 1). It should be noted, however, that PP-1 activity measurements were performed using the conventional substrate phosphorylase a. This may not be the ideal substrate for adipocyte PP-1, as the major function of adipose tissue is to store lipids in the form of triglycerides and to mobilize fat during certain physiological and pathophysiological states when a cell is challenged with excessive cAMP levels. Further studies are needed to identify the ideal in vitro and in vivo substrates of adipocyte PP-1.

Studies with P-labeled adipocyte membranes suggest that insulin effect was mediated by increased phosphorylation of the putative regulatory subunit of PP-1. Based on the results of the kinetics of PP-1 activation, the dose response data and studies with the inhibitors, viz. SpcAMP and ML-9, the effects of insulin on PP-1 appear to be mediated via the activation of MAP kinase cascade, which in turn phosphorylates and activates 90-kDa ribosomal S6 kinase II. This enzyme has been previously reported to be homologous to insulin-stimulated protein kinase isolated from the skeletal muscles of rabbits(8) . Insulin-stimulated protein kinase activates PP-1 by increasing site-1 phosphorylation on its regulatory subunit(7) . Recent studies from this laboratory have also shown that insulin stimulates PP-1 activity in cultured rat skeletal muscle cells by increasing phosphorylation of a 160-kDa regulatory subunit of PP-1(25) . A similar mechanism appears to be responsible for the activation of PP-1 by insulin in adipocyte membranes.

The above findings are in agreement with the concept that a large proportion of PP-1 in many eukaryotic cells and tissues is particulate (4) . Recent in vitro studies with the bacterially expressed three isoforms of the catalytic subunit of PP-1 suggest that inhibitor 2 is critical for the correct folding of nascent PP-1C molecules, and it acts as a cytosolic reservoir of PP-1C molecules that could be directed to various subcellular locations when required, following the synthesis of specific targeting subunits(33) . The function of inhibitor 2 in the adipocytes cannot be determined from the results of present studies.

A number of recent studies indicate that p21/MAP kinase pathway plays a pivotal role in insulin's effects on mitogenesis (11, 12, 13, 14, 15) . However, the role of p21/MAP kinase in metabolic effects of insulin on glucose transport and glycogen synthesis remains controversial(34, 35, 36) . Given the fact that in the rabbit skeletal muscle, PP-1 activation is due to phosphorylation of site-1 of the G subunit by an insulin-stimulated protein kinase, we began studies to identify the upstream activators of PP-1 and the insulin signaling pathway(s) responsible for phosphorylation and activation of PP-1. Since insulin rapidly activates both PI3 kinase and MAP kinase pathways, experiments were designed to evaluate the contribution of each of these pathways. As seen in Fig. 6, inhibition of PI3 kinase with a specific inhibitor, wortmannin, did not block PP-1 activation. However, treatment of adipocytes with a GTP antagonist, GDPbetaS, completely blocked insulin stimulation of MAP kinase/S6 kinase (29) as well as PP-1 (Fig. 7). In addition, treatment of permeabilized adipocytes with GTPS mimicked insulin's effect on PP-1 activation as well as MAP kinase activation reported earlier. The findings that PP-1 activation is mediated in rat adipocytes via the activation of Ras/MAP kinase pathway is further supported by experiments with cAMP agonist, SpcAMP. Treatment of adipocytes with SpcAMP not only blocked MAP kinase activation by insulin but also inhibited insulin effect on PP-1 activation (data not shown). The antagonism between cAMP and MAP kinase pathway in different cell systems is well documented (37, 38, 39, 40, 41) . The present findings on inhibition of insulin's effect on PP-1 by a cAMP agonist lend further support to the earlier observations of the counterregulatory effects of adrenalin on insulin. Whether it is due to site-specific phosphorylation on PP-1 as well as MAP kinase inhibition is not known.

In summary, the present study indicates that insulin rapidly activates a membranous form of adipocyte PP-1 that is similar to PP-1G, and the activation is mediated via Ras/MAP kinase pathway.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Diabetes Research Laboratory, Winthrop University Hospital, 259 First St., Mineola, NY 11501. Tel.: 516-663-3915; Fax: 516-663-2798.

(^1)
The abbreviations used are: PP-1, phosphatase-1; MAP kinase, mitogen-activated protein kinase; CF, cytosolic fraction; PF, particulate fraction; STI, soya trypsin inhibitor; GDPbetaS, guanosine 5`-O-2(thio)diphosphate; PMSF, phenylmethylsulfonyl fluoride; GTPS, guanosine 5`-3-O-(thio)triphosphate; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; OA, okadaic acid.


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