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Volume 270, Number 35, Issue of September 01, pp. 20801-20807, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mitogen-activated Protein Kinase Kinase Inhibition Does Not Block the Stimulation of Glucose Utilization by Insulin (*)

(Received for publication, April 21, 1995; and in revised form, June 8, 1995)

Dan F. Lazar ,  Russell J. Wiese ,  Matthew J. Brady ,  Cynthia Corley Mastick ,  Steven B. Waters (1),  Keishi Yamauchi (1),  Jeffrey E. Pessin (1),  Pedro Cuatrecasas ,  Alan R. Saltiel (§)

From the Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Ann Arbor, Michigan 48105 and the Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Insulin stimulates the activity of mitogen-activated protein kinase (MAPK) via its upstream activator, MAPK kinase (MEK), a dual specificity kinase that phosphorylates MAPK on threonine and tyrosine. The potential role of MAPK activation in insulin action was investigated with the specific MEK inhibitor PD98059. Insulin stimulation of MAPK activity in 3T3-L1 adipocytes (2.7-fold) and L6 myotubes (1.4-fold) was completely abolished by pretreatment of cells with the MEK inhibitor, as was the phosphorylation of MAPK and pp90, and the transcriptional activation of c-fos. Insulin receptor autophosphorylation on tyrosine residues and activation of phosphatidylinositol 3`-kinase were unaffected. Pretreatment of cells with PD98059 had no effect on basal and insulin-stimulated glucose uptake, lipogenesis, and glycogen synthesis. Glycogen synthase activity in extracts from 3T3-L1 adipocytes and L6 myotubes was increased 3-fold and 1.7-fold, respectively, by insulin. Pretreatment with 10 µM PD98059 was without effect. Similarly, the 2-fold activation of protein phosphatase 1 by insulin was insensitive to PD98059. These results indicate that stimulation of the MAPK pathway by insulin is not required for many of the metabolic activities of the hormone in cultured fat and muscle cells.

INTRODUCTION

Insulin is the most potent physiological anabolic agent known. It promotes the synthesis and storage of carbohydrates, lipids, and proteins and inhibits their degradation and release into the circulation. While the precise intracellular events that mediate insulin action are not well understood, regulation of protein phosphorylation is believed to play a critical role (Saltiel, 1994). The insulin receptor, a heterotetrameric protein complex, undergoes autophosphorylation on tyrosine residues upon binding of hormone, thereby increasing its tyrosine kinase activity and the tyrosine phosphorylation of specific intracellular proteins (Kasuga et al., 1982; Rees-Jones and Taylor, 1985). Distal to receptor activation, insulin regulates serine and threonine phosphorylation, paradoxically stimulating the phosphorylation of some proteins while causing the dephosphorylation of others (Czech et al., 1988; Rosen, 1987; Saltiel, 1990). Many of the serine/threonine phosphorylations induced by insulin are shared by other growth factors. In contrast, the dephosphorylation of proteins observed with insulin is unique. Indeed, many of the rate-limiting enzymes involved in glucose and lipid metabolism, such as glycogen synthase, hormone-sensitive lipase, and pyruvate dehydrogenase are regulated through dephosphorylation mechanisms. Thus, these dephosphorylations are likely to be critical to many of the metabolic effects of insulin, including stimulation of glycogen and lipid synthesis, and inhibition of lipolysis.

The best characterized pathway leading to insulin-dependent serine phosphorylation is the MAPK (^1)cascade. This pathway is initiated by tyrosine phosphorylation of insulin receptor substrate 1 or Shc proteins by the receptor kinase, inducing their association with the SH2 domain of the adapter protein Grb2 (Sasaoka et al., 1994b; Skolnik et al., 1993b). This association with phosphorylated Shc induces Grb2 to target the nucleotide exchange factor SOS, which in turn associates with and activates the GTP-binding protein p21 (Rozakis-Adcock et al., 1992; Sasaoka et al., 1994a; Skolnik et al., 1993a). p21 activation leads to the stimulation of Raf and other kinases (Thomas et al., 1992; Wood et al., 1992). These kinases can phosphorylate MAPK kinase, or MEK (Dent et al., 1992; Kyriakis et al., 1992; Zheng et al., 1994), a dual specificity kinase that catalyzes the phosphorylation of MAPK on threonine and tyrosine residues, causing its activation (Crews et al., 1992; Kosako et al., 1992). MAPK has a number of substrates, including transcription factors (Gille et al., 1992; Pulverer et al., 1991), phospholipase A(2) (Lin et al., 1993; Nemenoff et al., 1993), and other kinases, such as ribosomal S6 kinase II, or pp90 (Sturgill et al., 1988). Dent et al.(1990) have suggested that the phosphorylation and activation of pp90 by activated MAPK increases its activity toward site 1 on the regulatory glycogen-binding subunit (PP1G) of type 1 protein phosphatase (PP1), based on a series of reconstitution experiments. Increased phosphorylation of this site has also been detected in PP1G isolated from rabbit skeletal muscle following insulin treatment. The phosphorylation of this regulatory subunit produces a 2-fold increase in the PP1-catalyzed dephosphorylation of glycogen synthase and phosphorylase kinase, thereby increasing the overall rate of glycogen accumulation.

This model has provided an attractive link between the activation of MAPK by insulin and the stimulation of glycogen synthesis, resolving the apparent paradox of simultaneous stimulation of protein phosphorylation and dephosphorylation by insulin. However, there are inconsistencies with this model. We have further evaluated the role of the MAPK pathway in insulin action in the highly responsive 3T3-L1 adipocytes and L6 myotubes. Using a specific inhibitor of MEK, we demonstrate that MAPK activation is not required for insulin stimulation of PP1 activity and glucose metabolism, including glycogen synthesis, glucose uptake, and lipogenesis.

EXPERIMENTAL PROCEDURES

Materials

3T3-L1 and L6 cells were purchased from ATCC (Rockville, MD). DMEM and calf serum were purchased from Life Technologies, Inc., while fetal bovine serum (FBS) was obtained from Hyclone. Porcine insulin was a generous gift from Eli Lilly. [-P]ATP (3000 Ci/mmol), [U-^14C]glucose (298 mCi/mmol), and 2-[U-^14C]deoxyglucose (323 mCi/mmol) were from DuPont NEN, while UDP-[U-^14C]glucose (254 mCi/mmol) was from ICN. Mouse anti-phosphotyrosine monoclonal antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-MAPK antiserum used for immunoprecipitations was prepared from rabbits immunized with a C-terminal peptide (amino acids 425-445) of pp44 expressed as a GST fusion protein. Anti-ERK1/2 for immunoblotting was obtained from Zymed (San Francisco, CA). SOS polyclonal antibody was from Upstate Biotechnology Inc. For ECL detection, horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG were purchased from Life Technologies, Inc. Phosphatidylinositol was obtained from Avanti Polar Lipids. Anti-pp90 and anti-pp70 rabbit antisera were generous gifts of Dr. John Blenis (Harvard Medical School). Glycogen phosphorylase and phosphorylase kinase were purchased from Sigma, and okadaic acid was obtained from Calbiochem. Other reagents were from Sigma and were of the highest quality available.

Tissue Culture

3T3-L1 fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% calf serum prior to initiation of the differentiation protocol. Differentiation to adipocytes was induced by incubating confluent monolayers for 2 days in DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, and 0.4 µg/ml dexamethasone. Subsequently, cells were incubated for 2 days with 1 µM insulin in DMEM containing 10% FBS. One day after transfer to the same medium without insulin, greater than 85% of the cells expressed the adipocyte phenotype. L6 myoblasts were maintained in DMEM containing 10% FBS until approximately 50% confluent, at which point differentiation was initiated by conversion to DMEM with 2% FBS. Cell fusion was apparent at day 5, and greater than 85% of the culture expressed the myotube phenotype by day 12. Prior to assay, both adipocyte and myotube cultures were serum-starved for 3 h in Krebs-Ringer buffer with 30 mM Hepes (KRBH; pH 7.4) containing 0.5% bovine serum albumin and 2.5 mM glucose.

Immunoblots

After insulin treatment, cells (100-mm dishes) were washed twice with ice-cold phosphate-buffered saline (PBS), then lysed in HNTG buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl(2), 1 mM EDTA, 10 mM sodium pyrophosphate, 1 mM Na(3)VO(4), 30 mMp-nitrophenyl phosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 mM NaF, and 1 mM phenylmethylsulfonyl fluoride (Margolis et al., 1990). Cell lysates were centrifuged (10,000 g; 10 min) to preclear insoluble material, then diluted directly into Laemmli sample buffer. Lysates were then resolved by sodium dodecyl sulfate (SDS), 8% polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose paper, and immunoblotted with anti-phosphotyrosine, anti-pp90, anti-ERK1/2, and anti-SOS antibodies, followed by horseradish peroxidase-goat anti-mouse or horseradish peroxidase-goat anti-rabbit IgG, respectively. pp44 was immunoprecipitated from denatured cell lysates, as described previously (Mastick et al., 1994), prior to anti-phosphotyrosine immunoblotting.

Assay of MAPK Activity

Following insulin treatment of cultures in 12-well dishes, cells were washed twice in ice-cold PBS, then lysed in 50 µl of buffer containing 50 mM beta-glycerol phosphate, 10 mM Hepes, pH 8.0, 70 mM NaCl, 1 mM Na(3)VO(4), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride. Determination of MAPK activity in lysates was performed as described previously (Pang et al., 1993a). After centrifugation (10,000 g; 10 min) to preclear insoluble material, 10-µl aliquots of cell lysate (5-10 µg of protein) were incubated with approximately 5 µg of microtubule-associated protein 2 (MAP2) for 15 min at 25 °C in a final volume of 25 µl containing 50 mM Tris-HCl, pH 7.4, 2 mM EGTA, 10 mM MgCl(2), and 40 µM [-P]ATP (2 µCi). After termination of activity by the addition of 4 Laemmli sample buffer, phosphorylation of MAP2 was determined by resolution on SDS, 6% PAGE, Coomassie Blue staining, excision of MAP2 protein from the gel, and Cerenkov counting of incorporated radioactivity.

Transcriptional Activation of c-fos

3T3-L1 adipocytes were transfected using the calcium phosphate co-precipitation method with CsCl double-banded DNA as described previously (Yamauchi et al., 1993). Briefly, 10-day fully differentiated 3T3-L1 adipocytes were transfected with 15 µg of the serum response element-luciferase (SRE-Luc) and 5 µg of the Rous sarcoma virus-beta-galactosidase reporter plasmid DNAs. Twelve hours after transfection, the cells were placed into serum-free Ham's F12 medium for 12 h and incubated with or without PD98059 for 1 h prior to the addition of 100 nM insulin. Whole cell extracts were prepared at various times for the determination of luciferase (Luc) and beta-galactosidase activities. To correct for differences in transcription efficiencies between plates within an experiment, the luciferase activity in each extract was normalized to beta-galactosidase activity.

Assay of Phosphatidylinositol (PI) 3`-Kinase Activity

This was determined as described previously (Ohmichi et al., 1992). Cells were lysed in 0.5 ml of buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 1 mM Na(3)VO(4), 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. Cell lysates were precleared of insoluble material by centrifugation (10,000 g, 10 min, 4 °C), then preincubated with Pansorbin cells and rabbit IgG-agarose before being subjected to immunoprecipitation with anti-phosphotyrosine antibody and Protein A-agarose. The resulting pellets were thoroughly washed, and associated PI 3`-kinase activity was determined by the incubation of immunoprecipitates with [P]ATP and phosphatidylinositol. Phosphatidylinositol 3-phosphate produced was resolved by thin layer chromatography and visualized by autoradiography.

Determination of 2-Deoxyglucose Uptake

Cells in 12-well dishes were serum-deprived and insulin-treated as above. Assay of glucose uptake was initiated by the addition of 2-[U-^14C]deoxyglucose (1 µCi/well) and 5 mM glucose. After a 15-min incubation at 37 °C, cells were washed three times with ice-cold PBS containing 10 mM glucose, then solubilized in 0.5 M NaOH. Samples were then assessed for radioactivity by scintillation counting in Ready Gel (Beckman).

Glycogen Synthesis and Lipogenesis Assays

The accumulation of glycogen in intact cells was determined by an adaptation of the method of Lawrence et al.(1977), as described previously (Hess et al., 1991). After serum deprivation and pretreatment with or without PD98059, cells in 6-well dishes were incubated in the presence or absence of insulin for an additional 15 min. Subsequently, cells were incubated with 5 mMD-[U-^14C]glucose (2 µCi per well) for 60 min at 37 °C. Cells were then washed three times with ice-cold PBS, solubilized in 30% KOH, and radiolabeled glucose incorporation into glycogen was determined. Alternatively, in 3T3-L1 adipocytes (6-well dishes), radiolabeled glucose incorporation into lipid was assessed by scraping cells into 1 ml of PBS and shaking vigorously with 5 ml of Betafluor scintillant (National Diagnostics, Manville, NJ). After samples settled overnight, radioactivity which partitioned into the organic phase was determined by scintillation counting.

Assay of Glycogen Synthase Activity

Insulin stimulation of glycogen synthase activity was determined as described previously (Robinson et al., 1993; Thomas et al., 1968) with some modifications. Cells (100-mm dishes) were serum-deprived in KRBH with 0.5% bovine serum albumin in the absence of glucose for 3 h, pretreated with or without PD98059 for an additional 30 min, then incubated with insulin for 20 min at 37 °C. After three washes with ice-cold PBS, cells were scraped into 500 µl of glycogen synthase assay buffer (50 mM Tris-HCl, pH 7.8, 10 mM EDTA, and 100 mM KF) and homogenized with a glass-glass dounce homogenizer prior to centrifugation (10,000 g, 20 min). To measure glycogen synthase activity, 50 µl of the supernatant (50-200 µg of protein) was added to an equal volume of original buffer containing 10 mM UDP-[^14C]glucose (0.05-0.15 µCi/µmol) and 15 mg/ml glycogen, in the presence or absence of 10 mM glucose 6-phosphate. After a 15-min incubation at 37 °C, assay tubes were chilled for 15 min in an ice bath. Tube contents were then spotted on prelabeled Whatman filter papers (GF/A; 2.4 cm) which were immediately immersed in 500 ml of 70% ethanol (4 °C), mixed 40 min, then washed two more times in 250 ml of 70% ethanol (15 min and 60 min, respectively) to remove unincorporated substrate from precipitated glycogen. Filters were air-dried, and radioactivity was counted with 5 ml of Ready Gel scintillant. For optimal stimulation of glycogen synthase in 3T3-L1 adipocytes, cells were used 8-12 days post-differentiation.

Assay of PP1 Activity

Following insulin treatment, cell extracts were prepared in homogenization buffer (25 mM Hepes, pH 7.2, 2 mM EDTA, 0.2% beta-mercaptoethanol, 2 mg/ml glycogen, 40 µM leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin) as described previously (Srinivasan and Begum, 1994). PP1 activity in cell extracts (1-3 µg of protein) was determined against 20 µg of substrate ([P]phosphorylase a) for 7 min at 30 °C in 60 µl of homogenization buffer containing 3 nM okadaic acid to block type 2A protein phosphatase activity. After resolution by SDS, 8% PAGE, substrate was excised from the gel, and residual radioactivity was determined by scintillation counting. P-Labeled phosphorylase a was prepared as described (Cohen et al., 1988). Free [-P]ATP was removed from radiolabeled substrate by the use of Bio-Gel P-6 spin columns (Bio-Rad). As per glycogen synthase activation, optimal activation of PP1 by insulin was observed in 3T3-L1 adipocytes that were 8-12 days post-differentiation.

RESULTS

PD98059 Inhibits the Activation of MAPK by Insulin

The structure of PD98059 is shown in Fig. 1. This compound is a selective inhibitor of MAPK kinase, or MEK, and is noncompetitive with respect to ATP binding to MEK. (^2)3T3-L1 adipocytes and L6 myotubes were incubated for 30 min in the presence or absence of PD98059 prior to treatment with insulin. MAPK activity was assessed in cell extracts by examining in vitro phosphorylation of the specific substrate MAP2. Insulin treatment of 3T3-L1 adipocytes resulted in a 2.7-fold increase in MAP2 phosphorylation (Fig. 2A) which was maximal at 5 min and gradually decreased thereafter (Wiese et al., 1995). Pretreatment of adipocytes with PD98059 completely abolished the stimulation of MAPK by insulin, with an IC of approximately 1 µM and a maximal inhibitory effect obtained with 10 µM (Fig. 3A). 10 µM PD98059 prevented stimulation of MAPK activity by insulin up to 60 min after hormone treatment (data not shown). Insulin produces a more modest (35%) stimulation of MAPK activity in L6 myotubes. This increase was also completely blocked upon pretreatment of cells with 10 µM inhibitor (Fig. 2B). Basal activities were not significantly affected by incubation with inhibitor alone in either cell line. Ion-exchange chromatography of lysates from 3T3-L1 adipocytes revealed ERK-1 and ERK-2 as the only MAPK family members activated by insulin. PD98059 completely blocked activation by insulin of both forms of the enzyme. (^3)

Figure 1: Structure of PD98059.


Figure 2: PD98059 blocks the activation of MAPK by insulin. Serum-deprived 3T3-L1 adipocytes (A) and L6 myotubes (B) were treated with (shaded bars) and without (hatched bars) 10 µM PD98059 for 30 min prior to the addition of insulin (A, 100 nM; B, 300 nM) for 5 min. Cells were lysed, and MAPK activity was assayed as described under ``Experimental Procedures.'' Shown are the means + S.E. of three separate experiments, each performed in duplicate. Basal activities were 76 and 127 Cerenkov/µg of protein for 3T3-L1 adipocytes and L6 myotubes, respectively.


Figure 3: Concentration-dependent blockade of MAPK phosphorylation and activation by PD98059. 3T3-L1 adipocytes were treated for 30 min with increasing concentrations of PD98059 followed by 100 nM insulin for 5 min. A, MAPK activity in cell lysates was determined. Shown are the means + S.E. from three separate experiments, each performed in duplicate. B, anti-MAPK immunoprecipitates were resolved on SDS, 8% PAGE and subjected to Western blotting with anti-phosphotyrosine antibody. C, following pretreatment with 100 µM PD98059 for 60 min, cells were treated with insulin. Cell lysates (75 µg of protein) were resolved by SDS-PAGE, then immunoblotted with anti-ERK1/2 antisera.


Activation of MAPK is known to involve increased tyrosine and threonine phosphorylation of the kinase. To verify the inhibitory effect of PD98059 on MAPK activation by insulin, we examined the tyrosine phosphorylation of the enzyme. 3T3-L1 adipocytes were treated with 100 nM insulin in the presence or absence of PD98059. MAPK was immunoprecipitated, and tyrosine phosphorylation was evaluated by SDS-PAGE followed by immunoblotting with anti-phosphotyrosine antibody (Fig. 3B). Insulin treatment produced a significant increase in the tyrosine phosphorylation of pp44. This increased phosphorylation was inhibited in a concentration-dependent manner by pretreatment of cells with PD98059, with negligible tyrosine phosphorylation of MAPK remaining after incubation with 10 µM concentration of the compound. The activation of MAPK also results in a characteristic change in its SDS-PAGE mobility due to threonine and tyrosine phosphorylation (de Vries-Smits et al., 1992). Pretreatment of 3T3-L1 adipocytes with PD98059 completely blocked the insulin-induced gel shift of MAPK (Fig. 3C).

Treatment of cells with insulin has also been shown to increase the activities of other kinases, including pp90 (Erikson, 1991) and pp70 (Thomas, 1992). Activation of these kinases requires serine/threonine phosphorylation, also reflected by reduced mobility on SDS-PAGE (Blenis et al., 1991). pp90 is thought to be directly activated by a MAPK-catalyzed phosphorylation (Sturgill et al., 1988). To evaluate the role of MAPK in pp90 phosphorylation, 3T3-L1 adipocytes were treated with insulin, and pp90 and pp70 were detected by Western blotting. Insulin caused a shift in electrophoretic mobilities of both pp90 and pp70. Prior incubation of cells with 10 µM PD98059 completely prevented the insulin-stimulated shift in pp90 mobility (Fig. 4A), consistent with the successful blockade of MAPK activation in vivo. The mobility shift of pp70 was unaffected by inhibitor pretreatment (data not shown), confirming a lack of involvement of MAPK in this particular response to insulin (Ballou et al., 1990; Blenis et al., 1991). In addition to pp90, the guanine nucleotide exchange factor SOS is also believed to be a direct substrate of MAPK (Waters et al., 1995; Cherniack et al., 1994). Pretreatment of 3T3-L1 adipocytes with the MEK inhibitor completely blocked the insulin-stimulated SOS gel shift characteristic of serine/threonine phosphorylation (Fig. 4B).

Figure 4: PD98059 blocks insulin-stimulated phosphorylation of both pp90 and SOS and insulin stimulation of c-fos transcription. A, serum-deprived 3T3-L1 adipocytes were treated with or without 10 µM PD98059 for 30 min, followed by 100 nM insulin for 5 min. Cell lysates were resolved by SDS, 8% PAGE then immunoblotted with anti-pp90. B, serum-deprived 3T3-L1 adipocytes were treated with 100 µM PD98059 for 60 min, followed by 100 nM insulin for 15 min. Cell lysates were resolved by SDS, 5-10% PAGE and immunoblotted with anti-SOS. C, 3T3-L1 adipocytes were transfected with SRE-Luc and RSV-beta-galactosidase as described under ``Experimental Procedures,'' serum-deprived for 12 h, then treated with (solid bars) or without (hatched bars) 100 µM PD98059 for 60 min. Following the treatment of cells with 100 nM insulin for the indicated times, luciferase and beta-galactosidase activities were determined in cell extracts. Shown are the means + S.E. of two independent determinations, each performed in triplicate.


Previous studies have demonstrated that the c-fos serum response element (SRE) mediates the insulin-stimulated transcription of the c-fos gene (Stumpo et al., 1988). This is generally believed to occur via MAPK-dependent phosphorylation of the TCF/Elk-1 and SRF transcription factors (Gille et al., 1992). We therefore examined the effect of the MEK inhibitor on c-fos transcription using the SRE-luciferase (Luc) reporter gene construct (Yamauchi et al., 1993). 3T3-L1 adipocytes transfected with this construct demonstrate 1.6-fold and 1.8-fold increases in luciferase activity following insulin treatments of 1 and 2 h, respectively (Fig. 4C). Pretreatment of cells with PD98059 completely blocked the stimulation of luciferase activity at these time points, in agreement with insulin stimulation of c-fos transcription by a MAPK-dependent pathway.

Specificity of the Inhibitory Effects of PD98059

PD98059 inhibits MEK activity in a manner which is not competitive with either substrate (MAPK) or ATP binding and has been shown to be without effect on MAPK activity itself, as well as the activity of other serine kinases.^2 Furthermore, nerve growth factor-, epidermal growth factor-, and platelet-derived growth factor-receptor tyrosine autophosphorylation is completely insensitive to PD98059 pretreatment of cells.^2 To evaluate the specificity of this compound in blocking MAPK activation in both 3T3-L1 adipocytes and L6 myotubes, insulin receptor autophosphorylation was evaluated. PD98059 treatment was without effect on insulin-dependent phosphorylation of the receptor, as determined by anti-phosphotyrosine immunoblotting (Fig. 5). In the same immunoblot of cell lysates, insulin-stimulated tyrosine phosphorylation of both the 42- and 44-kDa isoforms of MAPK was effectively blocked by pretreatment with 10 µM PD98059.

Figure 5: PD98059 differentially blocks insulin-stimulated tyrosine phosphorylation. Serum-deprived 3T3-L1 adipocytes and L6 myotubes were treated with or without 10 µM PD98059, followed by insulin for 5 min, as indicated. Cell lysates (100 µg of protein) were resolved by SDS, 8% PAGE and immunoblotted with anti-phosphotyrosine antibody. Shown are the predicted positions of the insulin receptor and the 42- and 44-kDa isoforms of MAPK protein.


Upon activation, the insulin receptor catalyzes the tyrosine phosphorylation of its major substrate, insulin receptor substrate 1, resulting in its selective association with proteins containing SH2 domains (Sun et al., 1991, 1993). One such protein, PI 3`-kinase, undergoes activation upon occupancy of the SH2 domains of its 85-kDa regulatory subunit (Myers et al., 1992). Pretreatment of 3T3-L1 adipocytes with 10 µM PD98059 did not reduce activation of PI 3`-kinase by insulin, as detected in anti-phosphotyrosine immunoprecipitates (Fig. 6). Moreover, the MEK inhibitor had no effect on PI 3`-kinase activity when added directly to the in vitro assay (data not shown).

Figure 6: PD98059 does not block activation of PI 3`-kinase by insulin. 3T3-L1 adipocytes were treated with and without PD98059, followed by insulin treatment for 5 min. PI 3`-kinase activity associated with anti-phosphotyrosine immunoprecipitates was determined as described under ``Experimental Procedures.'' Shown is a representative result obtained in two separate determinations. PI3P, phosphatidylinositol 3`-phosphate.


Stimulation of Glucose Uptake and Lipid Synthesis by Insulin Does Not Require MAPK Activation

Differentiated 3T3-L1 cells respond to insulin treatment with marked increases in glucose uptake and metabolism. Incubation of cells with 100 nM insulin for 15 min produced a 9-fold increase in the uptake of the nonmetabolizable glucose analog, 2-deoxy-D-glucose in the presence of 5 mM unlabeled glucose (Fig. 7A). This stimulation of glucose transport was unaffected by prior treatment of cells with 10 µM PD98059. Similar results were obtained when 2-deoxy-D-glucose uptake was determined in the presence of 100 µM unlabeled glucose (data not shown). These data demonstrate that MAPK activation is not required for insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Insulin also increases the rate of lipid synthesis in these cells. Exposure of 3T3-L1 adipocytes to insulin produced a 7-fold increase in the conversion of radiolabeled glucose into lipid (Fig. 7B). This stimulation of lipogenesis was unaffected by pretreatment of cells with 10 µM PD98059. Moreover, the dose-response for insulin stimulation of lipid synthesis (EC approximately 3 nM) was not affected by the MEK inhibitor (data not shown). Basal activities were unaltered by the presence of inhibitor in both glucose uptake and lipogenesis assays.

Figure 7: Insulin-stimulated 2-deoxyglucose uptake and lipid synthesis are insensitive to PD98059. Insulin (100 nM) stimulation of 2-[U-^14C]deoxyglucose uptake (A) and [U-^14C]glucose incorporation into lipid (B) were determined in 3T3-L1 adipocytes following pretreatment with (solid bars) and without (hatched bars) 10 µM PD98059. Results are the means + S.E. from individual experiments performed in triplicate and are representative of three separate experiments.


The Stimulation of Glycogen Synthesis by Insulin Does Not Require MAPK Activation

The potential role of MAPK activation in the stimulation of glycogen synthesis by insulin was examined in both 3T3-L1 adipocytes and L6 myotubes by determining the incorporation of ^14C-labeled glucose into glycogen in the presence and absence of 10 µM PD98059 (Fig. 8A). Insulin stimulated the rate of glycogen synthesis in a dose-dependent manner in both cell lines, with an EC of approximately 5 nM in 3T3-L1 adipocytes and 30 nM in L6 myotubes. Concentrations of PD98059 sufficient to completely block insulin stimulation of MAPK activity had no effect on either the sensitivity or maximal stimulation of glycogen synthesis in these cells.

Figure 8: PD98059 does not affect insulin stimulation of glycogen synthesis. 3T3-L1 adipocytes and L6 myotubes were treated with (solid bars) and without (hatched bars) 10 µM PD98059 for 30 min, followed by insulin treatment. [U-^14C]Glucose incorporation into glycogen in intact cells (A) and glycogen synthase activity (±10 mM glucose 6-phosphate) in broken cell extracts (B) were determined as described under ``Experimental Procedures.'' Results are the means + S.E. of three separate experiments, each performed in triplicate.


The hormonal regulation of glycogen synthesis is primarily mediated by modulation of the activity of glycogen synthase. This enzyme is stimulated by its allosteric activator, glucose 6-phosphate, and by dephosphorylation. Glycogen synthase activity was assayed in lysates from 3T3-L1 adipocytes treated with 100 nM insulin for 20 min in the absence of extracellular glucose to eliminate the allosteric activation by glucose 6-phosphate that is produced upon insulin-stimulated glucose uptake. Insulin treatment produced a 3-fold increase in the glycogen synthase activity ratio, regardless of whether or not cells were pretreated with 10 µM PD98059 (Fig. 8B). Insulin (300 nM) produced a 1.7-fold increase in the glycogen synthase activity ratio in the myotubes, which also was unaffected by pretreatment with 10 µM PD98059 (Fig. 8B). Incubation with inhibitor alone had no effect on basal glycogen synthase activity, and total activity was not significantly altered by insulin and/or PD98059 treatment (data not shown). These results clearly demonstrate that MAPK activation is not required for insulin stimulation of glycogen synthase activity and the accumulation of glycogen in 3T3-L1 adipocytes and L6 myotubes.

Stimulation of Type 1 Protein Phosphatase Activity by Insulin Does Not Require MAPK Activation

Stimulation of PP1 activity is believed to be critical for many of the metabolic effects of insulin, including the stimulation of glycogen synthesis (Hess et al., 1991; Tanti et al., 1991). This enzyme was assayed by following the in vitro dephosphorylation of P-labeled glycogen phosphorylase in cell lysates (Fig. 9). In order to specifically assay PP1 activity, release of P was monitored in the presence of 3 nM okadaic acid, which completely inhibits type 2A protein phosphatase. Insulin treatment of 3T3-L1 adipocytes produced a 1.8-fold activation of PP1. This enzyme activity was similarly stimulated by insulin in L6 myotubes. In both cell lines, insulin stimulation of PP1 activity was unaffected by prior treatment of cells with 10 µM PD98059, and little or no effect was observed on basal activity.

Figure 9: PD98059 does not affect insulin stimulation of PP1 activity. Serum-deprived 3T3-L1 adipocytes and L6 myotubes were treated with (solid bars) and without (hatched bars) 10 µM PD98059 for 30 min prior to the addition of insulin (A, 100 nM; B, 10 nM) for 10 min. Cell extracts were prepared, and PP1 activity was assayed as described under ``Experimental Procedures.'' Shown are the means + S.E. of four separate experiments, each performed in triplicate.


DISCUSSION

The regulation of protein phosphorylation appears to be a central component in the pleiotropic actions of insulin (Saltiel, 1994). The insulin-dependent autophosphorylation of the receptor and activation of its tyrosine kinase activity leads to the subsequent tyrosine phosphorylation of several intracellular proteins, including insulin receptor substrate 1 (Sun et al., 1991) and Shc (Pronk et al., 1993). It is likely that the phosphorylation of these and other receptor substrates induces a series of protein-protein interactions, leading ultimately to changes in serine/threonine phosphorylation levels, paradoxically increasing the activities of both kinases and phosphatases that target numerous intracellular proteins (Czech et al., 1988; Rosen, 1987; Saltiel, 1990). Studies with mutant insulin receptors (McClain, 1990; Moller et al., 1991; Pang et al., 1993b; Pang et al., 1994; Rolband et al., 1993; Takata et al., 1991), wild-type receptors in particular cell lines (Ohmichi et al., 1993), or anti-receptor antibodies (Sung, 1991; Wilden et al., 1992) indicate that the activation of protein serine kinases and phosphatases may diverge at or near the receptor. One pathway leading to serine kinase activation which has been fairly well defined is activation of MAPK. The activity of this enzyme, first detected in insulin-treated 3T3-L1 cells (Ray and Sturgill, 1987), and later found to be activated by a number of other growth factors and mitogens, results from a well characterized cascade of events. While many of the molecular components involved in the activation of downstream serine/threonine kinases such as MAPK have been elucidated, less progress has been made in understanding the events that are more relevant to the metabolic effects of insulin, the activation of serine/threonine phosphatase activity. An attractive model has emerged (Dent et al., 1990) linking MAPK with stimulation of the type 1 protein phosphatase (PP1) responsible for activation of glycogen synthase and inactivation of phosphorylase kinase and glycogen phosphorylase. The MAPK-activated pp90 kinase can phosphorylate site 1 on the regulatory G subunit of PP1 in vitro, increasing the activity of the phosphatase toward glycogen synthase and phosphorylase kinase. However, evidence from several studies contradicts a central role for MAPK activation in this particular response. Agents such as phorbol esters or okadaic acid can activate MAPK, yet they antagonize the metabolic effects of insulin (Corvera et al., 1991; Hess et al., 1991). Moreover, platelet-derived growth factor and epidermal growth factor potently activate the MAPK pathway in 3T3-L1 adipocytes, but are ineffective in stimulating glycogen synthesis, suggesting that MAPK activation is not sufficient to produce this response (Robinson et al., 1993; Wiese et al., 1995). Furthermore, experiments in a number of cell lines expressing wild-type or mutant insulin receptors (Moller et al., 1991; Ohmichi et al., 1993; Pang et al., 1993b; Pang et al., 1994) or downstream effectors (Sakaue et al., 1995) have dissociated MAPK activation from metabolic responses, indicating that activation of this enzyme is not even required for insulin stimulation of glycogen synthesis. However, these latter studies were performed in cell lines not considered representative of the primary target tissues of insulin, especially with regard to glucose metabolism.

In order to determine whether MAPK activation is required for insulin stimulation of glucose metabolism in more classical insulin-responsive cell lines, we have studied insulin action and the involvement of MAPK activation in 3T3-L1 adipocytes and L6 myotubes. 3T3-L1 adipocytes are well-suited for the study of insulin-stimulated glucose metabolism. In addition to glycogen and lipid synthesis, glucose transport is insulin-sensitive in these cells due to expression of the insulin-responsive glucose transporter, Glut4 (Garcia de Herreros and Birnbaum, 1989). L6 myotubes are also a useful model system for studies of insulin action. Although these cells do not express Glut4, the regulation of glycogen synthesis by insulin via dephosphorylation of glycogen synthase resembles that observed in intact muscle. Using the specific MEK inhibitor PD98059, which blocks the phosphorylation and activation of MAPK in both cell-based and cell-free assays, we have found that complete blockade of MAPK activation and subsequent pp90 phosphorylation was without effect on insulin stimulation of glucose utilization, although both SOS phosphorylation and transcriptional activation of c-fos were completely inhibited. The stimulation of glucose uptake, lipogenesis, and glycogen synthesis were unaltered by blockade of MAPK activation. Moreover, stimulation of glycogen synthase and PP1 activities by insulin were also unaffected by MEK inhibition. The possibility remains that significant activation of PP1 via MAPK-activated pp90 does occur, but is not required due to the potential existence of an alternative pathway for the stimulation of PP1. In the event of such redundant signaling, one might expect the MEK inhibitor to produce decreased insulin sensitivity or maximal response for insulin stimulation of lipid or glycogen accumulation by insulin. However, the dose-response for insulin stimulation of glycogen synthesis was completely unaffected by the abolishment of MAPK activation in both 3T3-L1 adipocytes and L6 myotubes. These results, obtained in highly responsive fat and muscle cell lines, clearly demonstrate that activation of MAPK is not required for insulin stimulation of glycogen synthesis.

The molecular mechanisms by which metabolic enzymes such as glycogen synthase are regulated by insulin remains one of the crucial, unresolved issues in insulin action. While there is considerable evidence that these enzymes are modulated via dephosphorylation mechanisms likely to be catalyzed by protein phosphatase 1 activity, the precise pathway linking the insulin receptor to this activity requires further study.

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 and reprint requests should be addressed: Dept. of Signal Transduction, Parke-Davis Pharmaceutical Research/Warner-Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 313-996-3960; Fax: 313-996-5668.

(^1)
The abbreviations used are: MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; PP1, type 1 protein phosphatase; PAGE, polyacrylamide gel electrophoresis; SRE, serum response element; Luc, luciferase; PI, phosphatidylinositol; FBS, fetal bovine serum; MAP2, microtubule-associated protein 2; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium.

(^2)
Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A., in press.

(^3)
Lin, A. Y., Kong, X., Saltiel, A. R., Blackshear, P., and Lawrence, J. C. (1995) J. Biol. Chem.270, 18531-18538.

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