INTRODUCTION

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Insulin induces mitogenic and metabolic effects in most cell types (54). At the proximal level, phosphorylation of the insulin receptor substrates (IRSs) represents one of the earliest events involved in the eliciting these effects (54). In particular, the ubiquitous insulin receptor substrate 1 (IRS-1) protein appears to play a major role in transduction of insulin proliferative effects (2, 28, 51). IRS-1 possesses multiple tyrosine phosphorylation residues, which allow it to interact with other signaling molecules containing SH2 domains and conveying mitogenic signals further downstream (49, 54). These include phosphatidylinsitol 3-kinase (PI 3-kinase) regulatory subunits (1, 30) and Grb2 (43, 44), an adapter protein which interacts with the exchange factor Son of Sevenless (SOS) and induces Ras activation. Ras functions as an activator of Raf which, in turn, activates mitogen-activated protein kinase kinase (MAPKK) and enables phosphorylation and stimulation of MAPK (19). These events have been shown to be necessary for cell proliferation to occur in response to insulin in most cell types (6, 33). However, there is also evidence that insulin signal can be conveyed into the MAPK pathway independently of Ras (11).

Protein kinase C (PKC) comprises a multigene family that encodes at least 12 distinct isoforms differing in catalytic and regulatory properties (21, 26, 32). These PKC isoforms can be divided into three subgroups based on cofactor requirements: (i) conventional PKCs (such as , , and ) that are dependent on Ca²⁺ and diacylglycerol (DAG) for activity, (ii) novel PKCs (, , , and ) that are not dependent on Ca²⁺ but are activated by DAG, and (iii) atypical PKCs (, , and ) that are not dependent on Ca²⁺ and are not stimulated by DAG (20). PKC plays a pivotal role in controlling numerous cellular functions, including cell proliferation (26). PKC-mediated signaling systems have been shown to be activated following the stimulation of cell surface receptors by several growth factors such as epidermal growth factor (EGF) and platelet-derived growth factor (34, 35). Binding to these growth factor receptors leads to phospholipase activation and production of DAG (36) which, in turn, binds and activates PKC (32). PKCs may then provoke the activation of c-Ras and the Raf-MAPKK-MAPK pathway (24, 25, 41, 45). Also, DAG-regulated PKC may activate Raf independently of Ras (10, 24, 25).

Insulin has been shown to activate phospholipases (23, 46) and to stimulate the activity of several distinct PKC isoforms (3, 7, 18), but whether the activation of specific PKC isoforms is relevant to insulin mitogenic signal remains unknown. Also, the proximal events in insulin signaling leading to PKC activation are unclear. In the present report, we have addressed these issues by analyzing insulin mitogenic signaling in the L6 muscle cells. We have obtained evidence that PKC activation plays a major role in mediating insulin-dependent DNA synthesis in these cells, bypassing Ras and through the Raf-MAPKK-MAPK pathway.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. Media, sera, and antibiotics for cell culture were from Life Technologies, Inc. (Grand Island, N.Y.). The Lipofectamine reagent, rabbit polyclonal antibodies directed against specific PKC isoforms, and the PKC assay system (catalog no. 13161-013) were purchased from Life Technologies, Inc. Recombinant PKC, -, -, and - proteins, the PKC peptide substrate, and PKC pseudosubstrate were from Calbiochem-Novabiochem (La Jolla, Calif.). Polyclonal IR antibodies were from Oncogene Science (Manhasset, N.Y.), and p42/p44 MAPK and Raf-1 antibodies were from Transduction Laboratories, Inc. (Lexington, Ky.). Phosphorylated MAP kinase antibodies were from New England Biolabs (Beverly, Mass.). The IRS-1 ribozyme (IRS-1 rib) and control ribozyme (c-rib) were generous gifts of M. Quon (National Institutes of Health, Bethesda, Md.) (37). Phosphorothioate PKC and PKC antisense and control oligonucleotides have been previously described (12, 42) and were synthesized from PRIMM s.r.l. (Milan, Italy). The LY379196 inhibitor was a generous gift from Eli Lilly Co. (Indianapolis, Ind.) and has been described previously (4, 7, 22). PD98059 was purchased from ICN Biomedicals Inc. (Aurora, Ohio). Protein electrophoresis reagents were from Bio-Rad (Richmond, Va.), and Western blotting and enhanced chemiluminescence (ECL) reagents were from Amersham (Arlington Heights, Ill.). All other chemicals were from Sigma (St. Louis, Mo.).

Cell culture and transfection. The L6 muscle cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, 10,000 U of penicillin per ml, 10,000 µg of streptomycin per ml, and 2% L-glutamine, in a humidified CO₂ incubator by the method of Caruso et al. (12). Transient transfection of IRS-1 rib and c-rib, the phosphorothioate oligonucleotides, and of the L61,S186 dominant negative Ras mutant (40) was performed by the Lipofectamine method according to the manufacturer's instruction. For these studies, 50 to 80% confluent cells were washed twice with Optimem and incubated for 8 h with 12 µg of IRS-1 rib or antisense oligonucleotide and 45 µl of Lipofectamine. The medium was then replaced with DMEM with 10% fetal calf serum, and cells were incubated for 15 h before being assayed.

Western blot analysis and immunoprecipitation procedure. For these studies, the cells were solubilized in lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 10 mM EDTA, 10 mM Na₄P₂O₇, 2 mM Na₃VO₄, 100 mM NaF, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg of aprotinin per ml) for 2 h at 4°C. Lysates were centrifuged at 5,000 × g for 20 min and assayed by the method of Miele et al. (28). Briefly, solubilized proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.45-µm-pore-size Immobilon-P membranes (Millipore, Bedford, Mass.). Upon incubation with the primary and secondary antibodies, immunoreactive bands were detected by ECL according to the manufacturer's instructions. Immunoprecipitation of specific PKC isoforms, IRS-1 or -2, MAPK, Raf-1, and IRs were accomplished as previously described (28).

PKC assay. PKC activity was measured as previously described (18). Briefly, for these assays, cells were solubilized in extraction buffer (20 mM Tris [pH 7.5], 0.5 mM EDTA, 0.5 mM EGTA, 25 µg of aprotinin per ml, 25 µg of leupeptin) and then clarified by centrifugation at 5,000 × g for 20 min. Supernatants were further centrifuged at 60,000 × g for 2 h, and pellets were solubilized with extraction buffer containing 0.5% Triton X-100. Soluble pellets were supplemented with the lipid activators (10 mM phorbol 12-myristate 13-acetate (PMA), 0.28 mg of phosphatidylserine per ml, and 4 mg of dioleine per ml [final concentrations given]), and phosphorylation reactions were initiated by addition of the substrate solution, which contains 50 µM acetylated myelin basic protein (residues 4 to 14) [Ac-MBP(4-14)], 20 µM ATP, 1 mM CaCl₂, 20 mM MgCl₂, 4 mM Tris (pH 7.5), and 10 µCi of (3,000 Ci/mmol) [-³²P]ATP per ml (final concentrations given). The reaction mixtures were incubated for 10 min at room temperature, rapidly cooled on ice, and spotted on phosphocellulose discs. Disc-bound radioactivity was quantitated by liquid scintillation counting. Activity of the specific PKC isoforms was assayed as described above but using precipitates with specific antibodies (12). Determinations of PKC and - activities using the Ac-MBP(4-14) peptide as the substrate or the H-Arg-Phe-Ala-Val-Arg-Asp-Met-Arg-Gln-Thr-Val-Ala-Val-Gly-Val-Ile-Lys-Ala-Val-Asp-Lys-Lys-OH peptide (specific for PKC) or the pseudosubstrate region of PKC (specific for PKC) provided consistent results.

Insulin-mediated p21^ras-GTP formation. For the GTP overlay, 80% confluent L6 cells were serum and phosphate starved for 24 h and then exposed to 100 nM insulin for 15 min. Cell extracts were prepared, precipitated with Ras antibodies, and separated by SDS-PAGE. The gel was then soaked in 50 mM Tris-HCl (pH 7.5)-20% glycerol for 1 h and transferred onto nitrocellulose filters as described by Bucci et al. (9). After the transfer, the filters were rinsed twice for 10 min each time in a solution containing 50 mM NaH₂PO₄, 10 mM MgCl₂, 2 mM dithiothreitol, and 0.3% Tween 20 (pH 7.5) and incubated with 2 µCi of [-³²P]GTP per ml for 2 h. After six 5-min washes, the filters were autoradiographed. Quantitation was achieved by laser densitometry.

MAPK, Raf-1 kinase, and PI 3-kinase activities. Quantitation of MAPK and Raf-1 kinase activities was accomplished as described by Miele et al. (28). Briefly, cell lysates (200 µg of protein/assay) were immunoprecipitated for 18 h with either MAPK or Raf-1 antibodies and incubated with protein A-Sepharose for 2 h. Immobilized MAPK and Raf-1 kinases were washed three times with ice-cold TAT buffer (50 mM HEPES, [pH 7.5], 150 mM NaCl, 10 mM EDTA, 10 mM Na₄P₂O₇, 2 mM Na₃VO₄, 100 mM NaF, 10% glycerol, 1% Triton X-100) and two more times with HNTGVa buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 2 mM Na₃VO₄, 10% glycerol, 10% Triton X-100) and then resuspended in HNTGVa buffer supplemented with 60 mM Mg acetate, 30 µM ATP, 6 mM dithiothreitol, 1 µg of MBP per ml, and 0.5 µCi of [-³²P]ATP. Alternatively, Syntide 2 was also used as a Raf-1 substrate as described by Zou et al. (55). Upon incubation for 30 min at 25°C, reaction mixtures were spotted onto phosphocellulose discs and washed three times with 1% phosphoric acid and once more with ethanol. Disc-bound radioactivity was quantitated by liquid scintillation counting. For quantitation of PI 3-kinase activity, the cells were solubilized in TAN buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 10 mM EDTA, 10 mM Na₄P₂O₇, 1 mM Na₃VO₄, 10 µg of aprotinin per ml, 10 µg of leupeptin per ml, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride). Aliquots of the lysates were precipitated with IRS-1 or IRS-2 antibodies, and PI 3-kinase activity was determined as described by Miele et al. (28).

Thymidine incorporation. Cells were seeded in six-well plates. After 18 h, the cells were fed with DMEM supplemented with 0.25% bovine serum albumin. The cells were then maintained in this medium for an additional 16 h in the absence and presence of 100 nM insulin as indicated, followed by addition of 500 nCi of [³H]thymidine per ml. Four hours later, the cells were washed with ice-cold 0.9% NaCl and maintained in ice-cold 20% trichloroacetic acid for further 10 min. Cells were finally solubilized with 1 N NaOH, and radioactivity was quantitated by scintillation counting as described by Formisano et al. (17).

	RESULTS

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Insulin activation of PKC in L6 cells. Insulin action on PKC activity was compared in L6 cells overexpressing either wild-type human IRs or a kinase-deficient IR mutant in which tyrosines in the regulatory loop (Tyr₁₁₄₆, Tyr₁₁₅₀, and Tyr₁₁₅₁) had been substituted with phenylalanines. Both the L6 cell clones expressing the wild-type human IRs (L6hIR1, 3.2 × 10⁴ receptors/cell; L6hIR2, 3.7 × 10⁴ receptors/cell) and those expressing the mutant receptors (L6-3F1, 3.8 × 10⁴ receptors/cell; L6-3F2, 3.5 × 10⁴ receptors/cell) have been previously reported (12). In L6hIR cells, insulin (100 nM) induced a time-dependent increase in membrane PKC activity (Fig. 1, top panel). This effect was reduced by 90% in the L6-3F cell clones. Also, exposure of the L6hIR2 cells to insulin increased by two- to threefold the activity of PKC in PKC, -, -, and - immunoprecipitates (Fig. 1, bottom panel). Half-maximal insulin effect on each of these PKC isoforms was detected at 0.5 to 1 nM, and maximal effect was detected at 100 nM (data not shown). No insulin effect was measured for immunoprecipitates from the L6-3F2 cells. In contrast with the effect of insulin, 60 min of exposure to the phorbol ester PMA (1 µM) induced PKC, -, and - activities (but not PKC activity) in both L6hIR2 and L6-3F2 cells. Same results were obtained by comparing the L6hIR1 and L6-3F1 cell clones (data not shown).

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Insulin effect on PKC activity in L6 cells. (Top) Cells expressing wild-type human IRs (L6hIR1 and L6hIR2 clones) or kinase-deficient IRs (L6-3F1 and L6-3F2 clones) were exposed to 100 nM insulin for the indicated times. The cells were then solubilized and assayed for PKC activity as described in Materials and Methods. PKC activity is plotted as percent increase above that measured in cells not exposed to insulin. Data points represent the means ± standard deviations of triplicate determinations in four independent experiments. (Bottom) L6hIR2 and L6-3F2 cells were stimulated with insulin for 30 min or PMA for 60 min, solubilized, and immunoprecipitated with isoform-specific PKC antibodies (Ab) (200 µg of protein/sample). PKC activity in the immunoprecipitates was assayed as described in Materials and Methods and is plotted as radioactivity incorporated in the Ac-MBP(4-14) substrate peptide. Bars represent the means ± standard deviations of duplicate determinations in four independent experiments. The specificity of PKC isoform antibodies was controlled by Western blotting recombinant PKC, -, -, and - (rPKC, -, -, and -) with the individual antibodies as shown.

View larger version (37K): [in this window] [in a new window]   FIG. 2.   PKC activity in L6 cells transfected with IRS-1 rib. (Top) L6hIR2 cells were transfected with either IRS1-Rib or c-Rib, solubilized, separated by SDS-PAGE, and blotted with IRS-1 or IRS-2 antibodies as indicated (left). WB, Western blot. Alternatively (right), the cells were exposed to 100 nM insulin for 5 min, and the cell lysates were precipitated with IRS-1 or IRS-2 antibodies, subjected to SDS-PAGE, and Western blotted with phosphotyrosine antibodies (PTYR). Blots were revealed by ECL as described in Materials and Methods. The autoradiographs shown are representative of six (left) and four (right) independent experiments. IP, immunoprecipitation. (Bottom) PKC activity was assayed in lysates from L6hIR cells either in the absence or upon transfection of c-rib or IRS1-Rib as described in the legend to Fig. 1. Bars represent the means ± standard deviations of triplicate determinations in four independent experiments.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Activity of specific PKC isoforms in L6 cells transfected with IRS-1 rib. (Top) L6hIR2 cells transfected with either c-Rib or IRS1-Rib were stimulated with 100 nM insulin for 30 min, lysed, and precipitated with antibodies (Ab) to specific PKC isoforms as indicated. PKC activity was then assayed in the immunoprecipitates as described in Materials and Methods. Bars represent the means ± standard deviations of duplicate determinations in three independent experiments. (Bottom) For a control, lysates from the L6-3F2 cells and from the L6hIR2 cells (either those which were not transfected with ribozymes or those transfected with c-Rib or IRS1-Rib) were separated by SDS-PAGE and Western blotted with specific antibodies toward the PKC isoforms as indicated. Blots were revealed by ECL as described in Materials and Methods. The autoradiograph shown is representative of three independent experiments.

PKC action on MAPK. In muscle cells, the inhibition of insulin-dependent activation of PKC and - by IRS-1 rib was accompanied by an 80% inhibition (P < 0.001) of insulin-dependent MAPK activation (Fig. 4, top panel). As was the case for PKC and -, the control c-rib transfection was unable to inhibit MAPK activation. IRS-1-associated PI 3-kinase activity was barely detectable in cells transfected with IRS-1 rib compared to those transfected with c-rib and to those not transfected with the ribozymes (Fig. 4, middle panel). Consistent with the increased phosphorylation of IRS-2 in IRS-1 rib-transfected cells, IRS-2-associated PI 3-kinase activity was increased 60% (P < 0.001) compared to that in control cells (either cells transfected with c-rib or cells not transfected with the ribozyme) (Fig. 4, bottom panel). Interestingly, treatment of L6hIR cells with the PI 3-kinase inhibitor wortmannin (50 nM) inhibited insulin-stimulated PKC and - activities by only 20% (P < 0.05), which paralleled a similar inhibition of MAPK activation (Fig. 5). Wortmannin was much more effective on activation of PKC and - (45 and 95% inhibition, respectively; P < 0.01). Unlike wortmannin, the phospholipase inhibitor U73122 almost completely blocked insulin activation of PKC and - (P < 0.001) and MAPK but inhibited PKC induction by only 45% (P < 0.001) and showed no effect on activation of PKC.

View larger version (32K): [in this window] [in a new window]   FIG. 4.   MAPK and PI 3-K activities in L6 cells transfected with IRS-1 rib. (Top) L6hIR2 cells, either those transfected with IRS1-Rib or c-Rib and those which were not transfected, were exposed to 100 nM insulin for 30 min at 37°C. Cell lysates (200 µg of protein/sample) were then precipitated with MAPK antibodies and assayed for MAPK activity as described in Materials and Methods. MAPK activity is plotted as radioactivity incorporated in the MBP MAPK substrate. (Bottom) For determination of PI 3-K activity, the cells were exposed to 100 nM insulin for 10 min at 37°C and 200 µg of solubilized proteins precipitated with IRS-1 or IRS-2 antibodies (Ab) as indicated. IRS-1- and IRS-2-associated PI 3-K was then assayed as described in Materials and Methods. Bars represent the means ± standard deviations of duplicate determinations in four (top) and five (bottom) independent experiments.

View larger version (28K): [in this window] [in a new window]   FIG. 5.   Effects of wortmannin and U73122 on PKC isoform and MAPK activities. (Top) L6hIR2 cells were incubated with either 50 µM wortmannin or 25 µM U73122 for 30 min and then further exposed to 100 nM insulin for 30 min as indicated. The cells were then solubilized, and lysates (200 µg of protein/sample) were precipitated with isoform-specific PKC antibodies (top) or MAPK antibodies (bottom). PKC and MAPK activities were then assayed in the immunoprecipitates as described in Materials and Methods. Bars represent the means ± standard deviations of duplicate determinations in four (top) and five (bottom) independent experiments.

View larger version (38K): [in this window] [in a new window]   FIG. 6.   Blocking PKC and PKC activities in L6 cells with antisense oligonucleotides or LY379196. L6hIR2 cells were preincubated with 50 nM LY379196 or transfected with either specific PKC or PKC antisense oligonucleotides (ASPO and ASPO, respectively). Oligonucleotides with the same base composition as the PKC and - antisense oligonucleotides but with random sequence (PO and PO, respectively) were used for control. Cells were then exposed to 100 nM insulin for 30 min, solubilized, and precipitated with specific PKC (middle) or PKC (bottom) antibodies. PKC activity in the immunoprecipitates was assayed as described in Materials and Methods and is plotted as radioactivity incorporated in the Ac-MBP(4-14) substrate. Bars represent the means ± standard deviations of duplicate determinations in three independent experiments. For control (top panel), lysates (300 µg of protein/assay) were subjected to SDS-PAGE and Western blotted with PKC or PKC antibodies as indicated.

View larger version (46K): [in this window] [in a new window]   FIG. 7.   MAPK phosphorylation and activity in L6 cells upon blocking PKC and -. (Top) Antisense oligonucleotide and pharmacological blocking of PKC or PKC in L6hIR2 cells was achieved as described in the legend to Fig. 5. The cells were then exposed to 100 nM insulin or EGF as indicated, solubilized, and precipitated with MAPK antibodies. MAPK activity was assayed in the immunoprecipitates as described in the legend to Fig. 4. Bars represent the means ± standard deviations of duplicate determinations in four independent experiments. For control (middle), lysates (300 µg of protein/assay) were subjected to SDS-PAGE and Western blotted with PKC or PKC antibodies as indicated. Alternatively (bottom), proteins in the lysates were blotted with phosphorylated MAPK (pMAPK) or MAPK antibodies. The autoradiographs shown are representative of three (PKC and PKC) or five (pMAPK and MAPK) independent Western blot determinations.

Raf activation by PKC. To elucidate the mechanism by which PKC may affect insulin action on MAPK, we inhibited insulin-dependent p21^ras activation by transfection with the dominant negative mutant of Ras (L61,S186) or by preincubating the L6hIR2 cells with the HMG-coenzyme A-reductase inhibitor lovastatin (2 µg/ml). Both treatments blocked the abilities of insulin and EGF to stimulate p21^ras-GTP loading (Fig. 8, top panel). In contrast, the LY379196 PKC inhibitor exhibited no effect on p21^ras-GTP loading in response to insulin or EGF, both in the absence and presence of lovastatin or L61,S186 Ras. The lovastatin pretreatment and L61,S186 Ras expression inhibited insulin activation of MAPK by about 30% in these cells (P < 0.001) and EGF activation by almost 90% (Fig. 8, bottom panel). In contrast to these p21^ras inhibitors, LY379196 decreased insulin-stimulated MAPK activity by >80% despite its complete lack of p21^ras inhibitory action. EGF activation of MAPK was unaffected by LY379196. The inhibitory effects of insulin-stimulated MAPK activity by LY379196 and lovastatin or L61,S186 Ras were additive. Similar effects were observed by measuring MAPK phosphorylation instead of activity (data not shown). Thus, PKC seemed to control MAPK independently of Ras and at a step distal to Ras.

View larger version (36K): [in this window] [in a new window]   FIG. 8.   Insulin-dependent Ras function in L6 cells. (Top) L6hIR2 cells were treated with lovastatin or transfected with the dominant negative L61,S186 Ras mutant and/or exposed to LY379196 as indicated. The cells were then stimulated with insulin or EGF (100 nM) and solubilized, and the Ras GTP loading was estimated by the GTP overlay assay as described in Materials and Methods. Overlay filters were autoradiographed, and GTP loading was quantitated by densitometry. (Bottom) The cell lysates were precipitated with MAPK antibody, and MAPK activity was assayed as described in the legend to Fig. 4. Bars represent the means ± standard deviations of duplicate determinations in four (top) and three (bottom) independent experiments.

View larger version (57K): [in this window] [in a new window]   FIG. 9.   Insulin-dependent Raf-1 activation in L6 cells. L6hIR2 cells were treated with LY379196 or transfected with either specific PKC or PKC antisense oligonucleotides (ASPO and ASPO, respectively) or the control oligonucleotides specified in the legend to Fig. 5 (PO and PO). The cells were then exposed to 100 nM insulin for 30 min, solubilized, and precipitated with Raf-1 antibodies (200 µg of protein/sample). The immunoprecipitates were assayed for Raf-1 kinase activity as described in Materials and Methods. Bars represent the means ± standard deviations of duplicate determinations in four independent experiments. For control (top panel), parallel precipitates were blotted with Raf-1 antibodies and revealed by ECL as described in Materials and Methods.

View larger version (35K): [in this window] [in a new window]   FIG. 10.   Insulin-dependent PKC-Raf1 coprecipitation in L6 cells. (Top) L6hIR2 and L6-3F2 cells were exposed to the indicated concentrations of insulin for 30 min, solubilized, and precipitated (200 µg of protein/sample) with PKC antibodies as shown. Precipitates and whole-cell lysates from the two cell types were Western blotted with PKC antibodies, and filters were stripped and further probed with Raf-1 antibodies. For control (middle), L6hIR2 cells were stimulated with 100 nM insulin for 30 min, lysed, and precipitated with PKC, PKC, or Raf-1 antibodies as indicated. Precipitates were Western blotted with Raf-1 antibodies. Alternatively (bottom), the cells were transfected with PKC or PKC antisense oligonucleotides or treated with LY379196, stimulated with 100 nM insulin for 30 min, precipitated with PKC, PKC, or Raf-1 antibodies, and blotted with Raf-1 antibodies. All filters were revealed by ECL as described in Materials and Methods. The autoradiographs shown are representative of three (top and middle) and three (bottom) independent experiments. Abbreviations: I.P. and IP, immunoprecipitation; -PKC, antibody against PKC; WB, Western blotting.

View larger version (33K): [in this window] [in a new window]   FIG. 11.   Thymidine incorporation in L6 cells following insulin, EGF, or serum exposure. L6hIR2 cells were transfected with the IRS-1 or the control ribozymes (IRS1-Rib or C-Rib, respectively) or pretreated with the MAPK or PKC inhibitors PD98059 and LY379196 (top). Alternatively, the cells were transfected with either the specific PKC or PKC antisense oligonucleotide (ASPO or ASPO, respectively) or the control oligonucleotides specified in the legend to Fig. 5 (PO and PO). The cells were exposed to 100 nM insulin or to EGF or to 10% fetal calf serum (FCS) for 12 h, as indicated. [3H]thymidine incorporation into DNA was then assayed as described in Materials and Methods. Bars represent the means ± standard deviations of triplicate determinations in four independent experiments.

View larger version (10K): [in this window] [in a new window]   FIG. 12.   Schematic comparison of the mechanisms involved in insulin- and EGF-stimulated mitogenesis in L6 cells. Unlike the situation with EGF and other serum growth factors, insulin-induced MAPK activity in L6 cells appears to occur largely through Raf-1 rather than Ras activation. EGF-R, EGF receptor. The boldfaced pathway has been described for the first time in this paper.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Insulin activates PKC in a variety of cell types and tissues (3, 7, 18). In addition, certain PKC isoforms have been reported to play an important role in insulin action (13, 27, 47), regulation of IR kinase (5, 15) and intracellular sorting (18). However, the specific role of each individual PKC isoform in insulin signaling and the molecular mechanism causing PKC activation by insulin are still unclear. For instance, the role of PKC in insulin action has not been consistently reported (4, 7, 13, 48). In L6 skeletal muscle cells, we have shown that IR kinase activity is necessary to allow insulin activation of multiple PKC isoforms, including classical ( and ), novel (), and atypical () PKCs. In fact, mutant IRs featuring substitution of the regulatory tyrosines with phenylalanines simultaneously lose their abilities to undergo kinase activation in response to insulin (12, 29) and to transduce insulin activation of these different PKCs. In the muscle cells, however, the molecular mechanisms involved in receptor activation of the classical PKCs versus the other PKC isoforms appear to be distinct. Hence, we report that blocking IRS-1 expression with an IRS-1 ribozyme selectively impairs insulin activation of PKC and - without affecting PKC and -. Whether insulin activation of PKC and - is mediated by substrates other than IRS-1, such as IRS-2, and their specific role in insulin signaling, are presently under investigation in our laboratory.

We have also shown that, in L6 cells, the activation of PKC is necessary for insulin stimulation of MAPK function and for insulin proliferative responses. In fact, inhibition of PKC activity by either antisense oligonucleotide or the LY379196 specific agent (as well as the inhibition of IRS-1 expression) almost completely blocks the effects of insulin on MAPK and on DNA synthesis. The involvement of PKC in mitogenic signal transduction appears to be specific for the insulin mitogenic pathway, since neither the PKC antisense oligonucleotide nor LY379196 affected EGF- or serum-induced mitogenesis.

Brüning et al. (8) have recently reported that, in fibroblasts from animals with IRS-1 knockout, insulin-like growth factor I can still activate MAPK and induce DNA synthesis. However, the molecular mechanism remains elusive. The different roles of IRS-1 in transducing mitogenic signals in these fibroblasts and in the muscle cells may depend on diversity in the pathways used by insulin and insulin-like growth factor I for inducing cell proliferation. In addition, mitogenic signals may feature tissue specificity. In fact, while abundant in insulin-responsive tissues (7, 38) and necessary for insulin-induced mitogenesis in muscle cells (this study) and in mouse hepatocytes (data not shown), PKC is not even expressed in fibroblasts (20). This indicates diversity in the events responsible for insulin-induced mitogenesis in fibroblasts compared to insulin-responsive tissues. In addition, different cells may use different PKC isoforms to enter the MAPK pathway. Consistent with this, Sajan et al. (39) have recently reported that, in rat adipocytes, PKC plays a major role in insulin induction of MAPK activity. At variance, we report here that, in muscle cells, wortmannin elicits only slight inhibition of insulin-induced MAPK activity while completely blocking that of PKC.

Despite the relevance of PKC activation to insulin-regulated MAPK function, pharmacological inhibition of MAPK produced no effect on insulin-dependent PKC activity (data not shown). Thus, at least in the L6 muscle cells, activation of PKC by insulin seems to occur upstream of MAPK. Previous reports for 3T3-L1 adipocytes showed that PKCs may inhibit GTPase-activating proteins and activate Ras, conveying insulin signal through the MAPK cascade (41). In the L6 cells, however, selective block of Ras with lovastatin or a dominant negative Ras mutant causes only a slight inhibition of insulin-stimulated MAPK activation, which is an additive effect to that caused by the PKC inhibitor LY379196. This indicates that PKC activation of the MAPK pathway is largely independent of Ras in muscle cells. In fact, we showed that PKC inhibition almost completely blocked insulin activation of Raf-1 kinase in the L6 cells. Thus, in these cells, Raf-1 kinase represents at least one of the molecules conveying insulin mitogenic signals downstream of PKC. Most mitogenic signals initiated by tyrosine kinase receptors are believed to converge on MAPK mainly through direct Ras activation (19). However, Raf-1 kinase has been previously reported to be also phosphorylated and activated by PKCs in different cell types (24, 25, 45). In addition, Raf activation by PKC enables phorbol ester and vascular endothelial growth factor mitogenic signal transduction to MAPK independent of Ras (50, 52). Also, in 3T3-L1 adipocytes, insulin activates MAPK through an unknown mechanism independent of Ras (11). We now provide evidence that, in muscle cells, IRS-1-mediated PKC activation may directly activate Raf, thus inducing MAPK activity and the mitogenic pathway independently of Ras (Fig. 10). In addition, we provide evidence that PKC activation is necessary for driving its interaction with Raf. Hence, treatment of cells with the PKC inhibitor LY379196 prevents PKC-Raf coprecipitation similar to the antisense oligonucleotide block of PKC expression. To our knowledge, the data in the present report represent the first indication that insulin, unlike EGF and other serum growth factors, activates MAPK and mitogenesis mainly through PKC and largely bypasses Ras in insulin target tissues such as the muscle cells.

In agreement with the results of other investigators (16, 23, 46), the mechanism of IRS-1-dependent activation of PKC in L6 cells may involve insulin triggering of phospholipase activity which, in turn, activates PKC. In fact, we report that treatment of L6 cells with the phospholipase C inhibitor U73122 simultaneously blocks PKC and MAPK activation by insulin. In contrast, insulin-induced PI 3-kinase activation does not appear to be a major mechanism responsible for induction of PKC and mitogenesis in L6 cells. In fact, the PI 3-kinase inhibitor wortmannin elicits little effect on PKC activation in L6 cells, and in the same cells, insulin-stimulated PI 3-kinase activity persists in association with IRS-2 during treatment with IRS-1 ribozyme, a condition under which MAPK activity is inhibited.

Previous work in other laboratories has shown that, in different cell types, PKC overexpression results in increased serine/threonine phosphorylation of the IR with inhibition of its tyrosine kinase (5, 14). Based on these findings, it has been proposed that PKC regulates insulin signaling in cells at the receptor level. In the present report, we show that PKC enables insulin mitogenic signal, at least in part, by controlling Raf-1 kinase activation. Thus, these data identify PKC as a novel molecule in the insulin mitogenic pathway and indicate that PKC may be involved in insulin action both at the receptor and post-receptor levels.

Unlike PKC, blocking PKC by antisense oligonucleotide expression in L6 cells increased basal MAPK phosphorylation and activity while not affecting insulin stimulation of MAPK. In L6 cells, PKC is physically associated to the IR and phosphorylates the receptor on serine and threonine residues, inhibiting its kinase activity (12). We have previously shown that release of the PKC inhibitory action on the receptor, either by inhibiting PKC expression or by inducing its dissociation from the receptor, activates the receptor kinase and glucose uptake, independent of insulin (12). The block of PKC by antisense oligonucleotide is likely to be responsible for the constitutive activation of the insulin mitogenic pathway as well. It appears from these data that multiple PKC isoforms are embedded in the insulin mitogenic and metabolic pathways controlling the flow of signals either at the receptor level or at more distal step in its signaling cascade.