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J Biol Chem, Vol. 274, Issue 40, 28637-28644, October 1, 1999

In L6 Skeletal Muscle Cells, Glucose Induces Cytosolic Translocation of Protein Kinase C- and Trans-activates the Insulin Receptor Kinase^*

Matilde Caruso, Claudia Miele, Francesco Oriente, Alessandra Maitan, Giuseppe Bifulco^§, Francesco Andreozzi, Gerolama Condorelli, Pietro Formisano, and Francesco Beguinot^¶

From the Dipartimento di Biologia e Patologia Cellulare e Molecolare & Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Federico II University of Naples, Naples 80131, Italy

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In L6 skeletal muscle cells expressing human insulin receptors (L6_hIR), exposure to 25 mM glucose for 3 min induced a rapid 3-fold increase in GLUT1 and GLUT4 membrane translocation and glucose uptake. The high glucose concentration also activated the insulin receptor kinase toward the endogenous insulin receptor substrates (IRS)-1 and IRS-2. At variance, in L6 cells expressing kinase-deficient insulin receptors, the exposure to 25 mM glucose elicited no effect on glucose disposal. In the L6_hIR cells, the acute effect of glucose on insulin receptor kinase was paralleled by a 2-fold decrease in both the membrane and the insulin receptor co-precipitated protein kinase C (PKC) activities and a 3-fold decrease in receptor Ser/Thr phosphorylation. Western blotting of the receptor precipitates with isoform-specific PKC antibodies revealed that the glucose-induced decrease in membrane- and receptor-associated PKC activities was accounted for by dissociation of PKC but not of PKC or -. This decrease in PKC was paralleled by a similarly sized increase in cytosolic PKC. In intact L6_hIR cells, inhibition of PKC expression by using a specific antisense oligonucleotide caused a 3-fold increase in IRS phosphorylation by the insulin receptor. This effect was independent of insulin and accompanied by a 2.5-fold increase in glucose disposal by the cells. Thus, in the L6 skeletal muscle cells, glucose acutely regulates its own utilization through the insulin signaling system, independent of insulin. Glucose autoregulation appears to involve PKC dissociation from the insulin receptor and its cytosolic translocation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glucose represents a major energy fuel for most mammalian cells, and its utilization is affected by multiple regulatory factors (1, 2). These include insulin (3), hypoxia (4), and, in skeletal muscle, contraction (5). In addition, in muscle as well as in other tissues, glucose itself affects its own utilization and metabolism (6). Whereas prolonged exposure to glucose impairs its disposal by the cells, rapid increases in glucose concentration into the extracellular media are accompanied by opposite effects (7). The ability of glucose to increase acutely its own cellular uptake plays an important role in normal glucose homeostasis (8) and may affect the utilization of other energy sources as well (9). These regulatory actions have been attributed to the mass action effect of glucose rather than to activation of glucose transport by specific glucose transporters (8). More recently, however, acute hyperglycemia has also been shown to increase muscle membrane content of GLUT4, suggesting that glucose per se may activate its own glucose transport system (10, 11). The molecular mechanisms underlying these short term autoregulatory effects have not been completely elucidated yet.

Protein kinase C (PKC)¹ plays a key role in transducing signals generated by growth factors, hormones, and neurotransmitters (12-14) and represents major downstream targets for agents controlling glucose uptake and utilization (13). At least 10 distinct PKC isoforms have been described exhibiting differential regulation (15). These include the calcium-dependent conventional PKCs, the new calcium-independent PKCs, and the atypical PKC isoforms (16). In cultured muscle cells, pharmacological inhibition of PKC activity blocks glucose activation of the glucose transporter system, suggesting an important role of PKCs in glucose transport autoregulation (10). In rat skeletal muscle, exposure to high glucose concentrations has been reported to cause membrane translocation of PKC isoforms with no effect on , , , and , leading to the hypothesis that PKC might mediate glucose transport autoregulation in this tissue (10). In other cell types, however, glucose exposure acutely increases membrane translocation of PKC, -, -, and - as well as stimulation of glucose uptake (17). Thus, how glucose conveys its signal to the PKC system as well as which specific PKC isoform may play a role in autoregulation of glucose disposal in each cell type remain unclear.

In the present work, we have further investigated the mechanisms involved in glucose regulation of its own disposal by focusing on the immediate early events occurring in cells after exposure to glucose. We found that, within seconds after the exposure of L6 skeletal muscle cells to glucose, glucose utilization is increased by trans-activating the insulin signaling system. Evidence is presented that this effect is triggered by the rapid dissociation of PKC from the insulin receptor, accompanied by decreased phosphorylation of the receptor on serine and threonine.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Media and sera for tissue culture and the transfection reagent, (N-[1-(2,3-diomeoyloxy)-propyl]-N,N,N-trimethylammonium chloride/dioleoylphosphatidylethanolamine) were purchased from Life Technologies, Inc. Electrophoresis reagents were from Bio-Rad. Protein A-Sepharose beads and sulfo-hydroxysuccinimide long-chain biotin were from Pierce (Rockford, IL). Radiochemicals, Western blot and ECL reagents were from Amersham Pharmacia Biotech. Polyclonal GLUT1 (catalog number 4670-1701) and GLUT4 (catalog number 4670-170) antibodies were from Biogenesis (Biogenesis Sandown, NH). Monoclonal Ig2 phosphotyrosine (catalog number 05-321) and monoclonal IRS1 (catalog number sc-1555) antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal IRS2 antibodies (catalog number sc-1555) were from Santa Cruz Biotechnology (Santa Cruz, CA). mAb3IR antibody (catalog number GR07) was purchased from Oncogene Science (Manhasset, NY). Polyclonal antibodies toward PKC (catalog number 13191-010) and PKC (catalog number 13197-017) were from Life Technologies, Inc. (Grand Island, NY) and those toward PKC₂ (catalog number sc-210) from Santa Cruz Biotechnology. All other reagents were from Sigma (St. Louis, MO).

Mutant Construction, Transfection, and Cell Culture-- The L6 cell clones expressing the wild-type human insulin receptors have been previously characterized and described (18). The hIR cDNA (19) subcloned in the pSp65 vector was kindly provided by Dr. Steen Gammelfolt (Copenaghen, Denmark). Wild-type hIR cDNA (5.2-kilobase pair SalI fragment) was subcloned by linker insertion in the SacII-Xho site of the pCO11 expression vector containing the neo-selectable marker (20). To substitute tyrosines 1146, 1150, and 1151 with phenylalanines, the hIR cDNA fragment, BamHI-SalI (residues 1926-5200), derived from Sp65-hIR was subcloned in M13mp19. The single-stranded template was prepared, and the mutations were obtained by oligonucleotide-directed mutagenesis using the following primer, 5'-CGAGACATCTTTGAAACGGATTTCTTCCGGGAAAGGGG-3'. Mutagenesis was performed according to Taylor et al. (21) and confirmed by M13 dideoxy sequencing. The HincII fragment (residues 3187-3871) encoding the 3F mutant was cloned back in the pSp65-hIR and the mutant 3F cDNA cloned into PCO 11 vector. The final construct was sequenced to confirm the presence of the mutations. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum as described previously (18) and used at the myotube stage of differentiation.

Determinations of 2-Deoxy-D-glucose Uptake and Glucose Transporter Content-- For 2-deoxyglucose (2-DG) uptake studies, cells were rinsed and incubated in glucose-free buffer (25 mM Hepes, pH 7.4, 125 mM NaCl, 5 mM KCl, 2.5 mM MgCl₂, 1 mM CaCl₂, 0.25% bovine serum albumin) for 3 h. The cells were subsequently incubated for 3 min in the same buffer supplemented with the indicated concentrations of glucose, xylose, sucrose, or pyruvate, washed again, and incubated for further 10 min in glucose-free buffer containing 2-DG (final concentration 0.15 mM) and 0.5 µCi/assay [¹⁴C]2-DG, according to Refs. 18 and 22. The cells were finally lysed, and 2-DG uptake was determined by liquid scintillation counting. For determining the glucose transporter content, L6 cells were starved from serum for 18 h and maintained in glucose-free medium for an additional 3 h. The cells were then exposed for 1 h to Hepes buffer containing 25 mM glucose, xylose, or sucrose as indicated. Glucose transporter content was measured on total cell extracts by immunoblotting with specific Abs as described previously (18). For determining the glucose transporter recruitment to the plasma membrane, the cells were deprived from serum and glucose as described above and incubated for 10 min in Hepes buffer containing the indicated concentrations of glucose, xylose, sucrose, pyruvate, and/or insulin. Biotinylation of cell-surface transporters was determined by the method of Levy-Toledano et al. (23) modified as in Ref. 18.

Glucose Storage and Oxidation-- Glycogen content and glucose oxidation in L6 cells were determined as reported (18). Both assays were performed on myotubes maintained for 18 h in serum-free medium supplemented with 6 mM glucose. The cells were subsequently incubated with 25 mM glucose, xylose, sucrose, pyruvate, and insulin for the indicated times, thoroughly rinsed, and then assayed. Briefly, for glycogen content, L6 myotubes were collected in 0.6 N HClO₄, homogenized using a glass-Teflon potter, and centrifuged at 1500 rpm for 10 min at 4 °C. Aliquots of the homogenate were incubated with 0.33 M KHCO₃ and 9 mg/ml amyloglucosidase in 0.2 M acetate buffer, pH 4.8, for 2 h at 40 °C as reported previously (24). The reaction was stopped by addition of 0.6 N HClO₄ and centrifugation at 15,000 rpm at 4 °C for 15 min. Glucose concentration was determined with a Beckman glucose analyzer. For glucose oxidation, the cells were grown in 50-ml flasks. After the serum was withdrawn, the cells were further incubated in Joklik medium supplemented with 25 mM NaHCO₃, 5.55 mM [U-¹⁴C]glucose, 1.2 mM MgSO₄, 0.5 mM CaCl₂, 10 mM Hepes, pH 7.4, for 20 min at 30 °C after capping the flasks with rubber stoppers containing a hanging well filled with rolled filter paper. 0.4 ml of 1 M hyamine hydroxide in methanol were then injected through the rubber stoppers into the hanging wells followed by injection into the incubation medium of 0.4 ml of 10% HClO₄. The flasks were allowed to sit for 2 more h at 37 °C. The filter papers were removed and counted in a -counter. Glycogen synthase activity was assayed according to Thomas et al. (25). One unit of glycogen synthase was defined as the amount of enzyme that catalyzes the incorporation of 1 µmol of UDP-glucose into glycogen per min (26). PDH activity was assayed as described previously (18).

Phosphorylation of Poly (Glu-Tyr) and IRS by Insulin Receptors-- In vitro insulin receptor tyrosine kinase activity was investigated by assaying phosphorylation of the synthetic substrate poly-(Glu-Tyr) in the presence of [-³²P]ATP as described previously (27). The effect of glucose on IRS phosphorylation was measured on total cell extracts from L6 myotubes preincubated with 25 mM glucose for 5 min in the absence or the presence of 100 nM insulin. After the incubation, the cells were lysed in 50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 10 mM EDTA, 10 mM Na₄P₂O₇, 1 mM Na₃VO₄, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride (TAT buffer). Lysates were precipitated with IRS-1- or IRS-2-specific antibodies followed by Western blotting with phosphotyrosine antibodies (28). Detection of the bands was achieved by horseradish peroxidase-coupled anti-rabbit IgG (IRS1) or anti-goat IgG (IRS2) and ECL according to the manufacturer's instructions. Quantitation was obtained by laser densitometry.

PKC Assays-- Determination of PKC activity was achieved with a commercially available kit (Life Technologies Inc., catalog number 13161-013). This assay kit is based on measurement of phosphorylation of the synthetic peptide from myelin basic protein Ac-MBP-(4-14) by PKC (in the presence of activators) as described by Yasuda et al. (29). PKC specificity is confirmed by using the PKC pseudosubstrate inhibitor peptide PKC-(19-36), which acts as a potent inhibitor of this substrate (29). For this assay, L6 cells were deprived from serum and glucose as described above and then exposed to 25 mM glucose as indicated. PKC activity was then quantitated in total cell lysates or cell fractions or in immunoprecipitates as previously reported (30) and according to the manufacturer`s instructions. Briefly, for PKC activity determination, the cells were lysed with 20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 mg/ml aprotinin, and 25 mg/ml leupeptin (extraction buffer) and then clarified by centrifugation at 10,000 × g for 15 min at 4 °C. Upon protein quantitation, equal aliquots of the extract were added to the lipid activators (10 µM phorbol 12-myristate 13-acetate, 0.28 mg/ml phosphatidylserine, and 4 mg/ml dioleine, final concentrations) and the ³²P-substrate solution (50 mM Ac-MBP-(4-14), 20 µM ATP, 1 mM CaCl₂, 20 mM MgCl₂, 4 mM Tris, pH 7.5, and 10 mCi/ml (3,000 Ci/mM) [-³²P]ATP), in the presence or the absence of 25 µM of the PKC pseudosubstrate inhibitor peptide PKC-(19-36). The samples were incubated for 20 min at room temperature and rapidly cooled on ice, and 20-µl aliquots were spotted on phosphocellulose disc papers (Life Technologies, Inc.). Discs were washed twice with 1% H₃PO₄, followed by two additional washes in water, and the disc-bound radioactivity was quantitated by liquid scintillation counting. The dependence of the signal on time and amount of extract in the assay has been repeatedly demonstrated. For PKC co-precipitation studies, cell lysates were precipitated with protein A-Sepharose-bound antibodies to insulin receptor and the immunocomplexes were resuspended either in extraction buffer, for quantitation of PKC activity, or in Laemmli buffer for SDS-polyacrylamide gel electrophoresis protein separation. The resuspended proteins were then blotted on nitrocellulose filters, probed with isoform-specific PKC antibodies, and detected by ECL according to the manufacturer's instructions. Quantitation of the autoradiographs was obtained by laser densitometry. For detecting PKC isoforms in subcellular fractions, the cells were washed once with ice-cold phosphate-buffered saline and then lysed by resuspending in Buffer A (20 mM Tris-HCl, pH 7.4, 10 mM EDTA, 5 mM EGTA, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml leupeptin, 0.15 units/ml aprotinin) and passing through a 27-gauge needle. The cell lysates were centrifuged at 800 × g for 5 min at 4 °C. Supernatants were further centrifuged at 100,000 × g for 20 min at 4 °C. The final supernatants were collected and used as the cytosolic fraction. The membrane pellet was solubilized in Buffer A containing 1% Triton X-100 by bath sonication and centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was used as the membrane fraction according to Ref. 31. Based on the PKC assay system used in this work, it was estimated that the PKC activity ratio in the insulin receptor precipitates from the cells to that in the membranes is approximately 1/1000.

Phosphorylation of Insulin Receptors in Intact Cells-- For quantitating insulin receptor phosphoserine and phosphothreonine levels, L6 myotubes were labeled with [³²P]orthophosphate as previously reported (32). The cells were solubilized in 1 ml of a solution containing 50 mM Hepes, pH 7.4, 1% Triton X-100, 10 mM Na₄P₂O₇, 100 mM NaF, 4 mM EDTA, 2 mM Na₃VO₄, 2 mM phenylmethylsulfonyl fluoride, and 0.2 mg/ml aprotinin. Wheat germ-agarose purified receptors from the cell lysates were precipitated with insulin receptor antibodies and the immunoprecipitated phosphoproteins separated on 7.5% polyacrylamide gels. Phosphoserine/phosphothreonine content in the electrophoresed receptors was estimated (33).

PKC Antisense Studies-- For antisense studies a phosphorothioate PKC oligodeoxynucleotide (ASPO) was generated with the following sequence, 5'-CAGCCATGGTTCCCCCCAAC-3' (34). For control, a scrambled oligodeoxynucleotide (PO) with the sequence 5'-CCAGTCACTCGCACCATCGC-3' was also obtained. For antisense transient transfections, L6hIR cells were grown and allowed to differentiate in 6-well plates. The cells were then rinsed with 3 ml of serum-free Dulbecco's modified minimum essential medium and 3 ml of medium containing 2 µg/ml N-[1-(2,3-diomeoyloxy)-propyl]-N,N,N-trimethylammonium chloride/dioleoylphosphatidylethanolamine transfection reagent, and 4 µg/ml antisense or scrambled oligonucleotides were added for 16 h. The cells were washed with serum-free Dulbecco's modified minimum essential medium and incubated for 18 h in the same medium supplemented with 0.25% bovine serum albumin. Transfected cells were exposed to 25 mM glucose as indicated and assayed for 2-DG uptake and IRS phosphorylation as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Autoregulation of Glucose Uptake and Metabolism in L6 Myotubes-- We investigated the acute effect of glucose on its own metabolism in L6 myotubes expressing human insulin receptors (L6hIR). These cells express 3.2 × 10⁴ human insulin receptors/cell and have been previously characterized and described (18). Exposure of the L6hIR cells to increasing concentrations of glucose for 3 min was paralleled by a progressive increase in 2-deoxy-D-glucose uptake (Fig. 1). The same increase did not occur upon incubation of the cells with identical concentrations of either xylose or sucrose. Pyruvate was also unable to mimic glucose effect, indicating that glucose effect was not due to increased energy availability. Exposure of the L6hIR cells to 25 mM glucose for up to 60 min did not elicit any change in the total GLUT1 or GLUT4 content of the cells (Fig. 2A). However, based on biotinylation of cell-surface proteins followed by detection with horseradish peroxidase-conjugated streptavidin, glucose increased the transporter content of the plasma membrane (Fig. 2, B and C). This effect was not reproduced by either xylose or sucrose. Both in the case of GLUT1 and in the case of GLUT4 recruitment, the magnitude of 25 mM glucose stimulation in L6hIR cells (3.5- and 4-fold above basal levels, respectively) was similar and non-additive to that of a maximal insulin dose (10⁷ M). The increased glucose uptake in L6hIR cells exposed to 25 mM glucose was accompanied by 2.3- and 2-fold increases in glycogen synthase and pyruvate dehydrogenase activities, respectively (Fig. 3, A and B). Consistent with the higher enzyme activity levels, glycogen content and glucose oxidation also exhibited 2.5- and 2-fold increases upon cell exposure to the high glucose concentration (Fig. 3, C and D). As was the case for glucose uptake, these appeared specific and not matched by exposure to either xylose or sucrose or, in the case of glycogen content and synthase activity, to pyruvate. Again, glucose stimulation of glucose storage and oxidation were only slightly less effective than that of an optimal insulin dose and not additive to that evoked by insulin. These data suggested that, in L6 cells, similar events were involved in both the glucose and the insulin stimulation of glucose uptake and utilization.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   2-Deoxy-D-glucose uptake in L6 myotubes. Cells were deprived of glucose 3 h before being assayed and then exposed to increasing concentrations of glucose (), xylose (), sucrose (), or pyruvate () as indicated. The initial rate of 2-DG uptake was determined as described under "Experimental Procedures." Each data point represents the mean ± S.D. of triplicate determinations in four independent experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Glucose effect on GLUT1 and GLUT4 recruitment in L6 myotubes. A, the cells were incubated with 25 mM glucose, xylose, or sucrose for 1 h, as indicated. Cells were then lysed and Western-blotted with GLUT1 or GLUT4 mAbs. Transporters were revealed by chemiluminescence and autoradiography as described under "Experimental Procedures." The autoradiograph shown is representative of three independent experiments. For analyzing the recruitment of glucose transporters, the cells were deprived from glucose 3 h before being assayed and exposed for 10 min to glucose, xylose, sucrose, or insulin at the indicated concentrations. Cell-surface proteins were then biotinylated for 30 min at 4 °C, and the cells were lysed and precipitated with GLUT1 (B) or GLUT4 (C) mAbs. Upon Western blotting and incubation with peroxidized streptavidin, transporters were revealed by chemiluminescence and autoradiography as described under "Experimental Procedures." The autoradiograph shown is representative of five (GLUT1) and four (GLUT4) independent experiments.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Glycogen content, CO2 production, glycogen synthase, and pyruvate dehydrogenase activities in L6 myotubes. The cells were exposed to 25 mM glucose, xylose, sucrose, pyruvate, or to 100 nM insulin for 10 min (glycogen synthase and pyruvate dehydrogenase) or 3 h (glycogen synthesis and CO2 production), as indicated. Glycogen content, CO2 production, glycogen synthase, and pyruvate dehydrogenase activities were then assayed as described under "Experimental Procedures." Each bar represents the mean ± S.D. of triplicate determinations in four (glycogen content and glycogen synthase) and five (CO2 production and pyruvate dehydrogenase) independent experiments.

Glucose Activation of the Insulin Signaling System in L6 Myotubes-- The insulin signaling system is a major mechanism through which several agents may control nutrient utilization by the cells (35). We sought to investigate whether activation of this mechanism also plays a role in the glucose regulatory effects. As shown in Fig. 4A, exposure of L6hIR cells to 25 mM glucose for 3 min caused a 2.2-fold increase in the ability of purified insulin receptor kinase to phosphorylate the synthetic substrate poly(Glu-Tyr). No effect was measurable with identical concentrations of either sucrose or xylose. In comparison, exposure of the cells to 10⁷ M insulin led to a 3-fold increase in the insulin receptor kinase activity. Similarly, in intact L6 cells, phosphorylation of the endogenous substrates IRS-1 and IRS-2 increased by 5- and 7.5-fold in response to cell exposure to the high glucose concentration and to insulin, respectively, with no change in the total IRS levels of the cells (Fig. 4B). Phosphatidylinositol 3-kinase is a major molecule conveying IRS-mediated signals toward the glucose transporter system. In the L6 myotubes, treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin significantly inhibited GLUT4 membrane recruitment in response to both insulin and glucose (Fig. 4C).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Glucose effect on insulin receptor signaling in L6 myotubes. A, glucose-deprived L6 myotubes were exposed to 25 mM, xylose, sucrose, glucose, or to 100 nM insulin for 3 min. Insulin receptors were then partially purified from the cells, and poly(Glu-Tyr) phosphorylation was measured as described under "Experimental Procedures." Each bar represents the mean ± S.D. of triplicate determinations in five independent experiments. B, for IRS1 and IRS2 phosphorylation analysis, L6 myotubes were incubated with glucose, xylose, or insulin as above, and the extracts were precipitated with IRS-1 or IRS-2 mAbs followed by immunoblotting with phosphotyrosine Abs or with IRS-1/IRS-2 Abs (for detection of the total IRS levels), as indicated. The blotted proteins were revealed by chemiluminescence as described under "Experimental Procedures." The blots shown are representative of four independent experiments. C, myotubes were treated with 50 nM wortmannin for 30 min and incubated with the indicated concentrations of glucose or insulin for 10 min. GLUT4 membrane recruitment was analyzed as outlined in the legend to Fig. 2. Quantitation was achieved by laser densitometry of the bands. The autoradiograph shown is representative of four independent experiments.

To investigate whether the activation of these early steps of the insulin signaling mechanism is necessary for glucose regulation of its own metabolism in the L6 skeletal muscle cells, we have used a mutant insulin receptor featuring substitution of the three regulatory tyrosines (Tyr¹¹⁴⁶, Tyr¹¹⁵⁰, and Tyr¹¹⁵¹) with phenylalanines. This receptor is unable to undergo insulin-dependent activation and to mediate insulin responses in cells (36) and exhibits a dominant negative effect toward wild-type receptors (37). Consistently, L6 cells expressing 3.8 × 10⁴ and 3.5 × 10⁴ mutant receptors/cell (clones 3F1 and 3F2, respectively) did not respond to insulin in terms of receptor kinase and glycogen synthase activation (Fig. 5, A and B). Glucose was also unable to activate the mutant receptor kinase. Interestingly, glycogen synthase activation in response to glucose was completely blocked as well, indicating it is dependent upon an intact insulin signaling mechanism in these cells.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Glucose effect on insulin receptor kinase and glycogen synthase activities in L63F cells. The effects of glucose (25 mM) and insulin (100 nM) were compared in L6 myotubes expressing 3.2 × 104 wild-type human insulin receptors/cell (hIR1) and in two L6 cell clones expressing 3.4 × 104 or 3.8 × 104 kinase-defective human insulin receptors/cell (3F1 and 3F2, respectively). A, receptor kinase toward the poly(Glu-Tyr) peptide was determined as in the legend to Fig. 4. Each bar represents the mean ± S.D. of triplicate determinations in four independent experiments. To ensure equal amounts of insulin receptors in each sample, aliquots from each incubation mixture were blotted with insulin receptor antibodies. A representative blot is shown in the top panel of the figure. B, glycogen synthase activity in the hIR1, 3F1, and 3F2 cell clones was assayed as described under "Experimental Procedures." Each bar represents the mean ± S.D. of duplicate determinations in three independent experiments.

Glucose Regulation of PKC-insulin Receptor Association-- PKCs have been known to control the insulin receptor kinase activity (38, 39) and may have an important role in evoking responses in cells exposed to high glucose concentrations (10, 17, 40, 41). We have therefore investigated potential relationships between the activation of the insulin receptor and the PKC system in response to glucose in the L6hIR cells. Within 30 s upon exposure to 25 mM glucose, the PKC activity of the cells exhibited a 25% decline compared with the basal levels (p < 0.001) (Fig. 6A, circles). This effect reached a maximum (60% below the basal) 3 min after glucose exposure. By 7 min, however, PKC activity returned to the basal levels and showed a progressive increase at more prolonged exposure to glucose (maximum 2-fold above the basal upon 60 min exposure). The time course of glucose effect on the PKC activity that co-precipitated with insulin receptors was almost identical to that of glucose effect on total PKC (Fig. 6A, triangles). In intact L6hIR cells, the changes in PKC activity in response to glucose were accompanied by parallel changes in the insulin receptor phosphoserine and phosphothreonine content (Fig. 6B, squares). Interestingly, in the L6hIR cells, the time course of glucose effect on the tyrosine kinase activity of insulin receptors (Fig. 6B, IRTK) inversely correlated with that on receptor PKC association and receptor Ser/Thr phosphorylation (Fig. 6B, squares). Sucrose produced no effect comparable with those produced by glucose (Fig. 6C, filled symbols). Pyruvate (Fig. 6C, open symbols) was also unable to reproduce the early glucose-induced decrease in PKC association with the insulin receptor as well as the accompanying increase in receptor tyrosine kinase, although a slight increase in plasma membrane PKC activity became detectable upon >60 min exposure of the cells.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Glucose effects on PKC activity and on insulin receptor tyrosine kinase and serine phosphorylation in L6 myotubes, time course relationships. A, for determining membrane PKC activity levels in the myotubes (circles), extracts were first prepared from the membrane fraction of cells incubated with 25 mM glucose for the indicated times. The extracts were subsequently assayed for phosphorylation of the Ac-MBP-(4-14) substrate as described under "Experimental Procedures." The activity for PKC associated with the insulin receptor (triangles) is that obtained upon immunoprecipitating the receptor in 1 mg of cell lysate, and the activity for the membranes is the cpm/µg of protein. B, tyrosine kinase activity was assayed in vitro in partially purified insulin receptor preparations from cells exposed to 25 mM glucose for the indicated times (rhomboids). In the experiments shown, poly(Glu-Tyr) was used as insulin receptor substrate. The serine/threonine phosphorylation of insulin receptors (squares) was determined in 32P-labeled intact cells as described under "Experimental Procedures." Each data point represents the mean ± S.D. of triplicate (kinase and PKC activity) or duplicate (serine phosphorylation) determinations in four independent experiments. IRTK, insulin receptor tyrosine kinase. C, membrane and insulin receptor-associated PKC activities (left and middle panel, respectively) and receptor tyrosine kinase activity (right panel) were determined upon incubation of the cells with 25 mM sucrose (filled symbols) or pyruvate (open symbols) for the indicated times. Results are expressed as in A (PKC activity) or B (IR kinase activity). Each point represents the mean ± S.D. of duplicate determinations in three independent experiments.

To investigate whether the effects of glucose on IR-PKC co-precipitation simultaneously involved all of the major PKC isoforms expressed in the L6hIR cells, we assayed PKC activity in precipitates from the cells with PKC-, --, or --specific antibodies. As shown in Fig. 7, upon 60 min incubation with 25 mM glucose, PKC activity was increased by 2.5-fold in all of the precipitates. A slight increase in activity was also measured in precipitates with the PKC and - antibodies upon 3 min incubation with glucose. In contrast, this short exposure to glucose led to a 2-fold decrease in PKC activity in the precipitates with PKC antibodies. Since the activities of PKC and - in solubilized cell membranes increased with time but the total receptor-associated PKC activity decreased initially (Fig. 6A), the main contribution to the PKC activity of the insulin receptor appeared to be due to PKC. Accordingly, Western blotting with isoform-specific antibodies revealed a 2-fold decrease in PKC recovery in IR precipitates from cells exposed to 25 mM glucose for 3 min, whereas recovery of PKC and - in these same precipitates increased by 35 and 40%, respectively (Fig. 8A). Cell exposure to glucose for longer times was accompanied by a progressive increase in PKC co-precipitation with the IR and further increases in PKC and - co-precipitation as well. No PKC isoform was detected in the receptor precipitates by blotting with nonspecific IgG. The same results were obtained by precipitating the cells with PKC antibodies followed by blotting with receptor antibodies (data not shown). Interestingly, the changes in membrane- and IR-associated PKC upon exposure to 25 mM glucose were accompanied by opposite modifications of PKC levels in the cytosolic fraction of the cells, indicating cytosolic translocation of this PKC in response to glucose (Fig. 8B).

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Glucose effect on the activity of specific PKC co-precipitated with insulin receptors. L6 myotubes were exposed to 25 mM glucose for the indicated times, and PKC activity was determined in immunoprecipitates from solubilized cell membranes with PKC, -, and - antibodies as described under "Experimental Procedures." On the vertical axis, PKC activity is reported as percent change of that measured in insulin receptor precipitates from cells not exposed to glucose. Each value is the mean of triplicate determinations in four independent experiments.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Glucose effect on insulin receptor association with specific PKC isoforms. A, top, L6 myotubes were incubated with 25 mM glucose for the indicated times. Cell lysates were then precipitated with mAb3IR insulin receptor antibodies and Western-blotted with antibodies to PKC, -, or - or with nonimmune human IgG (hIgG). To ensure equal numbers of insulin receptors in each sample, filters were stripped and reprobed with anti-receptor antibodies (A, bottom). In parallel experiments, PKC, -, and -, were also immunoblotted from cytosolic preparations from the cells as described under "Experimental Procedures" (B, top). Normalization of these blots were controlled by re-probing the filters with a -actin antibody (B, bottom). Detection of the blots shown in the figure was achieved by chemiluminescence. The autoradiographs shown are representative of three independent experiments.

PKC Signaling of Glucose Disposal Autoregulation in L6 Myotubes-- If dissociation of PKC from the insulin receptor is responsible for the acute effects of glucose on its own disposal, one would predict that the inhibition of the endogenous expression of PKC would also mimic glucose action. We have addressed this issue using a specific PKC phosphorothioate antisense oligonucleotide (ASPO). Transient transfection of this antisense in L6hIR cells led to an 80% reduction in the endogenous PKC expression, with no changes in that of PKC2 and - (Fig. 9A). PKC expression was not affected by transfecting the nonspecific phosphorothioate oligonucleotide (PO). In the antisense-transfected cells, basal phosphorylations of IRS-1 and -2 were increased by 3.2- and 4-fold compared with that in the cells transfected with the nonspecific oligonucleotide, with no change in the total IRS levels of the cells (Fig. 9B). IRS phosphorylations did not further increase in response to 3 min exposure to 25 mM glucose in the antisense-transfected cells while increasing by 5- and 7-fold in the control cells. Similarly, insulin exposure increased phosphorylation of IRS-1 and IRS-2 by 8-fold in the control cells and by only 2-fold in those transfected with the specific antisense. In cells transfected with the specific PKC antisense, basal glucose uptake was increased to levels comparable to those observed in control cells after glucose exposure (Fig. 9C). Glucose exposure did not further affect its uptake in antisense cells, indicating a major role of PKC dissociation from the insulin receptor in the acute regulation of glucose disposal by glucose.

View larger version (28K): [in this window] [in a new window]   Fig. 9.   Antisense block of PKC expression in L6 cells, effects on IRS phosphorylation and 2-DG uptake. A, phosphorothioate antisense PKC oligonucleotides (ASPO) and control oligonucleotides (PO) were generated and transiently transfected in L6 cells as described under "Experimental Procedures." Untransfected cells (L6) and cells transfected with the antisense (ASPO) or the control oligonucleotides (PO) were solubilized and Western-blotted with PKC antibodies. The same filter was then re-probed with a PKC2 antibody. B, IRS-1 and IRS-2 phosphorylations were quantitated in antisense (ASPO) and in control cells (PO) upon preincubation with 100 nM insulin or 25 mM glucose as indicated. For control, total IRS levels were also quantitated as described in the legend to Fig. 4. The autoradiographs shown in the figure are representative of four independent experiments. C, ASPO and control cells were preincubated with 25 mM glucose as indicated. 2-DG uptake by the cells was then quantitated as described under "Experimental Procedures." Each bar represents the mean ± S.D. of triplicate determinations in four independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In addition to representing a fundamental energy source for most cells, glucose is an important regulatory molecule. Glucose concentration in the extracellular medium affects the expression of several genes (42), receptor signaling (17, 40, 41, 43), and the function of major effector molecules such as transporters (17, 44). For instance, recent reports indicate that acute hyperglycemia activates the glucose transport apparatus independently of insulin, contributing to an increase in the glucose uptake in peripheral tissues (10, 11, 17). There is evidence that the PKC system plays an important role in conveying glucose signals into the cells (17, 40, 45, 46). However, how glucose affects the PKC system and which PKC isoform is involved in mediating glucose signals have not been elucidated yet. In the present work, we have addressed these issues by investigating the mechanisms involved in autoregulation of glucose disposal by glucose in differentiated L6 skeletal muscle cells.

We found that acute exposure of the cells to increasing glucose concentrations determined a parallel increase in glucose uptake and intracellular metabolism. The increased glucose uptake caused by exposure to high glucose concentrations was accompanied by similar increases in the GLUT1 and GLUT4 plasma membrane content with no change in the total glucose transporters of the cells. Within seconds after raising the glucose concentration into the extracellular medium, the insulin receptor kinase also underwent activation, indicating trans-activation of the insulin receptor by glucose. In the L6 muscle cells, this early effect vanished upon 20 min of exposure to the high glucose concentrations. The immediate activation of the insulin receptor kinase by glucose likely accounted for the observed burst in glucose uptake and cellular metabolism. In fact, the expression of a dominant negative and functionally inactive kinase receptor in L6 myotubes almost completely blocked the ability of glucose to activate the glucose transport system and its further glucose metabolism. Thus, in muscle cells, the acute exposure to high glucose concentrations may stimulate the glucose transport apparatus and glucose intracellular metabolism by trans-activating the insulin signaling system.

Glucose activation of insulin receptor kinase in the L6 skeletal muscle cells was accompanied by a rapid fall in both the membrane and the IR-associated PKC activities. This reduction in receptor-associated PKC closely correlated with insulin receptor activation following glucose exposure of the cells. As was the case for the insulin receptor tyrosine kinase activity, the immediate glucose-induced decrease in plasma membrane- and receptor-associated PKC activities in L6 cells were followed by a more sustained rise in both the membrane-associated and the receptor-associated PKC activities when the exposure to high glucose concentrations was prolonged. Almost identical results were also attained in NIH-3T3 fibroblasts expressing low levels of human insulin receptors (data not shown). PKC is a likely candidate for the transient dissociation of PKC activity from the insulin receptor following exposure to high glucose levels. In fact PKC is one of the most abundant isoforms expressed in the L6 cells; based on co-precipitation with a panel of isoform-specific antibodies, PKC is the only isoform whose association with the insulin receptor transiently declined following acute exposure of the cells to high glucose levels; and in addition, specific inhibition of PKC expression with antisense oligonucleotides almost completely abolished the noted decrease in IR-coprecipitated PKC activity in response to glucose (data not shown). The same did not occur by inhibiting the expression or the function of PKC or - (data not shown).

The data presented in this report indicate that in L6 cells, in the absence of insulin, PKC is tonically associated to the insulin receptor under basal conditions. Acute glucose exposure of the cells, however, causes cytosolic translocation of PKC and induces its dissociation from the insulin receptor. This is followed by transient trans-activation of the receptor kinase and stimulation of the glucose metabolism apparatus. We suggest therefore that, in basal L6 cells, PKC may contribute to maintain the insulin receptor in an inactive state. Hence, specific inhibition of PKC expression with an antisense oligonucleotide was paralleled by increased insulin-independent receptor kinase activity and IRS phosphorylation. Concerning the mechanism through which the glucose-induced PKC dissociation from the insulin receptor might induce kinase trans-activation and signaling, decreased Ser/Thr phosphorylation of the insulin receptor itself by PKC may play an important role. Previous workers (37) have shown that PKCs phosphorylate the insulin receptor on serine and threonine residues and inhibit its tyrosine kinase activity and, conversely, that decreased Ser/Thr phosphorylation results in activation of the receptor kinase (39, 40, 46). In the present work, we showed that decreased Ser/Thr phosphorylation of the insulin receptor does occur in L6 cells following acute exposure to high glucose concentrations. In addition to affecting insulin receptor Ser/Thr phosphorylation, we have recently reported (30) that PKC association to the insulin receptor controls its cellular location and routing in different cell types. Thus, a sudden dissociation of PKC might reallocate insulin receptors in a specific cellular compartment improving its ability to interact with intracellular substrates and to initiate biological responses.

Recruitment to the plasma membrane is necessary for the activation of most PKCs in response to phorbol esters, hormones, and growth factors (12, 14). Very recently, however, N-acetylsphingosine (C₂-ceramide) has been shown to induce apoptosis by causing cytosolic translocation of the novel PKC isoforms  and  (31). This agent also inhibits PKC (47). Whereas ceramide inhibition of PKC is not achieved through translocation of the PKC, we now show that glucose can direct PKC toward the intracellular compartment releasing its control of the insulin receptor. It appears therefore that, in response to different signaling molecules, PKC functions are regulated by translocating them toward the cytosolic as well as toward the plasma membrane compartments.

PKCs are considered important physiological regulators of the insulin receptor kinase (48). These PKCs associate to the insulin receptor following prolonged glucose exposure, can phosphorylate the receptor on Ser/Thr residues (17, 40, 45), and inhibit insulin signaling (17, 45, 46, 49). PKC is also recruited to the plasma membrane compartment and associates to the insulin receptor upon insulin stimulation (30) and might elicit similar effects on insulin receptor signaling as PKC (39, 46, 49). However, by showing that PKC dissociates from the insulin receptor following exposure to high glucose concentrations, we have evidenced a novel mechanism through which classical PKCs may control the insulin signaling system and determine its trans-activation by glucose.

	ACKNOWLEDGEMENTS

We are grateful to Drs. E. Consiglio and G. Vecchio for their continuous support and advice during the course of this work. We also thank Dr. L. Beguinot (DIBIT, H. S. Raffaele, Milan, Italy) for advice and critical reading of the manuscript and Dr. D. Liguoro for the technical help.

	FOOTNOTES

^* This work was supported in part by the Biomed2 program of the European Community Grant BMH4-CT-0751 (to F. B.), Telethon Grant E.0896 (to F. B.), grants from the Associazione Italiana per la Ricerca sul Cancro (to F. B.), the Ministero dell' Università e della Ricerca Scientifica, and the CNR Target Project on Biotechnology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a fellowship of the Federazione Italiana per la Ricerca sul Cancro.

^§ Current address: Dept. di Ginecologia ed Ostetricia e Fisiopatologia della Riproduzione Umana, Federico II University of Naples, Medical School, Naples 80131, Italy.

^¶ To whom correspondence should be addressed: Dept. di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Via S. Pansini, 5, 80131 Naples, Italy. Tel.: 39 081 7463248; Fax: +39 081 7701016; E-mail beguino@unina.it.

	ABBREVIATIONS

The abbreviations used are: PKC, protein kinase C; Ab, antibody; mAb, monoclonal antibody; 2-DG, 2-deoxy-D-glucose; hIR, human insulin receptor; IR, insulin receptor; IRS, insulin receptor substrate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES