Insulin-stimulated Hydrogen Peroxide Reversibly Inhibits Protein-tyrosine Phosphatase 1B in Vivo and Enhances the Early Insulin Action Cascade

The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key post-receptor substrate proteins and balanced by the action of specific protein-tyrosine phosphatases (PTPases). PTPase activity, in turn, is highly regulated in vivo by oxidation/reduction reactions involving the cysteine thiol moiety required for catalysis. Here we show that insulin stimulation generates a burst of intracellular H2O2 in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity, as measured by a novel method using strictly anaerobic conditions. The specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of H2O2 as well as the inhibition of cellular PTPases, including PTP1B, and was associated with reduced insulin-stimulated tyrosine phosphorylation of its receptor and highM r insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other tyrosine phosphatases. The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key post-receptor substrate proteins and balanced by the action of specific protein-tyrosine phosphatases (PTPases). PTPase activity, in turn, is highly regulated in vivo by oxidation/reduction reactions involving the cysteine thiol moiety required for catalysis. Here we show that insulin stimulation generates a burst of intracellular H2O2 in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity, as measured by a novel method using strictly anaerobic conditions. The specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of H2O2 as well as the inhibition of cellular PTPases, including PTP1B, and was associated with reduced insulin-stimulated tyrosine phosphorylation of its receptor and highM r insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other tyrosine phosphatases. protein-tyrosine phosphatase insulin receptor substrate para-nitrophenyl phosphate 2-(N-morpholino)ethanesulfonic acid dithiothreitol 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate protein-tyrosine phosphatase 1B Protein-tyrosine phosphatases (PTPases)1 play a key role in the regulation of reversible tyrosine phosphorylation in the insulin action pathway. Insulin signaling is initiated by the phosphorylation of specific tyrosyl residues of the cell surface insulin receptor, which activates its exogenous kinase activity and promotes the phosphorylation of IRS proteins on specific tyrosine residues (1Withers D.J. White M. Endocrinology. 2000; 141: 1917-1921Crossref PubMed Scopus (94) Google Scholar). These activation steps are balanced, in turn, by specific cellular PTPases that dephosphorylate and inactivate the receptor kinase and reverse the adapter function of the receptor substrate proteins (2Goldstein B.J. LeRoith D. Olefsky J.M. Taylor S.I. Diabetes Mellitus: A Fundamental and Clinical Text. 2nd Ed. Lippincott, Philadelphia2000: 206-217Google Scholar). The cellular role of PTPases is apparent from the observation that highly purified insulin receptors and IRS proteins retain their tyrosine phosphorylation and activation state in vitro (3Hashimoto N. Feener E.P. Zhang W.R. Goldstein B.J. J. Biol. Chem. 1992; 267: 13811-13814Abstract Full Text PDF PubMed Google Scholar, 4Kozma L. Baltensperger K. Klarlund J. Porras A. Santos E. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4460-4464Crossref PubMed Scopus (89) Google Scholar), while in intact or permeabilized cells, receptor activation and substrate tyrosine phosphorylation are rapidly reversed (5Bernier M. Liotta A.S. Kole H.K. Shock D.D. Roth J. Biochemistry. 1994; 33: 4343-4351Crossref PubMed Scopus (26) Google Scholar, 6Mooney R.A. Kulas D.T. Bleyle L.A. Novak J.S. Biochem. Biophys. Res. Commun. 1997; 235: 709-712Crossref PubMed Scopus (37) Google Scholar, 7Calera M.R. Vallega G. Pilch P.F. J. Biol. Chem. 2000; 275: 6308-6312Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Since PTPases are high turnover number enzymes, physiological suppression of PTPase catalytic activity has been postulated to be a key feature of their regulation within the cellular environment to allow tyrosine phosphorylation to proceed in a balanced manner (8Hunter T. Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2269) Google Scholar). PTPases have in common a conserved ∼230-amino acid domain that contains the cysteine residue that catalyzes the hydrolysis of protein phosphotyrosine residues by the formation of a cysteinyl-phosphate intermediate (9Denu J.M. Dixon J.E. Curr. Opin. Chem. Biol. 1998; 2: 633-641Crossref PubMed Scopus (336) Google Scholar, 10Zhang Z.Y. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 1-52Crossref PubMed Scopus (251) Google Scholar). Several laboratories have recently provided evidence that reactive oxygen species, including H2O2, can oxidize and inactivate PTPasesin vivo (11Claiborne A. Yeh J.I. Mallett T.C. Luba J. Crane E.J. Charrier V. Parsonage D. Biochemistry. 1999; 38: 15407-15416Crossref PubMed Scopus (460) Google Scholar, 12Herrlich P. Bohmer F.D. Biochem. Pharmacol. 2000; 59: 35-41Crossref PubMed Scopus (141) Google Scholar). Since only the reduced form of the catalytic site is enzymatically active, stepwise and progressively irreversible oxidative inhibition is emerging as an important means by which PTPase activity can be suppressed in specific signal transduction pathways (13Denu J.M. Tanner K.G. Biochemistry. 1998; 37: 5633-5642Crossref PubMed Scopus (824) Google Scholar, 14Lee S.R. Kwon K.S. Kim S.R. Rhee S.G. J. Biol. Chem. 1998; 273: 15366-15372Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). In the present work, we show that insulin stimulation of hepatoma and adipose-like cells causes the rapid formation of intracellular H2O2, which is associated with significantly decreased overall PTPase activity as well as a reduction in the specific activity of PTP1B, a PTPase that has been strongly implicated in the regulation of the insulin signaling pathway. Inhibition of the insulin-stimulated production of H2O2 by catalase treatment blocks the PTPase inactivation and reduces insulin-stimulated receptor autophosphorylation and tyrosine phosphorylation of IRS proteins. These findings reveal a novel regulatory mechanism integral to the early steps in insulin signaling that contribute to the steady-state balance and propagation of the insulin action cascade. Murine 3T3-L1 preadipocytes were differentiated with insulin, dexamethasone, and isobutylmethylxanthine as described previously (15Gagnon A. Sorisky A. Obes. Res. 1998; 6: 157-163Crossref PubMed Scopus (35) Google Scholar). Cells were serum-starved overnight in medium containing 0.5% (w/v) bovine serum albumin prior to insulin stimulation. Intracellular H2O2 production was detected by fluorescence of 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA; catalog number C-6827, Molecular Probes) on confocal microscopy (Bio-Rad) at an excitation wavelength of 488 nm and emission at 515–540 nm. To avoid photo-oxidation, the fluorescence image was collected by a single rapid scan with identical parameters for all samples (16Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar). Endogenous PTPase activities as isolated from the cellular environment were measured using a novel anaerobic technique to avoid air oxidation as we described recently (41Zhu, L., Zilbering, A., Wu, X., Joseph, J. I., Jabbour, S., Deeb, W., and Goldstein, B. J. (2001) FASEB J., in press.Google Scholar). Briefly, an enclosed anaerobic work station (Forma Scientific number 901024) provided an oxygen-free environment (gas mixture of N2:H2:CO2 = 85:10:5) for cell homogenization, sealing tubes for centrifugations, immunoprecipitations, and PTPase enzyme assays. After the indicated treatments, cells were snap-frozen in liquid N2, introduced into the anaerobic chamber in a frozen state, and disrupted by scraping into ice-cold, deoxygenated homogenization buffer (150 mmNaCl, 5 mm EDTA, 5 mm EGTA, in 50 mm Hepes, pH 7.5, containing a protease inhibitor mixture (Sigma) followed by brief sonication. The whole cell lysate was prepared by adding 1% (v/v) Triton X-100, mixing on ice for 45 min, and clearing of the lysate by centrifugation at 15,000 ×g for 20 min. Prior to solubilization, the supernatant resulting from centrifugation of the homogenate at 100,000 ×g for 45 min at 4 °C was taken as the cytosol. The solubilized particulate fraction was prepared by adding Triton X-100 to the pellet from the ultracentrifugation step and incubating and clearing as above by centrifugation. Protein was measured using the method of Bradford (17Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216391) Google Scholar). PTPase activity was determined in cell fractions containing 30 μg of protein in a final volume of 100 μl at 30 °C for 30 min in reaction buffer containing 10 mm para-nitrophenyl phosphate (pNPP; Sigma) and 2 mm EDTA in 20 mm MES at pH 6.0. Where indicated, assay samples were incubated with 1 mm DTT on ice for 10 min prior to enzyme assay. The reaction was stopped by the addition of 50 μl of 1 m NaOH, and the absorption was determined at 410 nm (18Goldstein B.J. Bittner-Kowalczyk A. White M.F. Harbeck M. J. Biol. Chem. 2000; 275: 4283-4289Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). PTPase activity is reported as the optical density from hydrolysis of pNPP. Under strictly anaerobic conditions, PTP1B was immunoprecipitated from cell lysates with a monoclonal antibody directed at a C-terminal epitope that preserves its enzymatic activity (Oncogene Sciences; Ab-2) followed by adsorption to Trisacryl protein G (Pierce). PTPase activity was measured by the hydrolysis of pNPP in the anaerobic chamber in washed immunoprecipitates as described above. Control samples using non-immune mouse IgG showed minimal background PTPase activity (<5% of the activity with Ab-2). Samples of 3T3-L1 cell lysates containing 75 μg of protein were subjected to Western blot analysis using a monoclonal anti-phosphotyrosine antibody (4G10, Upstate Biotechnology) or polyclonal antibodies to IRS-1 or insulin receptor β-subunit proteins (Transduction Laboratories) as described previously (19Ahmad F. Li P.M. Meyerovitch J. Goldstein B.J. J. Biol. Chem. 1995; 270: 20503-20508Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Labeled proteins were visualized with horseradish peroxidase-conjugated anti-mouse IgG for 4G10 or with conjugated anti-rabbit IgG for the polyclonal antibodies, using conditions supplied by the manufacturer (Pierce). The blots were quantitated using an ImageStation 440 (Eastman Kodak Co.). An oxidant signal in response to insulin was demonstrated in 3T3-L1 adipocytes loaded with CM-H2DCF-DA, a redox indicator dye that is trapped intracellularly after cleavage by cellular esterases (Fig. 1). When oxidized in situ, DCF generates a signal that can be visualized by fluorescence confocal microscopy (16Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar). Following stimulation of 3T3-L1 adipocytes with 100 nm insulin, a strong oxidant signal was detected by DCF fluorescence within 1 min, peaked at 5 min, and began to dissipate by 10 min (Fig. 1 A). The oxidant generated was shown to be H2O2, since preincubation of the cells with catalase completely obliterated the fluorescent signal. A dose-response study using insulin exposure for 5 min showed that H2O2 production was detectable between 0.1 and 1 nm and was maximal at 10 nm (Fig. 1 B). Similar results were observed in human HepG2 hepatoma cells, with an insulin-stimulated H2O2 signal evident at 1 min that increased through 10 min of incubation and was completely blocked by catalase preincubation (Fig. 1 C). To determine whether the insulin-stimulated generation of intracellular H2O2 affected the endogenous activity of cellular PTPases, we applied a novel approach recently established in our laboratory that involves sample handling and analysis under anaerobic conditions (41Zhu, L., Zilbering, A., Wu, X., Joseph, J. I., Jabbour, S., Deeb, W., and Goldstein, B. J. (2001) FASEB J., in press.Google Scholar). This method preserves the activity of PTPases as isolated from the cultured cells and avoids oxidation and artifactual enzyme inhibition that occurs on exposure to air. Treatment of HepG2 cells with 100 nm insulin for 5 min resulted in a 32–52% reduction in overall PTPase activity in the cell homogenate, the cytosol, and the solubilized particulate fraction (p < 0.001) (Fig.2 A). Biochemical reduction of the enzyme samples with DTT prior to PTPase assay had no significant effect on the control samples prior to insulin treatment, but fully restored the reduced PTPase activity of the insulin-treated samples, indicating that they had been reversibly oxidized and inactivated by insulin exposure. A similar but more striking effect was observed using 3T3-L1 adipocytes, where insulin treatment caused a 62% drop in PTPase activity in the cell lysate (p < 0.001), which was restored to within control levels by treatment of the assay samples with DTT (Fig. 2 B). To determine the role of insulin-induced H2O2in the oxidative inhibition of cellular PTPase activity, cells were preincubated with catalase prior to insulin stimulation and PTPase assay. Catalase had no significant effect on the basal level of PTPase activity. However, the presence of catalase blocked the reduction of PTPase activity in the 3T3-L1 adipocyte cell lysate induced by insulin to a level that was not significantly different from the control samples (Fig. 2). This important finding indicated that H2O2 mediated the oxidative inhibition of cellular PTPase activity associated with insulin stimulation. To explore whether the insulin-stimulated generation of H2O2 affected the specific activity of PTP1B itself as isolated from intact cells, we immunoprecipitated PTP1B from snap-frozen HepG2 cell lysates under anaerobic conditions and assayed its activity within the anaerobic chamber. Following insulin treatment for 2 or 5 min, the activity of immunoprecipitated PTP1B was reduced to 46 and 29% of control, respectively (Fig.3 A). In the continued presence of insulin, this effect was sustained for at least 10 min (not shown). For comparison, direct treatment of HepG2 cells with 0.5 mmH2O2 for 5 min prior to cell lysis caused a 66% reduction of PTP1B enzyme activity (Fig. A). into the induced in PTP1B following insulin stimulation of HepG2 cells was by the immunoprecipitated enzyme with DTT prior to the PTPase assay (Fig.3 B). in the insulin stimulation reduced the PTP1B enzyme activity to of Treatment of the isolated enzyme with DTT restored the activity to of control, indicating that the insulin-induced oxidative inactivation of PTP1B was reversible by In treatment of the cells with H2O2 reduced the catalytic activity of immunoprecipitated PTP1B to a similar of but this effect was reversible in vitro with DTT to only of the control level (Fig.3 B). H2O2 itself causes a of PTP1B catalytic thiol oxidation with cell treatment with Treatment of immunoprecipitated PTP1B from the control HepG2 cells with DTT increased the enzyme activity but significantly by (p = situ, only a fraction of the enzyme is present in an oxidized state that is by reduction in In 3T3-L1 adipocytes, insulin treatment also reduced the activity of immunoprecipitated PTP1B to of control, which was reversible to of control by preincubation of the immunoprecipitated enzyme with DTT prior to PTPase assay (Fig.3 C). To the role of H2O2 in the reduced activity of PTP1B from the insulin-treated cells, 3T3-L1 adipocytes were preincubated with which completely blocks the insulin-stimulated generation of H2O2 in cells, as shown above in the Insulin stimulation of the 3T3-L1 adipocytes reduced PTP1B activity in the control cells by which was completely by the cells with catalase (Fig. Catalase the insulin-stimulated reduction in PTP1B activity in HepG2 cells (not indicating that in of cell the insulin-induced oxidative inhibition of PTP1B activity, mediated by H2O2, was the in overall PTPase activity by insulin in the cell A effect of insulin on PTP1B activity was also by treatment of HepG2 cells with G. Li J. A. Biophys. 1995; PubMed Scopus Google Scholar), which blocks insulin receptor autophosphorylation in the HepG2 cells (not and reduced the insulin inhibition of PTP1B enzyme activity by (Fig. 4 A). To determine the of the insulin insulin to the HepG2 cells was with a acid after of insulin and after an 10 min, the cellular PTP1B activity was determined by under anaerobic conditions (Fig. 4 B). continued exposure to insulin to a reduction in PTP1B of the insulin fully reversed the inhibition of PTP1B activity following with insulin of up to 30 min. we the effect of the insulin-induced production of H2O2 with catalase on insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS proteins in 3T3-L1 adipocytes Catalase treatment had no effect on the basal level of insulin receptor or IRS phosphorylation in the serum-starved However, catalase reduced the autophosphorylation of the insulin receptor by and and reduced the insulin-stimulated tyrosine phosphorylation of IRS proteins by and at 1 or 5 min of insulin The protein of IRS-1 and the insulin receptor β-subunit was by incubation of the cells with catalase (Fig. 5 A). data that by the insulin-induced inhibition of PTP1B, catalase treatment results in a sustained level of PTP1B activity, which to be to insulin-stimulated autophosphorylation of its receptor and the propagation of the early insulin signal to IRS proteins. involving oxidative inactivation of PTPases and the signaling pathways they has evident in work that reactive oxygen can be generated by stimulation of cells with and P. Bohmer F.D. Biochem. Pharmacol. 2000; 59: 35-41Crossref PubMed Scopus (141) Google Scholar, T. 2000; PubMed Scopus Google Scholar). H2O2 has been implicated in the activation of tyrosine phosphorylation in a manner that the effect of as and H2O2 can as an integral of signal transduction (16Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar, K. T. 1995; 270: PubMed Scopus Google Scholar, Google Scholar, M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Biol. 1997; PubMed Scopus Google Scholar, FASEB J. 1997; PubMed Scopus Google Scholar, Seo Kwon H.K. S. K.S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). data has also that signal transduction pathways by reactive oxygen associated with the action of and the stepwise oxidation and inactivation of PTPase (11Claiborne A. Yeh J.I. Mallett T.C. Luba J. Crane E.J. Charrier V. Parsonage D. Biochemistry. 1999; 38: 15407-15416Crossref PubMed Scopus (460) Google Scholar, 12Herrlich P. Bohmer F.D. Biochem. Pharmacol. 2000; 59: 35-41Crossref PubMed Scopus (141) Google Scholar). The of in the PTPase to oxidative inactivation in the site which the catalytic cysteine moiety to be in a reduced state Z.Y. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 1-52Crossref PubMed Scopus (251) Google Scholar). The catalytic thiol in to acid to the enzyme site that strongly its its a to more that in a cysteine and its at physiological pH (9Denu J.M. Dixon J.E. Curr. Opin. Chem. Biol. 1998; 2: 633-641Crossref PubMed Scopus (336) Google Scholar, D. M.P. Rev. Biophys. 1998; PubMed Scopus Google Scholar). This cysteine residue is to oxidation to progressively more this the which is reversible and to reduction by cellular or by in steps of oxidation to and and can to irreversible PTPase This a regulatory mechanism for PTPases within the cellular PTP1B, in to be a cellular for oxidative inactivation possibly followed by with that are at least balanced by cellular (13Denu J.M. Tanner K.G. Biochemistry. 1998; 37: 5633-5642Crossref PubMed Scopus (824) Google Scholar, 14Lee S.R. Kwon K.S. Kim S.R. Rhee S.G. J. Biol. Chem. 1998; 273: 15366-15372Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, S. Zhang Z.Y. Chock P.B. Biochemistry. 1999; 38: PubMed Scopus Google Scholar). PTP1B was of the specific PTPases to be implicated in the regulation of insulin receptor autophosphorylation and post-receptor insulin signaling M.R. Vallega G. Pilch P.F. J. Biol. Chem. 2000; 275: 6308-6312Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, B.J. Bittner-Kowalczyk A. White M.F. Harbeck M. J. Biol. Chem. 2000; 275: 4283-4289Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, M.F. J.E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, E. Olefsky J.M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Kim S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). The compelling evidence for a physiological role of PTP1B in insulin action has been the of insulin and of insulin-stimulated protein-tyrosine phosphorylation in PTP1B M. P. E. S. D. A. J. 1999; PubMed Scopus Google Scholar, Kim J.M. N. M. Kim A. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). in the intracellular enzymatic activity of PTP1B also insulin action in adipose from A. J. D. D. A. M. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). PTP1B has been as a for to insulin signaling through protein-tyrosine phosphorylation in the insulin action pathway Biochem. Pharmacol. 2000; PubMed Scopus Google Scholar), and specific of PTP1B have been reported M.S. J. B. L. D. J. Chem. 2000; PubMed Scopus Google Scholar). In the present work, we provide evidence for a between the insulin-induced generation of cellular the oxidative inactivation of the catalytic activity of In early of insulin was that H2O2 at least of the of insulin M.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and more work has demonstrated that H2O2 is physiological insulin signal transduction in adipose cells J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Meyerovitch and J. J.M. P. Biochemistry. 1992; PubMed Scopus Google the observation that stimulation of hepatoma cells with of insulin a of caused a significant reduction in PTPase activity measured an insulin receptor the novel findings in our present study is the that insulin stimulation generates a burst of H2O2in of insulin-sensitive In addition to the of the specific activity of cellular PTP1B by by insulin, is that this burst of H2O2 also insulin signaling, as shown in response to in other cell P. Bohmer F.D. Biochem. Pharmacol. 2000; 59: 35-41Crossref PubMed Scopus (141) Google Scholar, T. 2000; PubMed Scopus Google FASEB J. 1997; PubMed Scopus Google Scholar). the of this pathway for insulin signaling in cell are In we the novel observation that in 3T3-L1 adipocytes and hepatoma cells, the of reactive oxygen by insulin is associated with reversible oxidative inactivation of overall cellular PTPase activity and of This in turn, insulin-stimulated receptor autophosphorylation and the tyrosine phosphorylation of IRS proteins. the of evidence that PTP1B is in the regulation of insulin signaling, this regulation of the balance of protein-tyrosine phosphorylation play a role in signal transduction in the insulin action pathway. the of this work with of of and for with confocal