Produção Nacional
Revisado por pares
Early Steps of Insulin Action in the Skin of Intact Rats
2001; Elsevier BV; Volume: 117; Issue: 4 Linguagem: Inglês
10.1046/j.0022-202x.2001.01473.x
ISSN1523-1747
AutoresFabiana F.F. Pelegrinelli, Ana C.P. Thirone, Alessandra L. Gasparetti, Eliana P. Araújo, Lı́cio A. Velloso, Mário J.A. Saad,
Tópico(s)Cellular transport and secretion
ResumoInsulin is an important regulator of growth and initiates its action by binding to its receptor, which undergoes tyrosyl autophosphorylation and further enhances its tyrosine kinase activity towards other intermediate molecules, including insulin receptor substrate 1, insulin receptor substrate 2, and Shc. Insulin receptor substrate proteins can dock various src-homology-2-domain-containing signaling proteins, such as the 85 kDa subunit of phosphatidylinositol 3 kinase and growth-factor-receptor-bound protein 2. The serine-threonine kinase is activated downstream to phosphatidylinositol 3 kinase. Shc protein has been shown to directly induce the association with growth-factor-receptor-bound protein 2 and downstream the activation of the mitogen-activated protein kinase. In this study we investigated insulin signal transduction pathways in skin of intact rats by immunoprecipitation and immunoblotting with specific antibodies, and also by immunohistochemistry with anti-insulin-receptor antibody. Our results showed that skin fragments clearly demonstrated the presence of insulin receptor in cell bodies of the epidermis and hair follicles and some faint staining was also detected in fibroblasts of the dermis. It was also observed that acute stimulation with insulin can induce tyrosyl phosphorylation of insulin receptor, that the insulin receptor substrates and Shc proteins serve as signaling molecules for insulin in skin of rats, and that insulin is able to induce association of insulin receptor substrate 1/phosphatidylinositol 3 kinase and Shc/growth-factor-receptor-bound protein 2 in this tissue, as well as phosphorylation of mitogen-activated protein kinase and serine-threonine kinase, demonstrating that proteins involved in early steps of insulin action are expressed in skin of intact rats and are quickly activated after insulin stimulation. Insulin is an important regulator of growth and initiates its action by binding to its receptor, which undergoes tyrosyl autophosphorylation and further enhances its tyrosine kinase activity towards other intermediate molecules, including insulin receptor substrate 1, insulin receptor substrate 2, and Shc. Insulin receptor substrate proteins can dock various src-homology-2-domain-containing signaling proteins, such as the 85 kDa subunit of phosphatidylinositol 3 kinase and growth-factor-receptor-bound protein 2. The serine-threonine kinase is activated downstream to phosphatidylinositol 3 kinase. Shc protein has been shown to directly induce the association with growth-factor-receptor-bound protein 2 and downstream the activation of the mitogen-activated protein kinase. In this study we investigated insulin signal transduction pathways in skin of intact rats by immunoprecipitation and immunoblotting with specific antibodies, and also by immunohistochemistry with anti-insulin-receptor antibody. Our results showed that skin fragments clearly demonstrated the presence of insulin receptor in cell bodies of the epidermis and hair follicles and some faint staining was also detected in fibroblasts of the dermis. It was also observed that acute stimulation with insulin can induce tyrosyl phosphorylation of insulin receptor, that the insulin receptor substrates and Shc proteins serve as signaling molecules for insulin in skin of rats, and that insulin is able to induce association of insulin receptor substrate 1/phosphatidylinositol 3 kinase and Shc/growth-factor-receptor-bound protein 2 in this tissue, as well as phosphorylation of mitogen-activated protein kinase and serine-threonine kinase, demonstrating that proteins involved in early steps of insulin action are expressed in skin of intact rats and are quickly activated after insulin stimulation. serine-threonine kinase growth-factor-receptor-bound protein 2 insulin receptor insulin receptor substrate mitogen-activated protein kinase protein kinase that activates MAP kinases phosphatidylinositol 3-kinase a serine-threonine kinase activated by Ras one member of a large family of small molecular weight GTP binding proteins src homology 2/α-collagen-related src homology 2 domain a guanine nucleotide exchange factor for Ras Insulin exerts important metabolic and cellular mitogenic effects mediated through the insulin receptor (IR) that is present in virtually all vertebrate tissues (Kahn, 1985Kahn C.R. The molecular mechanism of insulin action.Ann Rev Med. 1985; 36: 429-451Crossref PubMed Scopus (265) Google Scholar). IR, a transmembrane glycoprotein with intrinsic tyrosine kinase activity, undergoes tyrosyl autophosphorylation and is activated in response to insulin binding to the extracellular α-subunit (Kasuga et al., 1982Kasuga M. Karlsson F.A. Kahn C.R. Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor.Science. 1982; 215: 185-187Crossref PubMed Scopus (669) Google Scholar;White and Kahn, 1994White M.F. Kahn C.R. The insulin signaling system.J Biol Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). This interaction further enhances the tyrosine kinase activity of the receptor towards other intermediate molecules, including insulin receptor substrate 1 (IRS-1), IRS-2, and Shc (White et al., 1985White M.F. Maron R. Kahn C.R. Insulin rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cell.Nature. 1985; 318: 183-186Crossref PubMed Scopus (440) Google Scholar;Sun et al., 1991Sun X.J. Rothenberg P.L. Kahn C.R. et al.Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein.Nature. 1991; 352: 73-77Crossref PubMed Scopus (1250) Google Scholar;Pelicci et al., 1992Pelicci G. Lanfrancone L. Grignani F. et al.A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1112) Google Scholar;Pronk et al., 1993Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. Insulin-induced phosphorylation of the 46- and 52-kDa Shc proteins.J Biol Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar;Araki et al., 1994Araki E. Lipes M.A. Patti M.A. Bruning J.C. Haag B. 3rd Johnson R.S. Kahn C.R. Alternative pathway of insulin signaling in mice with target disruption of the IRS-1 gene.Nature. 1994; 372: 186-190Crossref PubMed Scopus (1074) Google Scholar;Tamemoto et al., 1994Tamemoto H. Kadowaki T. Tobe K. et al.Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1.Nature. 1994; 372: 182-186Crossref PubMed Scopus (880) Google Scholar). These molecules, rather than the IR itself, then couple to a downstream signaling pathway by serving as binding sites for Src homology 2 (SH2) domain-containing signaling molecules (Kahn, 1994Kahn C.R. Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes.Diabetes. 1994; 43: 1066-1084Crossref PubMed Scopus (0) Google Scholar). IRS-1 and IRS-2 bind to the 85 kDa subunit of phosphatidylinositol 3-kinase (PI 3-kinase), growth-factor-receptor-bound protein 2 (Grb2), and other SH2-containing proteins (Lavan and Lienhard, 1993Lavan B.E. Lienhard G.E. The insulin-elicited 60 kDa phosphotyrosine protein in rat adipocytes is associated with phosphatidylinositol 3-kinase.J Biol Chem. 1993; 268: 5921-5928Abstract Full Text PDF PubMed Google Scholar). Downstream to PI 3-kinase, the serine-threonine kinase Akt is activated and its phosphorylation appears to be the primary mechanism by which enzymatic activity is regulated (Burgering and Coffer, 1995Burgering B.M. Coffer P.J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction.Nature. 1995; 376: 599-602Crossref PubMed Scopus (1845) Google Scholar). In contrast to IRS-1 and IRS-2, which can associate with a wide variety of downstream effector molecules, the tyrosine phosphorylation of Shc protein leads to a specific association with the small 23 kDa adapter protein, Grb2 (Skolnik et al., 1993Skolnik E.Y. Lee C.H. Batzer A. et al.The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signaling.EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (595) Google Scholar). It has also been demonstrated that insulin stimulates the mitogen-activated protein kinases (MAPK) (Roberts, 1992Roberts T.M. Cell biology. A signal chain of events.Nature. 1992; 360: 534-535Crossref PubMed Scopus (155) Google Scholar) and that one of the pathways that leads the IR to MAPK involves Shc, Grb2, son of Sevenless (SOS), Ras, Raf, and MAP-ERK kinase (MEK) (Aronheim et al., 1994Aronheim A. Engelberg D. Li N. al-Alawi N. Schlessinger J. Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway.Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (412) Google Scholar;Hill and Treisman, 1995Hill C.S. Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity.Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1178) Google Scholar). Much is known about the regulation of glucose transport and metabolism by the insulin signaling pathway in insulin-responsive tissues such as muscle, fat, and liver (Saad et al., 1992Saad M.J. Araki E. Miralpeix M. Rothenberg P.L. White M.F. Kahn C.R. Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin recistance.J Clin Invest. 1992; 90: 1839-1849Crossref PubMed Scopus (277) Google Scholar;Páez-Espinosa et al., 1998Páez-Espinosa V. Carvalho C.R.O. Alvarez-Rojas F. Janeri L. Velloso L.A. Boschero A.C. Saad M.J.A. Insulin induces tyrosine phosphorylation of Shc and stimulates Shc/Grb2 association in insulin-sensitive tissues of the intact rat.Endocrine. 1998; 8: 193-200Crossref PubMed Scopus (17) Google Scholar). Several facts suggest an active role for insulin in skin. It has been shown that keratinocyte cell lines express IR and insulin-like growth factor receptor (IGFR) (Verrando and Ortonne, 1985Verrando P. Ortonne J.P. Insulin binding properties of normal and transformed human epidermal cultured keratinocytes.J Invest Dermatol. 1985; 85: 328-332Crossref PubMed Scopus (37) Google Scholar;Misra et al., 1986Misra P. Nickoloff B.J. Morhenn V.B. Hintz R.L. Rosenfeld R.G. Characterization of insulin-like growth facttor-1/somatomedin-C receptors on human keratinocytes monolayers.J Invest Dermatol. 1986; 87: 264-267Crossref PubMed Scopus (56) Google Scholar;Hodak et al., 1996Hodak E. Gottlieb A.B. Anzilotti M. Krueger J.G. The insulin-like growth factor-1 receptor expressed by epithelial cell with proliferative potential in human epidermis and skin appendages: correlation of increased expression with epidermal hyperplasia.J Invest Dermatol. 1996; 106: 564-570Crossref PubMed Scopus (109) Google Scholar), that functional IR are expressed in cultured murine skin keratinocytes (Wertheimer et al., 2000Wertheimer E. Trebicz M. Eldar T. Gartsbein M. Nofeh-Moses S. Tennenbaum T. Differnetial roles of insulin receptor and insulin-like growth factor-1 receptor in differentiation of murine skin keratinocytes.J Invest Dermatol. 2000; 115: 24-29Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and that human keratinocytes are dependent on insulin for their growth (Tsao et al., 1982Tsao M.C. Walthall B.J. Ham R.G. Clonal growth of normal human epidermal keratinocytes in a defined medium.J Cell Physiol. 1982; 110: 219-229Crossref PubMed Scopus (350) Google Scholar). Recently it was demonstrated in IR null mouse that insulin regulates, via the IR, the differentiation and glucose transport of skin keratinocytes (Wertheimer et al., 2001Wertheimer E. Spravchikov N. Trebicz M. et al.The regulation of skin proliferation and differentiation in the IR null mouse: implications for skin complications of diabetes.Endocrinology. 2001; 142: 1234-1241Crossref PubMed Scopus (74) Google Scholar). Whether the downstream insulin signaling pathways are activated in skin by insulin, however, has not yet been investigated. Therefore, the aim of this study was to examine whether acute insulin stimulation could induce IR phosphorylation and the effects of such stimulation on IRS-1, IRS-2, Shc, MAPK, and Akt phosphorylation, besides the association of IRS-1/PI 3-kinase and Shc/Grb2 in the skin of rats. The reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were from Bio-Rad (Richmond, CA). Tris, phenylmethylsulfonyl fluoride (PMSF), aprotinin, dithiothreitol (DTT), Triton X-100, Tween 20, diaminobenzidine (DAB), H2O2, and glycerol were from Sigma (St. Louis, MO). Sodium amobarbital and human recombinant insulin (Humulin R) were from Eli Lilly. 125I-protein A was from Amersham (Amersham, U.K.) and Protein A Sepharose 6 MB was from Pharmacia (Uppsala, Sweden). Nitro cellulose (BA85, 0.2 µm) was from Schleicher & Schuell. Male Wistar rats were from the Unicamp Central Breeding Center. Antiphospho tyrosine (αPY), anti-IRS-1 (αIRS-1/C-20), anti-IRS-2 (αIRS-2/A-19), anti-Shc (αShc/C-20), anti-PI3K (αPI3K/pα85 Z-8), anti-Grb2 (αGrb2/C-23), anti-IR, anti-IGFR, anti-phospho-MAPK (p42–44), anti-phospho-Akt (serine 473), and goat antirabbit IgG peroxidase conjugated antibodies were from Santa Cruz Technology (Santa Cruz, CA). Six-week-old male Wistar rats were provided with standard rodent chow and water ad libitum. Food was withdrawn 12–14 h before the experiments. All experiments involving animals were approved by the University of Campinas ethical committee. Rats were anesthetized with sodium amobarbital (15 mg per kg body weight, intraperitoneally), and were used 10–15 min later, i.e., as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The abdominal cavity was opened, the cava vein exposed, and 0.5 ml of normal saline with or without 2 µg of insulin was injected. At 1, 3, 5, and 15 min after insulin injection a fragment of intact skin was removed. For the dose–response experiments, doses from 0.002 µg to 2 µg were infused. Skin fragments of about 1 cm2 were coarsely minced and immediately homogenized in extraction buffer (1% Triton-X 100, 100 mM Tris, pH 7.4, containing 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM ethylenediamine tetraacetic acid, 10 mM sodium vanadate, 2 mM PMSF, and 0.1 mg of aprotinin per ml) at 4°C with a Polytron PTA 20S generator (Brinkmann Instruments model PT 10/35) operated at maximum speed for 30 s. The extracts were centrifuged at 15,000 rpm and 4°C in a Beckman 70.1 Ti rotor (Palo Alto, CA) for 45 min to remove insoluble material, and the supernatant was used for immunoprecipitation with αIRS-1, αIRS-2, αShc, αIR, and αIGFR and Protein A Sepharose 6 MB or Protein A/G plus (Santa Cruz Technology). The precipitated proteins were treated with Laemmli sample buffer (Laemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (202559) Google Scholar) containing 100 mM DTT and heated in a boiling water bath for 4 min, after which they were subjected to SDS-PAGE (6% bis-acrylamide) in a Bio-Rad miniature slab gel apparatus (Mini-Protean). For total extracts, similar sized aliquots (200 µg protein) were subjected to SDS-PAGE. Electrotransfer of proteins from the gel to nitrocellulose was performed for 90 min at 120 V (constant) in a Bio-Rad miniature transfer apparatus (Mini-Protean) as described byTowbin et al., 1979Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA. 1979; 76: 4350-4354Crossref PubMed Scopus (44170) Google Scholar except for the addition of 0.02% SDS to the transfer buffer to enhance the elution of high molecular mass proteins. Non-specific protein binding to the nitrocellulose was reduced by preincubating the filter overnight at 4°C in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). The nitrocellulose blot was incubated with antiphosphotyrosine antibody, anti-PI3K antibody, anti-Grb2 antibody, anti-phospho-MAPK antibody, or anti-phospho-Akt antibody diluted in blocking buffer (0.3% bovine serum albumin instead of nonfat dry milk) overnight at 4°C and then washed for 60 min with blocking buffer without milk. The blots were subsequently incubated with 2 µCi of 125I-protein A (30 µCi per µg) in 10 ml of blocking buffer at room temperature for 2 h and then washed again for 30 min, as described above. 125I-protein A bound to the antiphosphotyrosine and antipeptide antibodies was detected by autoradiography using preflashed Kodak XAR film with Cronex Lightning Plus intensifying screens at -80°C for 12–48 h. Band intensities were quantitated by optical densitometry (Hoefer Scientific Instruments, San Francisco, CA; model GS 300) of the developed autoradiographs. For skin sample collection, rats were anesthetized as described above. Fragments of the skin were collected from the dorsal portion of the thoracic wall, fixed in 4% paraformaldehyde, and included in paraffin blocks for 5 µm sectioning. The sections were fixed to microscopy glass slides and used for IR-specific staining. Primary IR antibody (rabbit polyclonal, anti-IR beta subunit) was diluted 1:20 in phosphate-buffered saline to a final concentration of 1–2 µg per ml and incubated with the tissue section at 4°C for 12 h. Samples were rinsed three times with phosphate-buffered saline and incubated for 2 h at room temperature with secondary peroxidase-conjugated antibody (goat antirabbit IgG) to a final concentration of 0.5 µg per ml. Once more samples were rinsed three times and color development was obtained with DAB + H2O2. Finally glasses were mounted and results recorded by photomicrography. To investigate whether IR is phosphorylated in the skin of intact rats following stimulation by insulin, we infused insulin into the cava vein of rats and then removed and homogenized the skin fragments and immunoprecipitated the proteins with a polyclonal IR antiserum. The IR immuno precipitates were analyzed for tyrosyl phosphorylation by immunoblotting with a monoclonal antiphosphotyrosine antibody. IR was rapidly tyrosine phosphorylated, reaching maximal level after 1 min and almost vanishing after 5 min of insulin exposure Figure 1a. The insulin-stimulated phosphorylation of IR, as determined by anti-IR immunoprecipitates of skin extracts, was dose dependent Figure 1b. The presence of phosphorylated IR was detectable after the injection of as little as 2 ηg of insulin, and half maximal stimulation occurred with 200 ηg of the hormone Figure 1b. Although we could not determine cava insulin levels, in preliminary experiments peripheral insulin levels obtained 90 s after an intraportal injection of 400 ηg of insulin ranged between 40 and 70 microunits per ml and were similar to the normal physiologic postprandial range in rats. Maximal stimulation was observed with 2 µg of insulin and was seven to 12 times greater than the basal levels. Even after the infusion of such very high doses of insulin as 2 µg, however, there is no tyrosine phosphorylation of insulin-like growth factor 1 (IGF-1) receptor in the skin of the intact rat Figure 1c. Skin fragments obtained for immunohistochemistry of IR contained cellular elements from the epidermis and dermis, includ ing hair follicles and glandular structures. Immunoperoxidase staining of IR in skin fragments clearly demonstrated the presence of IR in cell bodies of the epidermis and hair follicles Figure 2. Some faint staining was also detected in fibroblasts of the dermis Figure 2. To ascertain whether IRS-1 protein was tyrosyl phosphorylated by insulin stimulation in the skin of rats, we performed a Western blot assay employing anti-IRS-1 and antiphosphotyrosine antibodies. We infused insulin into the cava vein of fasted rats and then removed and homogenized the skin; whole tissue lysates were immunoprecipitated with αIRS-1. When the blots were probed with αPY, insulin-dependent tyrosyl phosphorylation of a protein with an Mr of 170,000 appropriate for IRS-1 was detectable within 1 min after exposure to insulin, being maximal at this time Figure 3a. There is a relatively high affinity interaction between IRS-1 and the 85 kDa regulatory subunit of PI 3-kinase following insulin stimulation (Sun et al., 1993Sun X.J. Crimmins D.L. Myers Jr, M.G. Miralpeix M. White M.F. Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1.Mol Cell Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar), so that both proteins are coprecipitated by antibodies to either protein. When blots previously immunoprecipitated with αIRS-1 antibody were subsequently incubated with antibodies against the 85 kDa subunit of PI 3-kinase, we observed that the intensity of the bands increased after insulin stimulation. This increase paralleled that of IRS-1 phosphorylation, and was consistent with a stable association of IRS-1 with PI 3-kinase Figure 3b. The association was maximal 1 min after insulin infusion. IRS-2 has substantial structural similarity to IRS-1, including multiple potential tyrosyl phosphorylation sites that can be tyrosyl phosphorylated following insulin stimulation (Sun et al., 1995Sun X.J. Wang L.M. Zhang Y. et al.Role of IRS-2 in insulin and cytokine signalling.Nature. 1995; 337: 173-177Crossref Scopus (751) Google Scholar). To investigate whether IRS-2 is tyrosyl phosphorylated in the skin of intact rats, we infused insulin into the cava vein of rats and removed and homogenized the skin, immunoprecipitated with αIRS-2 antibody, and immunoblotted with αPY. Increased tyrosyl phosphorylation of a protein with an Mr appropriate for IRS-2 (180,000–190,000) was detected within 1 min postinsulin Figure 3c. As the involvement of Shc in tyrosine kinase signaling pathways appears to require its phosphorylation, and considering that insulin has already been demonstrated to promote the tyrosyl phosphorylation of Shc (Pelicci et al., 1992Pelicci G. Lanfrancone L. Grignani F. et al.A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1112) Google Scholar;Pronk et al., 1993Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. Insulin-induced phosphorylation of the 46- and 52-kDa Shc proteins.J Biol Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar), we examined whether insulin could induce Shc tyrosine phosphorylation in the skin of rats. Skin extracts were removed and homogenized after infusion of insulin into the cava vein. The solubilized proteins were immuno precipitated with αShc and the presence of phosphorylated tyrosines was assessed by Western blotting with αPY Figure 4a. The mammalian Shc gene encodes three overlapping proteins of 46, 52, and 66 kDa. We have already demonstrated that in liver, muscle, and fat the 52 kDa Shc isoform has a higher level of tyrosine phosphorylation than the 46 Da species when stimulated by insulin, probably as a consequence of the higher amounts of the former compared with those of other Shc isoforms in rat tissues (Páez-Espinosa et al., 1998Páez-Espinosa V. Carvalho C.R.O. Alvarez-Rojas F. Janeri L. Velloso L.A. Boschero A.C. Saad M.J.A. Insulin induces tyrosine phosphorylation of Shc and stimulates Shc/Grb2 association in insulin-sensitive tissues of the intact rat.Endocrine. 1998; 8: 193-200Crossref PubMed Scopus (17) Google Scholar). We observed similar results in the skin of rat, as an increased tyrosyl phosphorylation of a protein migrating at Mr ≈ 52,000 (appropriate for Shc) was observed within 1 min, being maximal at this time. Shc tyrosine phosphorylation decreased 3 min after insulin infusion. As Shc, after insulin stimulation, can associate with Grb2, blots with samples that had been previously immunoprecipitated with anti-Shc were incubated with anti-Grb2. A protein recognized by αGrb2 in Western blots, migrating with the appropriate size for Grb2 (Mr 23,0000), was precipitated by αShc in an insulin-dependent fashion Figure 4b. The association of Shc/Grb2 was maximal 1 min after insulin stimulation. The insulin-stimulated phosphorylation of Shc in skin was dose dependent Figure 4c. The phosphorylated Shc was detectable after injection of as little as 0.02 µg, and half-maximal stimulation occurred with 0.2 µg of the hormone. MAPK activation appears to require the recruitment of Grb2, which has been shown to bind phosphorylated tyrosines in Shc (Lowenstein et al., 1992Lowenstein E.J. Daly R.J. Batzer A.G. et al.The SH2 and SH3 domain-containing proteins GRB2 links receptor tyrosine kinases to ras signaling.Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1307) Google Scholar;Roberts, 1992Roberts T.M. Cell biology. A signal chain of events.Nature. 1992; 360: 534-535Crossref PubMed Scopus (155) Google Scholar;Skolnik et al., 1993Skolnik E.Y. Lee C.H. Batzer A. et al.The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signaling.EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (595) Google Scholar;Aronheim et al., 1994Aronheim A. Engelberg D. Li N. al-Alawi N. Schlessinger J. Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway.Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (412) Google Scholar;Hill and Treisman, 1995Hill C.S. Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity.Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1178) Google Scholar). To estimate the rate of insulin-induced MAPK phosphoryl ation in the skin, we performed a time-course experiment after administration of insulin into the cava vein. We assessed whole tissue extract and performed the immunoblotting with anti-phospho-MAPK, which identifies phosphorylated MAPK. As shown in Figure 5(a), 1 min after exposure to insulin there was already a substantial increase in the phosphorylation of MAPK. Serine-threonine phosphorylation of Akt appears to be the primary mechanism by which its enzymatic activity is regulated (Kohn et al., 1995Kohn A.D. Kovacina K.S. Roth R.A. Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase.EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (313) Google Scholar). We have performed similar experiments to evaluate the insulin-induced serine phosphorylation of Akt in the skin. By using whole tissue extract, we performed the immunoblotting with anti-phospho-Akt, which identifies Akt phosphorylated on serine 473. We observed that 1 min after insulin infusion there was an increase in Akt phosphorylation Figure 5b. This increase paralleled that of IRS-1/PI 3-kinase association and was consistent with activation of this protein downstream of PI 3-kinase. Several clinical and experimental studies have shown that wound healing is impaired in patients with diabetes mellitus (Bouter et al., 1993Bouter K.P. Storm A.J. de Groot R.R. Uitslager R. Erkelens D.W. Diepersloot R.J. The diabetic foot in Dutch hospitals: epidemiological features and clinical outcome.Eur J Med. 1993; 2: 215-218PubMed Google Scholar;Verhofstad et al., 1998Verhofstad M.H. Bisseling T.M. Haans E.M. Hendriks T. Collagen synthesis in rat skin and ileum fibroblasts is affected differently by diabetes-related factors.Int J Exp Pathol. 1998; 79: 321-328Crossref PubMed Scopus (16) Google Scholar) and that diabetic rat serum stimulated collagen synthesis to a significantly lesser extent than normal rat serum (Backer et al., 1992Backer J.M. Myers Jr, M.G. Shoelson S.E. et al.The phosphatidylinositol 3-kinase is activated by association with IRS-1 during insulin stimulation.Embo J. 1992; 11: 3469-3479Crossref PubMed Scopus (807) Google Scholar). On the other hand, topical use of insulin improves wound healing (Hanam et al., 1983Hanam S.R. Singleton C.E. Rudek W. The effect of topical insulin on infected cutaneous ulceration in diabetic and non-diabetic mice.J Foot Surg. 1983; 22: 298-301PubMed Google Scholar), and it is known that insulin stimulates [3H]thymidine incorporation into human skin fibroblasts (Chaiken et al., 1986Chaiken R.L. Moses A.C. Usher P. Flier J.S. Insulin stimulation of aminoisobutyric acid transport in human skin fibroblasts is mediated through both insulin and type I insulin-like growth factor receptors.J Clin Endocrinol Metab. 1986; 63: 1181-1185Crossref PubMed Scopus (36) Google Scholar;Flier et al., 1986Flier J.S. Usher P. Moses A.C. Monoclonal antibody to the type I insulin-like growth factor (IGF-1) receptor blocks IGF-1 receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-1 and insulin in human skin fibroblasts.Proc Natl Acad Sci USA. 1986; 83: 664-668Crossref PubMed Scopus (207) Google Scholar). Also, insulin strongly and specifically stimulated collagen synthesis in skin fibroblasts. The exact molecular mechanism behind this stimulatory growth effect of insulin on skin cells is still unclear. In this study we investigated whether insulin induces the tyrosine phosphorylation of IR in the skin of animals and whether the downstream insulin signaling pathways are activated in this tissue by this hormone. Our results showed that acute stimulation with insulin can rapidly induce tyrosyl phosphorylation of this target protein. We also demonstrated the presence of the IR in intact skin, localized in keratinocytes of the epidermis and in some few fibroblasts of the dermis. The most staining was obtained in keratinocytes of the hair follicles, however. According toWinter et al., 1972Winter G.D. Epidermal regeneration studied in the domestic pig.in: Maibach H.I. Rovee D.T. Epidermal Wound Healing. Year Book. Medical Publishers, Chicago, IL1972: 74-108Google Scholar, keratinocytes present in the hair follicle participate actively in the regeneration processes occurring in wounded skin. Thus, we believe that signaling events described in this report are mostly occurring in hair follicle and epidermal keratinocytes and to a lesser extent in dermal fibroblasts. Although the acute stimulation with insulin can rapidly induce tyrosyl phosphorylation of IR, this effect of insulin is not shared with IGF-1 receptor, because we could not demonstrate insulin-induced IGF-1 receptor tyrosine phosphorylation in the skin of intact rats. This is in accordance with a recent report that showed that in proliferating keratinocytes both insulin and IGF-1 induced phosphorylation of both receptors, whereas insulin stimulation of terminally differentiated cells resulted in phosphorylation of the IR alone (Wertheimer et al., 2000Wertheimer E. Trebicz M. Eldar T. Gartsbein M. Nofeh-Moses S. Tennenbaum T. Differnetial roles of insulin receptor and insulin-like growth factor-1 receptor in differentiation of murine skin keratinocytes.J Invest Dermatol. 2000; 115: 24-29Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The tyrosyl phosphorylation of the IR plays a key role in insulin signal transduction by coordinating the assembly of multicomponent signaling complexes around the activated receptor or docking proteins (Kahn, 1985Kahn C.R. The molecular mechanism of insulin action.Ann Rev Med. 1985; 36: 429-451Crossref PubMed Scopus (265) Google Scholar;Geffner and Golde, 1988Geffner M.E. Golde D.W. Selective insulin action on skin, ovary, and heart in insulin-resistant states.Diabetes Care. 1988; 11: 500-505Crossref PubMed Scopus (82) Google Scholar). IRS-1 is a major cytoplasmic substrate of the IR (White and Kahn, 1994White M.F. Kahn C.R. The insulin signaling system.J Biol Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar) and our results show that insulin treatment leads to rapid changes in IRS-1 tyrosine phosphorylation in intact skin, in agreement with studies that demonstrated that insulin stimulates tyrosine phosphorylation of a 185 kDa protein in all cells and in a variety of tissues (White et al., 1985White M.F. Maron R. Kahn C.R. Insulin rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cell.Nature. 1985; 318: 183-186Crossref PubMed Scopus (440) Google Scholar;Sun et al., 1991Sun X.J. Rothenberg P.L. Kahn C.R. et al.Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein.Nature. 1991; 352: 73-77Crossref PubMed Scopus (1250) Google Scholar). IRS-1 binds several SH2 proteins through its multiple tyrosine phosphorylated sites (Kahn, 1994Kahn C.R. Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes.Diabetes. 1994; 43: 1066-1084Crossref PubMed Scopus (0) Google Scholar). The PI 3-kinase was the first SH2 protein found to be associated with IRS-1 (Lavan and Lienhard, 1993Lavan B.E. Lienhard G.E. The insulin-elicited 60 kDa phosphotyrosine protein in rat adipocytes is associated with phosphatidylinositol 3-kinase.J Biol Chem. 1993; 268: 5921-5928Abstract Full Text PDF PubMed Google Scholar). In insulin-stimulated cells, the association of PI 3-kinase with tyrosyl phosphorylated IRS-1 activates this enzyme (Backer et al., 1992Backer J.M. Myers Jr, M.G. Shoelson S.E. et al.The phosphatidylinositol 3-kinase is activated by association with IRS-1 during insulin stimulation.Embo J. 1992; 11: 3469-3479Crossref PubMed Scopus (807) Google Scholar). Thus, the ability of insulin to stimulate the association of IRS-1 to the 85 kDa regulatory subunit in intact skin suggests that insulin activates PI 3-kinase in this tissue. PI 3-kinase plays an important role in many insulin-regulated mitogenic and metabolic processes, including glucose uptake, general and growth-specific protein synthesis, and glycogen synthesis (White, 1998White M.F. The IRS-signaling system: a network of docking proteins that mediate insulin action.Mol Cell Biochem. 1998; 182: 3-11Crossref PubMed Scopus (617) Google Scholar); this can possibly be one pathway by which insulin can induce cell proliferation in skin. We also demonstrated that acute stimulation with insulin can induce Akt phosphorylation in intact skin, which is in accordance with previous studies that showed that this protein could be activated by insulin (Kohn et al., 1995Kohn A.D. Kovacina K.S. Roth R.A. Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase.EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (313) Google Scholar), being a downstream effector of PI 3-kinase (Lavan and Lienhard, 1993Lavan B.E. Lienhard G.E. The insulin-elicited 60 kDa phosphotyrosine protein in rat adipocytes is associated with phosphatidylinositol 3-kinase.J Biol Chem. 1993; 268: 5921-5928Abstract Full Text PDF PubMed Google Scholar). The results of this study showing that IRS-2 is tyrosyl phosphorylated in response to insulin suggests that both IRS family members have a role in insulin signaling in skin. Clearly, there is some overlap in function between IRS-1 and IRS-2 as both bind PI 3-kinase, Grb2, and SHP2 in response to insulin. Our results for skin tissue demonstrate that after treatment with insulin Shc was tyrosyl phosphorylated. To our knowledge, this is the first demonstration of insulin-induced tyrosine phosphorylation in this specific tissue. This result is in accordance with other reports showing that in other tissues there is an increase in Shc tyrosine phosphorylation after insulin treatment (Páez-Espinosa et al., 1998Páez-Espinosa V. Carvalho C.R.O. Alvarez-Rojas F. Janeri L. Velloso L.A. Boschero A.C. Saad M.J.A. Insulin induces tyrosine phosphorylation of Shc and stimulates Shc/Grb2 association in insulin-sensitive tissues of the intact rat.Endocrine. 1998; 8: 193-200Crossref PubMed Scopus (17) Google Scholar). Shc is thought to function as an adaptor molecule to recruit Grb2-mSos1 complexes to the activated receptor (Skolnik et al., 1993Skolnik E.Y. Lee C.H. Batzer A. et al.The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signaling.EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (595) Google Scholar). The nucleotide exchange factor mSos1 then promotes the formation of p21 Ras (GTP), thereby initiating a cascade of phosphorylation events that culminates with the phosphorylation of specific transcription factors in the nucleus (Aronheim et al., 1994Aronheim A. Engelberg D. Li N. al-Alawi N. Schlessinger J. Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway.Cell. 1994; 78: 949-961Abstract Full Text PDF PubMed Scopus (412) Google Scholar;Hill and Treisman, 1995Hill C.S. Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity.Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1178) Google Scholar). The finding in this study that Grb2 coprecipitates with Shc protein is consistent with insulin activation of MAPK, which was also observed in this study. These results suggest that this may be another pathway for the mitogenic effects that are present following stimulation with insulin and that insulin can also play a direct pivotal role in the regulation of cellular growth and differentiation. In summary, our results show that the IRS and Shc proteins serve as signaling molecules for insulin in intact skin of rats and that insulin induced association of IRS-1/PI 3-kinase and Shc/Grb2 in this tissue, as well as phosphorylation of MAPK and Akt, describing signaling pathways by which insulin can induce growth-promoting effects in skin. This work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and Conselho Nacional de Pesquisa (PRONEX). The authors wish to thank Luiz Janeri for his technical assistance.
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