RESUMO
BACKGROUND: Wound healing is impaired in diabetes mellitus, but the mechanisms involved in this process are virtually unknown. Proteins belonging to the insulin signaling pathway respond to insulin in the skin of rats. OBJECTIVE: The purpose of this study was to investigate the regulation of the insulin signaling pathway in wound healing and skin repair of normal and diabetic rats, and, in parallel, the effect of a topical insulin cream on wound healing and on the activation of this pathway. RESEARCH DESIGN AND METHODS: We investigated insulin signaling by immunoblotting during wound healing of control and diabetic animals with or without topical insulin. Diabetic patients with ulcers were randomized to receive topical insulin or placebo in a prospective, double-blind and placebo-controlled, randomized clinical trial (NCT 01295177) of wound healing. RESULTS AND CONCLUSIONS: Expression of IR, IRS-1, IRS-2, SHC, ERK, and AKT are increased in the tissue of healing wounds compared to intact skin, suggesting that the insulin signaling pathway may have an important role in this process. These pathways were attenuated in the wounded skin of diabetic rats, in parallel with an increase in the time of complete wound healing. Upon topical application of insulin cream, the wound healing time of diabetic animals was normalized, followed by a reversal of defective insulin signal transduction. In addition, the treatment also increased expression of other proteins, such as eNOS (also in bone marrow), VEGF, and SDF-1α in wounded skin. In diabetic patients, topical insulin cream markedly improved wound healing, representing an attractive and cost-free method for treating this devastating complication of diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT01295177.
Assuntos
Complicações do Diabetes/tratamento farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Insulina/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Administração Tópica , Idoso , Animais , Medula Óssea/metabolismo , Quimiocina CXCL12/metabolismo , Cromonas/farmacologia , Complicações do Diabetes/metabolismo , Diabetes Mellitus Experimental/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Morfolinas/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Ratos Wistar , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The phosphatidylinositol 3-kinase-independent pathway to induce glucose transport may involve the tyrosine phosphorylation of the protooncogene c-Cbl. In the present study, we examined whether acute exposure to insulin stimulates the tyrosine phosphorylation of Cbl and its association with Cbl-associated protein (CAP) in muscle and adipose tissue of rats in vivo. We report herein that insulin induces Cbl tyrosine phosphorylation and association with CAP in adipose tissue but not in muscle. We also examined the expression and tyrosyl-phosphorylation state of Cbl and CAP/Cbl association in adipose tissue of rats submitted to prolonged fasting and in monosodium glutamate (MSG)-insulin-resistant rats. An increase in Cbl phosphorylation is observed in the fat of MSG rats, parallel with an increase in association of CAP-Cbl as well as an augment in CAP and Cbl protein expression in the adipose tissue of these animals. These events are accompanied by a decrease in insulin-stimulated insulin receptor/ insulin receptor substrate (IRS)-1 tyrosine phosphorylation and an increase in the IRS-2/phosphatidylinositol 3-kinase/Akt/Foxo1 pathway. In adipocytes of fasted rats, there is a decrease in CAP and Cbl protein expression, insulin-induced Cbl phosphorylation, and the association with CAP. In parallel, there is also a decrease in the insulin receptor/IRSs/Akt/Foxo1 pathway. Thus, insulin is able to induce Cbl tyrosine phosphorylation and its association with CAP in the adipose tissue of normal rats. In addition, our data provide evidence that the CAP-Cbl pathway may have a role in the modulation of adiposity in fasting and in MSG-treated rats.
Assuntos
Tecido Adiposo/metabolismo , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases , Adipócitos/metabolismo , Tecido Adiposo/citologia , Animais , Modelos Animais de Doenças , Jejum/fisiologia , Hiperinsulinismo/metabolismo , Hipoglicemiantes/sangue , Hipoglicemiantes/farmacologia , Insulina/sangue , Insulina/farmacologia , Masculino , Fosforilação , Proteínas Proto-Oncogênicas c-cbl , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Glutamato de Sódio/farmacologia , Tirosina/metabolismoRESUMO
Growth hormone (GH) and IGFs have a long distinguished history in diabetes, with possible participation in the development of renal complications. The implicated effect of GH in diabetic end-stage organ damage may be mediated by growth hormone receptor (GHR) or postreceptor events in GH signal transduction. The present study investigates the effects of diabetes induced by streptozotocin (STZ) on renal GH signaling. Our results demonstrate that JAK2, insulin receptor substrate (IRS)-1, Shc, ERKs, and Akt are widely distributed in the kidney, and after GH treatment, there is a significant increase in phosphorylation of these proteins in STZ-induced diabetic rats compared with controls. Moreover, the GH-induced association of IRS-1/phosphatidylinositol 3-kinase, IRS-1/growth factor receptor bound 2 (Grb2), and Shc/Grb2 are increased in diabetic rats as well. Immunohistochemical studies show that GH-induced p-Akt and p-ERK activation is apparently more pronounced in the kidneys of diabetic rats. Administration of G120K-PEG, a GH antagonist, in diabetic mice shows inhibitory effects on diabetic renal enlargement and reverses the alterations in GH signal transduction observed in diabetic animals. The present study demonstrates a role for GH signaling in the pathogenesis of early diabetic renal changes and suggests that specific GHR blockade may present a new concept in the treatment of diabetic kidney disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Diabetes Mellitus Experimental/fisiopatologia , Hormônio do Crescimento/fisiologia , Rim/fisiopatologia , Proteínas Proto-Oncogênicas , Receptores da Somatotropina/antagonistas & inibidores , Transdução de Sinais/fisiologia , Substituição de Aminoácidos , Animais , Glicemia/metabolismo , Peso Corporal , Diabetes Mellitus Experimental/patologia , Proteína Adaptadora GRB2 , Hormônio do Crescimento/genética , Imuno-Histoquímica , Proteínas Substratos do Receptor de Insulina , Janus Quinase 2 , Rim/patologia , Masculino , Tamanho do Órgão , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Ratos , Ratos Wistar , Valores de Referência , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Domínios de Homologia de srcRESUMO
After receptor binding, growth hormone (GH) induces GH receptors (GHR) dimerization and JAK2 is activated after its association with a dimerized GHR, stimulating the tyrosyl phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2 and Shc proteins. G120K-PEG, a GH antagonist is produced by a mutation that blocks GH action by preventing the GHR dimerization. This study shows that the inhibitory effect of G120K-PEG was maximal with a GH:G120K-PEG ratio of 1:100, as no increase in JAK2 tyrosyl phosphorylation was observed with this dose of GH. When the dose of GH was increased and with a GH:G120K-PEG ratio of 1:10 some tyrosyl phosphorylation of JAK2 could be observed. Additionally, GH-induced IRS-1, IRS-2 and SHC tyrosyl phosphorylation was inhibited approximately 50% at equimolar concentrations of the antagonist of GH and almost abolished with a GH:G120K-PEG ratio of 1:100. The results clearly show that G120K-PEG inhibits GH signal transduction in mouse liver.