RESUMEN
Insulin-induced glucose uptake by skeletal muscle results from Akt2 activation and is severely impaired during insulin resistance. Recently, we and others have demonstrated that BMP9 improves glucose homeostasis in diabetic and non-diabetic rodents. However, the mechanism by which BMP9 modulates insulin action remains unknown. Here we demonstrate that Smad5, a transcription factor activated by BMP9, and Akt2, are upregulated in differentiated L6 myotubes. Smad5, rather than Smad1/8, is downregulated "in vivo" and "in vitro" by dexamethasone. Smad5 knockdown decreased Akt2 expression and serine phosphorylation and insulin-induced glucose uptake, and increased the expression of the lipid phosphatase Ship2. Additionally, binding of Smad5 to Akt2 gene is decreased in dexamethasone-treated rats and increased in L6 myotubes compared to myoblasts. The present study indicates that Smad5 regulates glucose uptake in skeletal muscle by controlling Akt2 expression and phosphorylation. These finding reveals Smad5 as a potential target for the therapeutic of type 2 diabetes.
Asunto(s)
Glucosa/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Smad5/metabolismo , Análisis de Varianza , Animales , Western Blotting , Dexametasona/farmacología , Glucocorticoides/farmacología , Inmunoprecipitación , Inositol Polifosfato 5-Fosfatasas , Insulina/farmacología , Masculino , Fibras Musculares Esqueléticas/efectos de los fármacos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación/fisiología , Interferencia de ARN/fisiología , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Proteína Smad5/genética , Proteína Smad8/metabolismo , TransfecciónRESUMEN
The Msx1 gene in mice has been proven to be induced by BMP (bone morphogenetic protein) proteins, and three binding sites for SMAD, an intracellular BMP signalling transducer, have already been identified in its promoter. Gel shift analyses were performed and they demonstrated that the consensus found very near the transcription start site, a region designed BP (basal promoter), is functional for binding nuclear proteins from 10.5, 11.5 and 13.5 dpc (days post-coitum) embryos. Notably, this binding occurs only when the SMAD-binding consensus sequence is maintained, suggesting that it is required for the formation of a protein complex over BP. Binding of purified SMAD 1 and SMAD 4 as well as supershift assay with SMAD 1/SMAD 5/SMAD 8 antibody proved that a SMAD protein is present in this complex. Transfection assays in cell cultures with fragments from BP driving the expression of luciferase confirmed that only in the presence of the SMAD consensus site is Msx1 expression activated. A proteomic analysis of the complex components after immunoprecipitation identified several proteins necessary to activate transcription including SMAD 8. Our results suggest that BMP2/BMP4 signalling through SMAD 8 is required for transcriptional activation of the mouse Msx1 gene.