Using siRNA-mediated gene silencing in cultured adipocytes, we have dissected the insulin-signalling pathway leading to translocation of GLUT4 glucose transporters to the plasma membrane. RNAi (RNA interference)-based depletion of components in the putative TC10 pathway (CAP, CrkII and c-Cbl plus Cbl-b) or the phospholipase Cγ pathway failed to diminish insulin signalling to GLUT4. Within the phosphoinositide 3-kinase pathway, loss of the 5′-phosphatidylinositol 3,4,5-trisphosphate phosphatase SHIP2 was also without effect, whereas depletion of the 3′-phosphatase PTEN significantly enhanced insulin action. Downstream of phosphatidylinositol 3,4,5-trisphosphate and PDK1, silencing the genes encoding the protein kinases Akt1/PKBα, or CISK(SGK3) or protein kinases Cλ/ζ had little or no effect, but loss of Akt2/PKBβ significantly attenuated GLUT4 regulation by insulin. These results show that Akt2/PKBβ is the key downstream intermediate within the phosphoinositide 3-kinase pathway linked to insulin action on GLUT4 in cultured adipocytes, whereas PTEN is a potent negative regulator of this pathway.
Analysis of insulin signalling by RNAi-based gene silencing
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Q.L. Zhou, J.G. Park, Z.Y. Jiang, J.J. Holik, P. Mitra, S. Semiz, A. Guilherme, A.M. Powelka, X. Tang, J. Virbasius, M.P. Czech; Analysis of insulin signalling by RNAi-based gene silencing. Biochem Soc Trans 1 November 2004; 32 (5): 817–821. doi: https://doi.org/10.1042/BST0320817
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