Analysis of insulin signalling by RNAi-based gene silencing

Abstract

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 Cgamma 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/PKBalpha, or CISK(SGK3) or protein kinases Clambda/zeta had little or no effect, but loss of Akt2/PKBbeta significantly attenuated GLUT4 regulation by insulin. These results show that Akt2/PKBbeta 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.

Efficacy of and resistance to anti-IGF-1R therapies in Ewing's sarcoma is dependent on insulin receptor signaling

“…(Belfiore et al, 2009). In our cells we observed exclusive expression of the IR-A isoform and increased activation of ERK1/2, AKT and STAT-3, all potentially associated with the conserved malignant phenotype of these resistant cells and associated to insulin signaling proliferation pathways (Zhou et al, 2004). Thus, cells resistant to IGF-1R HAb show a switch towards insulin pathways sustaining proliferation and malignancy, rather than metabolism.…”

The molecular basis of insulin-stimulated glucose uptake: signalling, trafficking and potential drug targets

“…In a study by Chang et al (2007), knockdown of TC10a but not TC10b in 3T3-L1 adipocytes leads to a partial inhibition of both insulin-stimulated glucose uptake and GLUT4 translocation. However, the siRNAmediated ablation of Cbl, CAP and CrkII do not have any significant effect on insulin-stimulated glucose uptake or GLUT4 translocation , Zhou et al 2004. Furthermore, while the generation of Cbl knockout mice does not result in a significant difference in insulin sensitivity (Molero et al 2004), APS knockout mice show enhanced insulin sensitivity and hypoinsulinaemia (Minami et al 2003).…”

The glucose-lowering agent sodium tungstate increases the levels and translocation of GLUT4 in L6 myotubes through a mechanism associated with ERK1/2 and MEF2D

“…In response to the hormone or to physical exercise, the amount of GLUT4 located at the plasma membrane increases, which accounts for the augmented transport capacity [1]. Insulin-induced GLUT4 translocation is dependent on the activation of phosphatidylinositol 3-kinase (PI3K) [2,3], while physical exercise enhances translocation by the activation of 5′AMP-activated protein kinase (AMPK) [4]. A novel mechanism of glucose signalling through a PI3K-independent mechanism, but dependent on the activation of extracellular signalregulated kinases 1 and 2 (ERK1/2) and phospholipase D, has also been described and may be relevant to GLUT translocation [2,5].…”