Analysis of insulin signalling by RNAi-based gene silencing.

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.

Expression of ARF6 mutants in neuroendocrine cells suggests a role for ARF6 in synaptic vesicle biogenesis.

ARF6 regulates membrane trafficking between the plasma membrane and endosomes. We investigated the role of ARF6 in synaptic vesicle biogenesis as this process occurs both at the plasma membrane and at endosomes. We used a synaptic vesicle marker protein, p-selectin-horseradish peroxidase (HRP), to follow the effects of ARF6 expression on synaptic vesicle biogenesis in PC12 neuroendocrine cells. Expression of a constitutively active ARF6 mutant increased, while expression of a nucleotide-free ARF6 mutant decreased, p-selectin-HRP levels in the synaptic vesicle peak. These results provide the first direct evidence for a role for ARF6 in synaptic vesicle biogenesis.