Analysis of insulin signalling by RNAi based gene silencing

  • Zhou Q
  • Park J
  • Jiang Z
 et al. 
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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

Author-supplied keywords

  • Adipocyte
  • Analysis
  • GLUT4
  • Gene
  • Glucose
  • Glucose transporter
  • Insulin
  • Kinase
  • Membrane
  • Negative
  • Pathway
  • Phosphatase
  • Phosphatidylinositol
  • Phosphoinositide
  • Phospholipase
  • Plasma
  • Plasma membrane
  • Preadipocyte
  • Protein
  • Protein kinase
  • RNA
  • RNAi
  • Regulation
  • Regulator
  • Review
  • Signalling
  • effect
  • translocation
  • transporter

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Authors

  • Q L Zhou

  • J G Park

  • Z Y Jiang

  • J J Holik

  • P Mitra

  • S Semiz

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