RNAi-based analysis of CAP, Cbl, and CrkII function in the regulation of GLUT4 by insulin.
Stimulation of glucose transport by insulin in cultured adipocytes through translocation of intracellular GLUT4 glucose transporters to the plasma membrane has been suggested to require phosphatidylinositol (PI) 3-kinase-dependent and independent mechanisms. To test the involvement of a PI 3-kinase-independent pathway leading to activation of the TC10 GTPase, the putative intermediates CAP, c-Cbl, Cbl-b, and CrkII were selectively depleted in 3T3-L1 adipocytes using highly efficient small interfering (si) RNAs. Simultaneous depletion of the ubiquitination factors c-Cbl plus Cbl-b in cultured adipocytes had the expected effect of delaying dephosphorylation of EGF receptors upon removal of EGF. However, siRNA-mediated gene silencing of both Cbl isoforms or CAP or CrkII in these cells failed to attenuate insulin-stimulated deoxyglucose transport or Myc-tagged GLUT4-GFP translocation at either sub-maximal or maximal concentrations of insulin. The dose-response relationship for insulin stimulation of deoxyglucose transport in primary adipocytes derived from c-Cbl knock-out mice was also identical to insulin action on adipocytes from wild type mice. These data are consistent with the hypothesis that CAP, Cbl iso-forms, and CrkII are not required components of insulin signaling to GLUT4 transporters.
Mitra
Prasenjit
P
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Zheng
Xuexiu
X
Czech
Michael P
MP
D016475:3T3 Cells; D000818:Animals; D003598:Cytoskeletal Proteins / Q000502:physiology*; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009124:Muscle Proteins / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D050721:Proto-Oncogene Proteins c-cbl; D050737:Proto-Oncogene Proteins c-crk; D034622:RNA Interference; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000502:physiology*
D003598:Cytoskeletal Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; C495693:Slc2a4 protein, mouse; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C493849:Cbl protein, mouse