Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing.
Glucose homeostasis is controlled by insulin in part through the translocation of intracellular glucose transporter 4 to the plasma membrane in muscle and fat cells. Akt/protein kinase B downstream of phosphatidylinositol 3-kinase has been implicated in this insulin-signaling pathway, but results with a variety of reagents including Akt1-/- and Akt2-/- mice have been equivocal. Here we report the application of small interfering RNA-directed gene silencing to deplete both Akt1 and Akt2 in cultured 3T3-L1 adipocytes. Loss of Akt1 alone slightly impaired insulin-mediated hexose transport activity but had no detectable effect on glycogen synthase kinase (GSK)-3 phosphorylation. In contrast, depletion of Akt2 alone by 70% inhibited approximately half of the insulin responsiveness. Combined depletions of Akt1 plus Akt2 in these cells even more markedly attenuated insulin action on glucose transporter 4 movements, hexose transport activity, and GSK-3 phosphorylation. These data demonstrate a primary role of Akt2 in insulin signaling, significant functional redundancy of Akt1 and Akt2 isoforms in this pathway, and an absolute requirement of Akt protein kinases for regulation of glucose transport and GSK-3 in cultured adipocytes.
Jiang
Zhen Y
ZY
Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA.
Zhou
Qiong L
QL
Coleman
Kerri A
KA
Chouinard
My
M
Boese
Queta
Q
Czech
Michael P
MP
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000493:pharmacokinetics; D018274:Electroporation; D005347:Fibroblasts / Q000378:metabolism; D020868:Gene Silencing; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors
D003847:Deoxyglucose; D005947:Glucose; D007328:Insulin; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; C494918:AKT1 protein, human; C494921:AKT2 protein, human; C494922:Akt2 protein, mouse