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.
Zhou
Q L
QL
Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA 01605, USA.
Park
J G
JG
Jiang
Z Y
ZY
Holik
J J
JJ
Mitra
P
P
Semiz
S
S
Guilherme
A
A
Powelka
A M
AM
Tang
X
X
Virbasius
J
J
Czech
M P
MP
D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D020868:Gene Silencing; D005821:Genetic Techniques; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes; D008954:Models, Biological; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction; D000099293:Protein Kinase C-lambda
D005947:Glucose; D051275:Glucose Transporter Type 4; D007328:Insulin; D007527:Isoenzymes; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D000099293:Protein Kinase C-lambda; C495692:SLC2A4 protein, human