Identification of wortmannin-sensitive targets in 3T3-L1 adipocytes. DissociationoOf insulin-stimulated glucose uptake and glut4 translocation.
The current studies investigated the contribution of phosphatidylinositol 3-kinase (PI3-kinase) isoforms to insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation. Experiments involving the microinjection of antibodies specific for the p110 catalytic subunit of class I PI3-kinases demonstrated an absolute requirement for this form of the enzyme in GLUT4 translocation. This finding was confirmed by the demonstration that the PI3-kinase antagonist wortmannin inhibits GLUT4 and insulin-responsive aminopeptidase translocation with a dose response identical to that required to inhibit another class I PI3-kinase-dependent event, activation of pp70 S6-kinase. Interestingly, wortmannin inhibited insulin-stimulated glucose uptake at much lower doses, suggesting the existence of a second, higher affinity target of the drug. Subsequent removal of wortmannin from the media shifted this dose-response curve to one resembling that for GLUT4 translocation and pp70 S6-kinase. This is consistent with the lower affinity target being p110, which is irreversibly inhibited by wortmannin. Wortmannin did not reduce glucose uptake in cells stably expressing Myr-Akt, which constitutively induced GLUT4 translocation to the plasma membrane; this demonstrates that wortmannin does not inhibit the transporters directly. In addition to elucidating a second wortmannin-sensitive pathway in 3T3-L1 adipocytes, these studies suggest that the presence of GLUT4 on the plasma membrane is not sufficient for activation of glucose uptake.
Hausdorff
S F
SF
Howard Hughes Medical Institute, the Cox Institute, and the Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Fingar
D C
DC
Morioka
K
K
Garza
L A
LA
Whiteman
E L
EL
Summers
S A
SA
Birnbaum
M J
MJ
D016475:3T3 Cells; D017667:Adipocytes; D000626:Aminopeptidases / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002867:Chromones / Q000494:pharmacology; D010122:Cystinyl Aminopeptidase; D003847:Deoxyglucose / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008845:Microinjections; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D000077191:Wortmannin
D000626:Aminopeptidases; D000730:Androstadienes; D002867:Chromones; D010122:Cystinyl Aminopeptidase; D003847:Deoxyglucose; D005947:Glucose; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D007527:Isoenzymes; D009004:Monosaccharide Transport Proteins; D009025:Morpholines; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D000077191:Wortmannin; C495693:Slc2a4 protein, mouse; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C105964:leucyl-cystinyl aminopeptidase