title: On the mechanism for neomycin reversal of wortmannin inhibition of insulin stimulation of glucose uptake.
abstract: Although a number of studies and approaches have indicated that activation of the Ser/Thr kinase called Akt/protein kinase B is critical for the insulin-stimulated increase of glucose uptake in adipocytes, other studies have indicated that this enzyme may play an ancillary role. For example, a recent study indicated that neomycin would allow insulin-stimulated Glut4 translocation and glucose transport in the presence of the phosphatidylinositol (PI) 3-kinase inhibitor, wortmannin, a known inhibitor of Akt activation (James, D. J., Salaun, C., Brandie, F. M., Connell, J. M. C., and Chamberlain, L. H. (2004) J. Biol. Chem. 279, 20567-20570). To better understand this observation, we examined a number of downstream targets of Akt. As previously reported, treatment of 3T3-L1 adipocytes with neomycin prevented the wortmannin inhibition of insulin-stimulated glucose transport. However, in the presence of neomycin, wortmannin did not inhibit the insulin-stimulated phosphorylation of several downstream targets of Akt including a proline-rich Akt substrate of 40 kDa, ribosomal protein S6, and glycogen synthase kinase-3. In addition, neomycin did not prevent the ability of a structurally unrelated PI 3-kinase inhibitor, LY294002, to inhibit the insulin-stimulated activation of glucose uptake. Moreover, neomycin reversed the inhibitory effect of wortmannin but not LY294002 on insulin stimulation of Akt kinase activity. Finally, neomycin was found to inactivate in vitro the PI 3-kinase inhibitory actions of wortmannin but not LY294002. These results indicate that the effects of neomycin in adipocytes are not mediated via its ability to sequester phosphatidylinositol 4,5-bisphosphate but are instead caused by the ability of neomycin to inactivate wortmannin.
authors.affiliation: Department of Molecular Pharmacology, Stanford University School of Medicine, 269 Campus Drive, Stanford, CA 94305, USA.
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Shimaya
authors.forename: Kristina S
authors.initials: KS
authors.lastname: Kovacina
authors.forename: Richard A
authors.initials: RA
authors.lastname: Roth
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016466:CHO Cells; D002867:Chromones / Q000494:pharmacology; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism / Q000493:pharmacokinetics*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009355:Neomycin / Q000494:pharmacology*; D010766:Phosphorylation; D011392:Proline / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D038601:Ribosomal Protein S6 / Q000378:metabolism; D013997:Time Factors; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D000900:Anti-Bacterial Agents; D002867:Chromones; D004791:Enzyme Inhibitors; D007328:Insulin; D009025:Morpholines; D038601:Ribosomal Protein S6; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D011392:Proline; D017346:Protein Serine-Threonine Kinases; D038362:Glycogen Synthase Kinase 3; D009355:Neomycin; D005947:Glucose; D000077191:Wortmannin