title: Vanadate and rapamycin synergistically enhance insulin-stimulated glucose uptake.
abstract: Tyrosine dephosphorylation, serine phosphorylation, and proteasomal degradation of insulin receptor substrates (IRSs) are implicated in the negative regulation of insulin action. Here we show that simultaneous inhibition of IRS-1 tyrosine dephosphorylation and proteasomal degradation synergistically augments insulin-responsive glucose uptake. L6 skeletal muscle cells (L6 cells) were treated with inhibitors of protein-tyrosine phosphatases, proteasomal degradation, and mammalian target of rapamycin (mTOR), and the effects of insulin on glucose uptake, IRS-1 tyrosine phosphorylation, phosphatidylinositol (PI) 3-kinase activity, and IRS-1 mass were examined. Pretreatment of L6 cells with sodium orthovanadate (Na(3)VO(4)) plus the mTOR inhibitor rapamycin caused a 5-fold increase in insulin-responsive glucose uptake at 2 hours when compared to insulin alone. Evaluation of IRS-1 associated PI 3-kinase activity, IRS-1-associated p85 mass, and IRS-1 tyrosine phosphorylation showed that 2 hours after insulin addition they were reduced by 70% from maximal activity. Likewise, IRS-1 mass was reduced by 50%. When L6 cells were pretreated with Na(3)VO(4) plus the proteasome inhibitor MG-132 or the mTOR inhibitor rapamycin prior to insulin addition, IRS-1 mass loss as well as IRS-1/PI-3 kinase complex decay was blocked at 2 hours and PI 3-kinase activity was increased 2.5-fold and 4-fold, respectively, over insulin alone. Finally, treatment of L6 cells with subtherapeutic amounts of vanadyl sulfate and rapamycin induced a synergistic 3-fold increase in insulin-induced glucose uptake at 2 hours. These findings indicate that vanadium and rapamycin synergize to enhance glucose uptake by preventing IRS-1 mass loss and IRS-1/PI 3-kinase complex decay and may offer a new approach to enhance glucose transport in diabetes.
authors.affiliation: Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Jason C
authors.initials: JC
authors.lastname: O'Connor
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Freund
mesh_terms: D000818:Animals; D002460:Cell Line; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004357:Drug Synergism; D005947:Glucose / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex; D020123:Sirolimus / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology*
chemical_list: D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D005947:Glucose; D007004:Hypoglycemic Agents; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D007976:Leupeptins; D009097:Multienzyme Complexes; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D046988:Proteasome Endopeptidase Complex; D020123:Sirolimus; D014443:Tyrosine; D014638:Vanadates; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde