title: Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes.
abstract: Increased flux of glucose through the hexosamine biosynthetic pathway (HSP) is believed to mediate hyperglycemia-induced insulin resistance in diabetes. The end product of the HSP, UDP beta-N-acetylglucosamine (GlcNAc), is a donor sugar nucleotide for complex glycosylation in the secretory pathway and for O-linked GlcNAc (O-GlcNAc) addition to nucleocytoplasmic proteins. Cycling of the O-GlcNAc posttranslational modification was blocked by pharmacological inhibition of O-GlcNAcase, the enzyme that catalyzes O-GlcNAc removal from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc). PUGNAc treatment increased levels of O-GlcNAc and caused insulin resistance in 3T3-L1 adipocytes. Insulin resistance induced through the HSP by glucosamine and chronic insulin treatment correlated with increased O-GlcNAc levels on nucleocytoplasmic proteins. Whereas insulin receptor autophosphorylation and insulin receptor substrate 2 tyrosine phosphorylation were not affected by PUGNAc inhibition of O-GlcNAcase, downstream phosphorylation of Akt at Thr-308 and glycogen synthase kinase 3 beta at Ser-9 was inhibited. PUGNAc-induced insulin resistance was associated with increased O-GlcNAc modification of several proteins including insulin receptor substrate 1 and beta-catenin, two important effectors of insulin signaling. These results suggest that elevation of O-GlcNAc levels attenuate insulin signaling and contribute to the mechanism by which increased flux through the HSP leads to insulin resistance in adipocytes.
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
authors.forename: Keith
authors.initials: K
authors.lastname: Vosseller
authors.forename: Lance
authors.initials: L
authors.lastname: Wells
authors.forename: M Daniel
authors.initials: MD
authors.lastname: Lane
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Hart
mesh_terms: D016475:3T3 Cells; D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism*; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005944:Glucosamine / Q000378:metabolism; D005947:Glucose / Q000493:pharmacokinetics; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006031:Glycosylation; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance; D051379:Mice; D010091:Oximes / Q000378:metabolism; D048448:Phenylcarbamates; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators; D014443:Tyrosine / Q000378:metabolism; D051176:beta Catenin
chemical_list: C495267:CTNNB1 protein, mouse; D003598:Cytoskeletal Proteins; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D010091:Oximes; D048448:Phenylcarbamates; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D051176:beta Catenin; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D014443:Tyrosine; D038341:Glycogen Synthase Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D005947:Glucose; D005944:Glucosamine; D000117:Acetylglucosamine