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	Keith Vosseller, Lance Wells, M Daniel Lane, Gerald W Hart
Journal	Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 99 Issue 8 Pg. 5313-8 (Apr 16 2002) ISSN: 0027-8424 [Print] United States
PMID	11959983 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
Chemical References	CTNNB1 protein, mouse Cytoskeletal Proteins Insulin Insulin Receptor Substrate Proteins Irs1 protein, mouse Oximes Phenylcarbamates Phosphoproteins Proto-Oncogene Proteins Trans-Activators beta Catenin N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime Tyrosine Glycogen Synthase Kinases Protein Serine-Threonine Kinases Proto-Oncogene Proteins c-akt Calcium-Calmodulin-Dependent Protein Kinases Glycogen Synthase Kinase 3 Glucose Glucosamine Acetylglucosamine
Topics	3T3 Cells Acetylglucosamine (analogs & derivatives, metabolism) Adipocytes (metabolism) Animals Blotting, Western Calcium-Calmodulin-Dependent Protein Kinases (metabolism) Catalysis Cell Line Cell Nucleus (metabolism) Cytoplasm (metabolism) Cytoskeletal Proteins (metabolism) Dose-Response Relationship, Drug Enzyme Activation Glucosamine (metabolism) Glucose (pharmacokinetics) Glycogen Synthase Kinase 3 Glycogen Synthase Kinases Glycosylation Insulin (metabolism, pharmacology) Insulin Receptor Substrate Proteins Insulin Resistance Mice Oximes (metabolism) Phenylcarbamates Phosphoproteins (metabolism) Phosphorylation Precipitin Tests Protein Binding Protein Processing, Post-Translational Protein Serine-Threonine Kinases Proto-Oncogene Proteins (metabolism) Proto-Oncogene Proteins c-akt Signal Transduction Time Factors Trans-Activators Tyrosine (metabolism) beta Catenin

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