title: High glucose and insulin promote O-GlcNAc modification of proteins, including alpha-tubulin.
abstract: Increased flux through the hexosamine biosynthesis pathway has been implicated in the development of glucose-induced insulin resistance and may promote the modification of certain proteins with O-linked N-acetylglucosamine (O-GlcNAc). L6 myotubes (a model of skeletal muscle) were incubated for 18 h in 5 or 25 mM glucose with or without 10 nM insulin. As assessed by immunoblotting with an O-GlcNAc-specific antibody, high glucose and/or insulin enhanced O-GlcNAcylation of numerous proteins, including the transcription factor Sp1, a known substrate for this modification. To identify novel proteins that may be O-GlcNAc modified in a glucose concentration/insulin-responsive manner, total cell membranes were separated by one- or two-dimensional gel electrophoresis. Selected O-GlcNAcylated proteins were identified by mass spectrometry (MS) analysis. MS sequencing of tryptic peptides identified member(s) of the heat shock protein 70 (HSP70) family and rat alpha-tubulin. Immunoprecipitation/immunoblot studies demonstrated several HSP70 isoforms and/or posttranslational modifications, some with selectively enhanced O-GlcNAcylation following exposure to high glucose plus insulin. In conclusion, in L6 myotubes, Sp1, membrane-associated HSP70, and alpha-tubulin are O-GlcNAcylated; the modification is markedly enhanced by sustained increased glucose flux.
authors.affiliation: Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors.forename: Jennie L E
authors.initials: JL
authors.lastname: Walgren
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Vincent
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Schey
authors.forename: Maria G
authors.initials: MG
authors.lastname: Buse
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D005947:Glucose / Q000494:pharmacology*; D006031:Glycosylation / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007333:Insulin Resistance / Q000502:physiology; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism; D032342:Muscle Cells / Q000166:cytology / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D007004:Hypoglycemic Agents; D007328:Insulin; D008565:Membrane Proteins; D016329:Sp1 Transcription Factor; D014404:Tubulin; D005947:Glucose; D000117:Acetylglucosamine