High glucose and insulin promote O-GlcNAc modification of proteins, including alpha-tubulin.

Walgren JL, Vincent TS, Schey KL, Buse MG
Am J Physiol Endocrinol Metab. 2003 284 (2): E424-34

PMID: 12397027 · DOI:10.1152/ajpendo.00382.2002

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.

MeSH Terms (14)

Acetylglucosamine Animals Cells, Cultured Glucose Glycosylation HSP70 Heat-Shock Proteins Hypoglycemic Agents Insulin Insulin Resistance Mass Spectrometry Membrane Proteins Muscle Cells Sp1 Transcription Factor Tubulin

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