High glucose and insulin promote O-GlcNAc modification of proteins, including alpha-tubulin.
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.
Walgren
Jennie L E
JL
Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Vincent
Timothy S
TS
Schey
Kevin L
KL
Buse
Maria G
MG
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*
D018840:HSP70 Heat-Shock Proteins; D007004:Hypoglycemic Agents; D007328:Insulin; D008565:Membrane Proteins; D016329:Sp1 Transcription Factor; D014404:Tubulin; D005947:Glucose; D000117:Acetylglucosamine