High glucose and insulin promote<i>O-</i>GlcNAc modification of proteins, including α-tubulin

American Journal of Physiology - Endocrinology and Metabolism - Tập 284 Số 2 - Trang E424-E434 - 2003
Jennie Walgren1,2, Timothy S. Vincent3, Kevin L. Schey1, Maria G. Buse4,2
1Departments of Pharmacology
2Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, and
3Pathology, and
4Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425

Tóm tắt

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 α-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 α-tubulin are O-GlcNAcylated; the modification is markedly enhanced by sustained increased glucose flux.

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American Journal of Physiology - Endocrinology and Metabolism

Tập 284 Số 2

E424-E434

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