Enhanced O-GlcNAc protein modification is associated with insulin resistance in GLUT1-overexpressing muscles

Maria G. Buse, Katherine A. Robinson, Bess A. Marshall, Richard C. Hresko, Mike M. Mueckler

Research output: Contribution to journalArticlepeer-review

87 Scopus citations

Abstract

O-linked glycosylation on Ser/Thr with single N-acetylglucosamine (O-GlcNAcylation) is a reversible modification of many cytosolic/nuclear proteins, regulated in part by UDP-GlcNAc levels. Transgenic (T) mice that overexpress GLUT1 in muscle show increased basal muscle glucose transport that is resistant to insulin stimulation. Muscle UDP-GlcNAc levels are increased. To assess whether GLUT4 is a substrate for O-GlcNAcylation, we translated GLUT4 mRNA (mutated at the N-glycosylation site) in rabbit reticulocyte lysates supplemented with [35S] methionine. O-GlcNAcylated proteins were galactosylated and separated by lectin affinity chromatography; >20% of the translated GLUT4 appeared to be O-GlcNAcylated. To assess whether GLUT4 or GLUT4-associated proteins were O-GlcNAcylated in muscles, muscle membranes were prepared from T and control (C) mice labeled with UDP-[3H]galactose and immunoprecipitated with anti-GLUT4 IgG (or nonimmune serum), and N-glycosyl side chains were removed enzymatically. Upon SDS-PAGE, several bands showed consistently two- to threefold increased labeling in T vs. C. Separating galactosylated products by lectin chromatography similarly revealed approximately threefold more O-GlcNAc-modified proteins in T vs. C muscle membranes. RL-2 immunoblots confirmed these results. In conclusion, chronically increased glucose flux, which raises UDP-GlcNAc in muscle, results in enhanced O-GlcNAcylation of membrane proteins in vivo. These may include GLUT4 and/or GLUT4-associated proteins and may contribute to insulin resistance in this model.

Original languageEnglish
Pages (from-to)E241-E250
JournalAmerican Journal of Physiology - Endocrinology and Metabolism
Volume283
Issue number2 46-2
DOIs
StatePublished - 2002

Keywords

  • Glucose transporter 4
  • Glucose transporter 4-associated proteins
  • O-linked glycosylation on serine/threonine with single N-acetylglucosamine of membrane proteins
  • Rabbit reticulocyte lysate
  • Transgenic mice overexpressing glucose transporter 1 in muscle

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