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Articles in PresS, published online ahead of print March 26, 2002
Am J Physiol Endocrinol Metab, 10.1152/ajpendo.00060.2002
Submitted on February 12, 2002
Accepted on March 18, 2002
1 Department of Medicine, Division of Endocrinology, Diabetes, and Medical Genetics, Medical University of South Carolina, Charleston, SC, USA; Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA
2 Department of Medicine, Division of Endocrinology, Diabetes, and Medical Genetics, Medical University of South Carolina, Charleston, SC, USA
3 Deptartment of Pediatrics, Division of Endocrinology and Metabolism, Washington University School of Medicine, St. Louis, MO, USA; Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA
4 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO, USA
* To whom correspondence should be addressed. E-mail: busemg{at}musc.edu.
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, which 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, immunoprecipitated with anti-GLUT4 IgG (or non-immune serum), and N-glycosyl side chains were removed enzymatically. Upon SDS-PAGE several bands showed consistently 2 - 3 fold increased labeling in T vs. C. Separating galactosylated products by lectin chromatography similarly revealed ~3-fold 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.
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