Enhanced O-GlcNAc protein modification is associated with insulin resistance in GLUT1-overexpressing muscles.
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 [(35)S]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-[(3)H]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.
Division of Endocrinology, Department of Medicine, Diabetes, and Medical Genetics, Medical University of South Carolina, Charleston, South Carolina 29425, USA. busemg@musc.edu
Maria G
MG
Buse
Katherine A
KA
Robinson
Bess A
BA
Marshall
Richard C
RC
Hresko
Mike M
MM
Mueckler
D000215:Acylation; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006801:Humans; D007333:Insulin Resistance / Q000502:physiology*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D013379:Substrate Specificity
D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495677:SLC2A1 protein, human; C495692:SLC2A4 protein, human; C495678:Slc2a1 protein, mouse; C495693:Slc2a4 protein, mouse