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.
Buse
Maria G
MG
Division of Endocrinology, Department of Medicine, Diabetes, and Medical Genetics, Medical University of South Carolina, Charleston, South Carolina 29425, USA. busemg@musc.edu
Robinson
Katherine A
KA
Marshall
Bess A
BA
Hresko
Richard C
RC
Mueckler
Mike M
MM
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