Increased flux through the hexosamine biosynthetic pathway (HBP) has been proposed as one mechanism by which hyperglycemia induces insulin resistance and complications associated with diabetes. Excess glucose entering the HBP is converted to UDP-GlcNAc, a sugar donor for the posttranslational glycosylation of Ser/Thr residues by O-linked N-acetylglucosamine monosaccharide (GlcNAc). This dynamic and reversible modification of cytosolic and nuclear proteins provides an alternative mechanism of protein regulation to phosphorylation. The purpose of this study was to assess whether increased O-GlcNAc modification of proteins precedes the development of glucose-induced insulin resistance in vivo.
Methods In male Wistar rats, blood glucose was maintained at 10-12 mM by variable glucose infusion for 3 or 5 hours, after which blood glucose was returned to basal over 45 minutes by stepwise cessation of iv glucose. A 2-hour euglycemic hyperinsulinemic clamp (0.25 U/kg/hr) was then performed in these and saline-infused control rats. Insulin resistance was present in both muscle and liver after 5 but not after 3 hours of glucose infusion. Soluble protein, isolated from liver and gastrocnemius muscle of rats infused with saline or glucose for 3 or 5 hours, was separated by SDS-PAGE and transferred to nitrocellulose. Global protein O-GlcNAc modification was quantitated using an anti-O-GlcNAc antibody and normalized for protein loading using α-tubulin.
Results Preliminary data suggest that in liver, but not in muscle, there is an increase in the global level of O-GlcNAc modification following 3 hours of glucose infusion. Analysis of the four most intense bands corresponding to O-GlcNAc modified proteins in the liver at 141 kDa, 130 kDa, 90 kDa, and 60 kDa showed an increase (range 20-500%) in the level of modification with glucose infusion. We found that the mean difference in protein modification between glucose- and saline-infused rats was borderline significant for the 141 kDa protein (p = .0751) and significant for the 90 kDa and 60 kDa proteins (p = .0479 and .0022, respectively) after adjusting for hour (3 vs 5) and accounting for correlation of repeated measures.
Conclusions In an in vivo model of insulin resistance, a global increase in protein O-GlcNAc modification in liver precedes the development of insulin resistance.
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