Prolonged incubation in PUGNAc results in increased protein O-Linked glycosylation and insulin resistance in rat skeletal muscle.
Increased flux through the hexosamine biosynthetic pathway and increased O-linked glycosylation (N-acetylglucosamine [O-GlcNAc]) of proteins have been implicated in insulin resistance. Previous research in 3T3-L1 adipocytes indicated that insulin-stimulated glucose uptake and phosphorylation of Akt were reduced after incubation with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc; 100 micromol/l), an inhibitor of the O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Therefore, in this study, we tested the effects of PUGNAc on skeletal muscle. Incubation of rat epitrochlearis muscles for 19 h with 100 micromol/l PUGNAc resulted in a marked increase in O-GlcNAcylation of multiple proteins. Incubation with PUGNAc reduced glucose transport with a physiologic insulin concentration without affecting glucose transport without insulin or with supraphysiologic insulin. PUGNAc did not significantly alter insulin-stimulated phosphorylation of Akt (serine and threonine) or its substrates glycogen synthase kinase (GSK)3 alpha and GSK3 beta. Insulin stimulated a dose-dependent (12.0 > 0.6 > 0 nmol/l) increase in the phosphorylation of a 160-kDa protein detected using an antibody against an Akt substrate phosphomotif. PUGNAc treatment did not alter phosphorylation of this protein. These results indicate that PUGNAc is an effective inhibitor of O-GlcNAcase in skeletal muscle and suggest that O-GlcNAc modification of proteins can induce insulin resistance in skeletal muscle independent of attenuated phosphorylation of Akt, GSK 3 alpha, GSK3 beta, and a 160-kDa protein with an Akt phosphomotif.
Arias
Edward B
EB
Department of Kinesiology, University of Wisconsin-Madison, Madison, Wisconsin, USA. edarias@umich.edu
Kim
Junghoon
J
Cartee
Gregory D
GD
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D007328:Insulin / Q000494:pharmacology*; D007333:Insulin Resistance / Q000502:physiology*; D007700:Kinetics; D008297:Male; D009124:Muscle Proteins / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology*; D010091:Oximes / Q000494:pharmacology*; D048448:Phenylcarbamates; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D051381:Rats; D017208:Rats, Wistar
D007328:Insulin; D009124:Muscle Proteins; D010091:Oximes; D048448:Phenylcarbamates; D010769:Phosphothreonine; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D010768:Phosphoserine; D005947:Glucose; D000117:Acetylglucosamine