Prolonged incubation in PUGNAc results in increased protein O-Linked glycosylation and insulin resistance in rat skeletal muscle

Diabetes. 2004 Apr;53(4):921-30. doi: 10.2337/diabetes.53.4.921.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylglucosamine / analogs & derivatives*
  • Acetylglucosamine / pharmacology*
  • Animals
  • Biological Transport / drug effects
  • Glucose / metabolism
  • Glycosylation
  • Insulin / pharmacology*
  • Insulin Resistance / physiology*
  • Kinetics
  • Male
  • Muscle Proteins / drug effects
  • Muscle Proteins / metabolism
  • Muscle, Skeletal / drug effects
  • Muscle, Skeletal / physiology*
  • Oximes / pharmacology*
  • Phenylcarbamates*
  • Phosphorylation
  • Phosphoserine / metabolism
  • Phosphothreonine / metabolism
  • Protein Processing, Post-Translational / drug effects*
  • Rats
  • Rats, Wistar

Substances

  • Insulin
  • Muscle Proteins
  • Oximes
  • Phenylcarbamates
  • Phosphothreonine
  • N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime
  • Phosphoserine
  • Glucose
  • Acetylglucosamine