Exp Mol Med.  2005 Jun;37(3):220-229.

O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes

• Park SY
• Ryu J
• Lee W

Abstract

It has been known that O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of proteins plays an important role in transcription, translation, nuclear transport and signal transduction. The increased flux of glucose through the hexosamine biosynthetic pathway (HBP) and increased O-GlcNAc modification of protein have been suggested as one of the causes in the development of insulin resistance. However, it is not clear at the molecular level, how O-GlcNAc protein modification results in substantial impairment of insulin signaling. To clarify the association of O-GlcNAc protein modification and insulin resistance in rat primary adipocytes, we treated the adipocytes with O-(2-acetamido-2deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), a potent inhibitor of O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Prolonged treatment of PUGNAc (100 micrometer for 12 h) increased O-GlcNAc modification on proteins in adipocytes. PUGNAc also drastically decreased insulin-stimulated 2-deoxyglucose (2DG) uptake and GLUT4 translocation in adipocytes, indicating that PUGNAc developed impaired glucose utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 and Akt2 was increased by PUGNAc, accompanied by a partial reduction of insulin-stimulated phosphorylations of IRS-1 and Akt2. The PUGNAc treatment has no effect on the expression level of GLUT4, whereas O-GlcNAc modification of GLUT4 was increased. These results suggest that the increase of O-GlcNAc modification on insulin signal pathway intermediates, such as IRS-1 and Akt2, reduces the insulin-stimulated phosphorylation of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes.

Keyword

Akt2; GLUT4; insulin resistance; IRS-1; O-GlcNAc; PUGNAc

MeSH Terms

Acetylglucosamine/*analogs & derivatives/metabolism/pharmacology
Adipocytes/*metabolism
Animals
Deoxyglucose/pharmacokinetics
Glycosylation
Immunoprecipitation
*Insulin Resistance
Male
Monosaccharide Transport Proteins/metabolism
Oximes/*pharmacology
Phenylcarbamates/*pharmacology
Phosphoproteins/*metabolism
Phosphorylation
Protein-Serine-Threonine Kinases/*metabolism
Proto-Oncogene Proteins/*metabolism
Rats
Rats, Sprague-Dawley
Research Support, Non-U.S. Gov't
Subcellular Fractions/metabolism
beta-N-Acetylhexosaminidase/antagonists & inhibitors