Elevation of Global O-GlcNAc Levels in 3T3-L1 Adipocytes by Selective Inhibition of O-GlcNAcase Does Not Induce Insulin Resistance

TY - JOUR

T1 - Elevation of Global O-GlcNAc Levels in 3T3-L1 Adipocytes by Selective Inhibition of O-GlcNAcase Does Not Induce Insulin Resistance

AU - Macauley, Matthew S.

AU - Bubb, Abigail K.

AU - Martinez-Fleites, Carlos

AU - Davies, Gideon J.

AU - Vocadlo, David J.

PY - 2008/12/12

Y1 - 2008/12/12

N2 - The O-GlcNAc post-translational modification is considered to act as a sensor of nutrient flux through the hexosamine biosynthetic pathway. A cornerstone of this hypothesis is that global elevation of protein O-GlcNAc levels, typically induced with the non-selective O-GlcNAcase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-D-glycopyranosylidene) amino-N-phenylcarbamate), causes insulin resistance in adipocytes. Here we address the potential link between elevated O- GlcNAc and insulin resistance by using a potent and selective inhibitor of O- GlcNAcase (NButGT (1,2-dideoxy-2'-propyl-alpha-D-glucopyranoso-[2,1-D]-Delta 2'-thiazoline), 1200-fold selectivity). A comparison of the structures of a bacterial homologue of O-GlcNAcase in complex with PUGNAc or NButGT reveals that these inhibitors bind to the same region of the active site, underscoring the competitive nature of their inhibition of O- GlcNAcase and the molecular basis of selectivity. Treating 3T3-L1 adipocytes with NButGT induces rapid increases in global O- GlcNAc levels, but strikingly, NButGT treatment does not replicate the insulin desensitizing effects of the non-selective O- GlcNAcase inhibitor PUGNAc. Consistent with these observations, NButGT also does not recapitulate the impaired insulin-mediated phosphorylation of Akt that is induced by treatment with PUGNAc. Collectively, these results suggest that increases in global levels of O-GlcNAc-modified proteins of cultured adipocytes do not, on their own, cause insulin resistance.

AB - The O-GlcNAc post-translational modification is considered to act as a sensor of nutrient flux through the hexosamine biosynthetic pathway. A cornerstone of this hypothesis is that global elevation of protein O-GlcNAc levels, typically induced with the non-selective O-GlcNAcase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-D-glycopyranosylidene) amino-N-phenylcarbamate), causes insulin resistance in adipocytes. Here we address the potential link between elevated O- GlcNAc and insulin resistance by using a potent and selective inhibitor of O- GlcNAcase (NButGT (1,2-dideoxy-2'-propyl-alpha-D-glucopyranoso-[2,1-D]-Delta 2'-thiazoline), 1200-fold selectivity). A comparison of the structures of a bacterial homologue of O-GlcNAcase in complex with PUGNAc or NButGT reveals that these inhibitors bind to the same region of the active site, underscoring the competitive nature of their inhibition of O- GlcNAcase and the molecular basis of selectivity. Treating 3T3-L1 adipocytes with NButGT induces rapid increases in global O- GlcNAc levels, but strikingly, NButGT treatment does not replicate the insulin desensitizing effects of the non-selective O- GlcNAcase inhibitor PUGNAc. Consistent with these observations, NButGT also does not recapitulate the impaired insulin-mediated phosphorylation of Akt that is induced by treatment with PUGNAc. Collectively, these results suggest that increases in global levels of O-GlcNAc-modified proteins of cultured adipocytes do not, on their own, cause insulin resistance.

KW - BETA-N-ACETYLGLUCOSAMINE

KW - SEQUENCE-BASED CLASSIFICATION

KW - SUBSTRATE-ASSISTED CATALYSIS

KW - SKELETAL-MUSCLE

KW - IN-VIVO

KW - STRUCTURAL INSIGHTS

KW - NAG-THIAZOLINE

KW - GLYCOSYLATION

KW - MECHANISM

KW - PROTEINS

UR - http://www.scopus.com/inward/record.url?scp=58049197848&partnerID=8YFLogxK

U2 - 10.1074/jbc.M804525200

DO - 10.1074/jbc.M804525200

M3 - Article

SN - 0021-9258

VL - 283

SP - 34687

EP - 34695

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 50

ER -