Cross-talk between GlcNAcylation and phosphorylation: roles in insulin resistance and glucose toxicity

Abstract

O-linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic posttranslational modification that, analogous to phosphorylation, cycles on and off serine and/or threonine hydroxyl groups. Cycling of O-GlcNAc is regulated by the concerted actions of O-GlcNAc transferase and O-GlcNAcase. GlcNAcylation is a nutrient/stress-sensitive modification that regulates proteins involved in a wide array of biological processes, including transcription, signaling, and metabolism. GlcNAcylation is involved in the etiology of glucose toxicity and chronic hyperglycemia-induced insulin resistance, a major hallmark of type 2 diabetes. Several reports demonstrate a strong positive correlation between GlcNAcylation and the development of insulin resistance. However, recent studies suggest that inhibiting GlcNAcylation does not prevent hyperglycemia-induced insulin resistance, suggesting that other mechanisms must also be involved. To date, proteomic analyses have identified more than 600 GlcNAcylated proteins in diverse functional classes. However, O-GlcNAc sites have been mapped on only a small percentage (<15%) of these proteins, most of which were isolated from brain or spinal cord tissue and not from other metabolically relevant tissues. Mapping the sites of GlcNAcylation is not only necessary to elucidate the complex cross-talk between GlcNAcylation and phosphorylation but is also key to the design of site-specific mutational studies and necessary for the generation of site-specific antibodies, both of which will help further decipher O-GlcNAc's functional roles. Recent technical advances in O-GlcNAc site-mapping methods should now finally allow for a much-needed increase in site-specific analyses to address the functional significance of O-GlcNAc in insulin resistance and glucose toxicity as well as other major biological processes.

Fig. 1.

Flux through the hexosamine biosynthetic pathway (HBP; A), regulation of O-linked β-N-acetylglucosamine (O-GlcNAc) cycling enzymes O-GlcNAc transferase (OGT)/O-GLcNAcase (OGA) in mediating insulin resistance (B), and Ca²⁺ cycling (C). Increased protein GlcNAcylation by 1) excess HBP flux due to elevated glucose, overexpression of glutamine:fructose-6-phosphate amidotransferase (GFAT), or glucosamine and 2) overexpression of OGT or inhibition of OGA {by O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino-N-phenylcarbamate (PUGNAc) or 1,2-dideoxy-2′-methyl-d-glucopyranoso[2,1-d]-2′-thiazoline (GT)} contributes to the establishment of insulin resistance. Overexpression of OGT in cardiomyocytes impairs Ca²⁺ cycling, whereas OGA overexpression promotes proper Ca²⁺ cycling. Glucosamine-6-P, glucosamine 6-phosphate; Fru-6-P, fructose 6-phosphate; Gln, glucosamine; DON, doxynorleucine; UDP-GlcNAc, uridine 5′-diphospho-N-acetylglucosamine. Red arrows represent increases in respective metabolites. Blue arrows represent unknown mechanisms.

Fig. 2.

O-GlcNAc-modified proteins directly involved in glucose-responsive transcription and signaling events. See main text for detailed description. IRS-1, insulin receptor substrate-1; GS, glycogen synthase; Sp1, transcription factor Sp1; PDX-1, pancreatic duodenal homeobox-1; NeuroD1a, neurogenic differentiation 1; STZ, streptozotocin; GLUT4, glucose transporter 4. Red arrows represent an increase or decrease in respective metabolites.

Fig. 3.

O-GlcNAc site-specific regulation of transducer of regulated cAMP response element-binding protein (CREB)2 (CRTC2; a.k.a TORC2) and forkhead box protein O1 (FoxO1) in mediating hepatic gluconeogenesis and “glucose toxicity.” High glucose-induced expression of gluconeogeneic genes is dependent on HBP flux and in part by GlcNAcylation of FoxO1 and CRTC2. Red arrows represent adenoviral overexpression of OGT and OGA. AZA, azaserine (GFAT inhibitor); 14-3-3, 14-3-3 protein family members.