Research Highlights

Insulin signaling: Modifying competition

Mechanisms of activation of Akt. From Nat. Rev. Mol. Cell Biol. 11, 9-22 (2010) doi:10.1038/ncpneuro0155

The attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine or threonine residues is a posttranslational modification that rivals phosphorylation as a regulatory switch in cell signaling pathways. Insulin signaling is dampened by O-GlcNAc, an effect thought to occur by direct competition with phosphorylation for the same or adjacent regulatory sites. However, it is not clear which insulin-responsive proteins are regulated by this simple sugar modification. Gerald Hart and colleagues examined the pathway upstream of AKT and found that O-GlcNAc inhibits phosphorylation of insulin receptor substrate 1 (IRS-1) at a kinase-binding motif, leading to insulin resistance. The study, published in the Journal of Biological Chemistry, implicates O-GlcNAc in the etiology of diabetes.

Insulin stimulates uptake of glucose by binding to the insulin receptor (IR) and initiating a well-studied signaling cascade that increases trafficking of the GLUT-4 glucose receptor to the plasma membrane. In this pathway, insulin receptor substrate proteins including IRS-1 are phosphorylated by the IR, and in turn activate phosphoinositide 3-kinase (PI3K), causing phosphorylation of phosphoinositides and activation of their dependent kinase, PDK1. AKT is phosphorylated by activated PDK1, which leads to the mobilization of GLUT-4 vesicles.

The authors immunopurified several proteins of the AKT pathway from adipocytes, with or without prior insulin stimulation, and incubated them with O-GlcNAc transferase (OGT) and labeled sugar. The results confirmed that IRS-1 is a good substrate for OGT, irrespective of insulin treatment. IRS-2 and PDK1, by contrast, were more highly modified by O-GlcNAc in the absence of insulin stimulation, suggesting that phosphorylation or O-GlcNAcylation within the cell in response to insulin blocks the subsequent in vitro transfer of O-GlcNAc.

Modification of IRS-1 and IRS-2 with O-GlcNAc has been reported before, but no effect was detected on their overall insulin-stimulated phosphorylation. Hart and colleagues examined a specific IRS-1 phosphorylation site, Tyr 608, from a domain crucial for the binding and activation of PI3K. They also measured the phosphorylation intensity of AKT at Thr 308, an indicator of AKT activation. An overabundance of O-GlcNAc – which significantly increased the O-GlcNAc modification of IRS-1 – reduced phosphorylation of IRS-1 at Tyr 608, and reduced AKT activity by 45%. Insulin-induced phosphorylation of IRS-1 was likewise reduced, and the interaction with PI3K was inhibited. The decrease in AKT activity reduced GLUT-4 glucose uptake, confirming the biological relevance of the increased O-GlcNAcylation of IRS-1.

The activity of OGT is responsive to insulin, and the authors tested whether the transferase interacts with insulin signaling proteins upstream of AKT. OGT interaction with PDK1, and O-GlcNAcylation of the kinase, increased with insulin stimulation. This interaction was blocked by elevated O-GlcNAc, which could result from modification of sites on OGT, PDK1 or other interacting proteins. The role of insulin stimulated OGT interaction with PDK1 is not yet clear.

These data support the importance of O-GlcNAcylation as a regulator of insulin signaling, and increase understanding of how this special glycosylation might contribute to the etiology of diabetes.

Author: Emma Leah