Detecting O-GlcNAc and understanding its role in insulin action
MetadataShow full item record
O-linked β-N-acetylglucosamine (O-GlcNAc) modification is a ubiquitous glycosylation found on the serine and threonine side chains of intracellular proteins. The spatial and temporal distribution of O-GlcNAc is orchestrated by a pair of cycling enzymes, O-GlcNAc transferase (OGT) and β-N-acetylglucosaminidase (OGA), in response to a variety of cellular and environmental stimuli. Given that UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP), is an obligatory donor substrate of OGT, O-GlcNAc is posed as an effector of excessive glucose flux through the HBP which in turn can lead to the development of insulin resistance, a hallmark of type 2 diabetes. In this dissertation, We have studied the impact of O-GlcNAc modification on insulin signaling, as well as its implication in the development of insulin resistance. Specifically, given that glucosamine inhibits the anti-apoptotic action of insulin, we wanted to evaluate whether this occurs via an O-GlcNAc-dependent mechanism. By comparing different OGA inhibitors to modulate intracellular O-GlcNAc levels, we found that increases in global O-GlcNAc levels do not correlate with inhibition of the anti-apoptotic action of insulin and demonstrated that PUGNAc, a non-selective but commonly used OGA inhibitor, has off-target effects that influence insulin signaling. We also characterized three pan-O-GlcNAc specific monoclonal IgG antibodies using a panel of biochemical and mass-spectrometry (MS) based approaches and identified 215 O-GlcNAc modified proteins, 140 of which for the first time. Lastly, we devised a novel workflow combining metabolic labeling, click chemistry, ammonia-based β-elimination (ABBE) and tandem MS for O-GlcNAc site determination. Thus, my work has established novel tools for the study of O-GlcNAc and disproved the hypothesis that elevated O-GlcNAc levels inhibit the anti-apoptotic action of insulin.