O-GlcNAc Regulation of Mitochondrial Function and Energy Metabolism

O-GlcNAc is a post-translational modification (PTM) of a single N-acetylglucosamine sugar attachment on serine or threonine residues of nuclear, cytoplasmic, and mitochondrial proteins. Two opposing enzymes facilitate the modification; O-GlcNAc transferase (OGT) adds the modification, while O-GlcNAcase (OGA) removes it. The addition and the removal of O-GlcNAc, termed O-GlcNAc cycling, is often a dynamic process sensitive to changes in the cellular environment. Disruptions in O-GlcNAcylation contribute to diseases such as diabetes, cancer, and neurodegeneration. Accumulative chronic dysfunctional mitochondria also lead to the development of disease; and importantly, O-GlcNAcylation regulates mitochondrial function. In order to test our first hypothesis that disruptions in O-GlcNAc cycling affect mitochondrial function by changing the mitochondrial proteome, we employed a proteomics screen using SH-SY5Y neuroblastoma cells. We found that OGT and OGA overexpression severely disrupted the mitochondrial proteome, including proteins involved in the respiratory chain and TCA cycle. Furthermore, mitochondrial morphology in the over-expressing cells had disorganized cristae and altered shape and size. Both cellular respiration and glycolysis is impaired. These data support that O-GlcNAc cycling was essential for the proper regulation of mitochondrial function. We next investigated how sustained elevations in cellular O-GlcNAc levels would alter the metabolic profile of the cell. We elevated cellular O-GlcNAc levels by either treating SH-SY5Y cells with low levels of glucosamine (GlcN), the metabolic substrate of OGT, or the OGA inhibitor Thiamet-G (TMG). We found cellular respiration was altered and ATP levels were lower in these cells with sustained elevated O-GlcNAc. Additionally, these cells produce significantly less reactive oxygen species (ROS). Both GlcN and TMG treated cells have elongated mitochondria, while mitochondrial fusion/fission protein expressions were decreased. RNA-sequencing analysis showed that the ...