O‐GlcNAc is Critical for the Regulation of Energy Metabolism

O‐linked N ‐acetylglucosamine (O‐GlcNAc) is the attachment of a single β‐N‐acetylglucosamine to serine/threonine amino acid residues of nuclear, cytoplasmic, and mitochondrial proteins. The modification is sensitive to changes in the cellular environment and is dynamically regulated by the opposing functions of two specific enzymes; O‐GlcNAc transferase (OGT) adds the modification, whereas, O‐GlcNAcase (OGA) removes it. Disruptions in O‐GlcNAc cycling contribute to diseases such as neurodegeneration. Dysfunctional mitochondria lead to neurodegenerative diseases, and importantly, O‐GlcNAcylation regulates mitochondrial function. Previously, we found in a proteomic‐based study that OGT or OGA overexpression disrupted mitochondrial protein expression, including electron transport chain (ETC) and TCA cycle proteins. Here, we hypothesized that prolonged elevations in cellular O‐GlcNAc levels would decrease mitochondrial function. We elevated cellular O‐GlcNAc levels by either treating SH‐SY5Y cells with low levels of Glucosamine (GlcN), the metabolic substrate of OGT, or OGA inhibitor Thiamet‐G (TMG). We found that mitochondrial respiration and ATP production rates are lower in these cells. Additionally, these cells produce significantly less reactive oxygen species (ROS). We found that mitochondria were longer and mitochondrial fusion/fission protein expressions were decreased. Next, we performed RNA‐Seq analysis and found that 21 mitochondrial‐encoded genes are affected by the treatment. We found the nuclear factor erythroid 2 (NRF2) pathway that controls cellular ROS response was down regulated in these O‐GlcNAc elevated cells. Next, we found that C57B/6J mice dosed with TMG display a similar phenotype as SY5Y cells. Mitochondria isolated from mice brain had lower respiration and ROS levels, and decreased NRF2 response compared to control. Altogether, our results point towards prolonged elevations of O‐GlcNAc modulating mitochondrial function by influencing mitochondrial transcriptional programs, and that O‐GlcNAc affects ROS production likely via adjusting ETC activity and skewing its substrate (NAD + /NADH) availability. Furthermore, prolonged elevations of O‐GlcNAc impacts mitochondrial ROS production influencing the NRF2 antioxidant response. Importantly, these findings demonstrate the critical role for O‐GlcNAc cycling in the regulation of metabolism and will provide new insights into metabolic diseases such as Alzheimer's. Support or Funding Information Biomedical Research Training Program Fellowship, Mabel A. Woodyard Fellowship, National Institute of Diabetes and Digestive and Kidney Diseases R01DK100595 to C. Slawson