O-GlcNAc Regulation of Mitochondrial Function and Energy Metabolism

Abstract

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 transcriptome is reprogrammed and NRF2 anti-oxidant response is down-regulated. Importantly, sustained O-GlcNAcylation in mice brain and liver validated the metabolic phenotypes seen in cells, whereas liver OGT knockdown elevated ROS levels, impaired mitochondrial respiration, and increased NRF2 anti-oxidant response. Furthermore, we discovered from an indirect calorimetric study that sustained elevated O-GlcNAc promoted weight loss and lowered respiration, skewing mice toward using carbohydrates as their main energy source. Here, our results demonstrated that sustained elevation in O-GlcNAcylation, coupled with increased OGA expression, reprograms energy metabolism and can potentially impact the development of metabolic diseases. Altogether, these studies provide new evidence supporting the role of O-GlcNAc as a critical regulator of mitochondrial function and energy metabolism.