1) The document examines how elevating O-GlcNAc levels through OGA inhibition or increasing OGT substrate availability affects mitochondrial function. 2) Experiments found that elevated O-GlcNAc levels decreased mitochondrial respiration and ATP production, while reducing reactive oxygen species levels. 3) Pathway analysis predicted that the NRF2-mediated oxidative stress response, a key regulator of antioxidants, was downregulated with elevated O-GlcNAc. Protein expression of antioxidant genes regulated by NRF2, such as TXNRD1, were also reduced. 4) Together, the results demonstrate that prolonged alterations to cellular O-GlcNAc homeostasis impact mitochondrial function and metabolism.