Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, and is crucial for efficient glycolysis–oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyses reversible O-GlcNAcylation, a post-translational modification influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of the regulatory domain of HK1, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances the mitochondrial association with HK1, orchestrating glycolytic and mitochondrial ATP production. Mutation in HK1’s O-GlcNAcylation site reduces ATP generation in multiple cell types, specifically affecting metabolic efficiency in neurons. This study reveals a previously unappreciated pathway that links neuronal metabolism and mitochondrial function through OGT and the formation of the glycolytic metabolon, providing potential strategies for tackling metabolic and neurological disorders.

葡萄糖是主要的细胞能量来源，通过限速酶己糖激酶 (HK) 引发的糖酵解进行代谢。在大脑等能量需求组织中，HK1 是主要的异构体，主要位于线粒体上，对于有效的糖酵解-氧化磷酸化偶联和最佳能量生成至关重要。这项研究揭示了由代谢传感器酶 O-GlcNAc 转移酶 (OGT) 介导的调节 HK1 活性、糖酵解和线粒体偶联动力学的独特机制。 OGT 催化可逆的 O-GlcNAc 酰化，这是一种受葡萄糖通量影响的翻译后修饰。 OGT 活性升高会诱导 HK1 调节域的动态 O-GlcNAc 酰化，随后促进糖酵解代谢物在线粒体外膜上的组装。这种修饰增强了线粒体与 HK1 的关联，协调糖酵解和线粒体 ATP 的产生。 HK1 的 O-GlcNAc 酰化位点的突变会减少多种细胞类型中 ATP 的生成，特别是影响神经元的代谢效率。这项研究揭示了一条以前未被重视的途径，该途径通过 OGT 和糖酵解代谢物的形成将神经元代谢和线粒体功能联系起来，为解决代谢和神经系统疾病提供了潜在的策略。