V ̇ O 2 max $\dot V_{\rm{O_{2max}}}$ -based all-out aerobic exercise attenuates hexosamine biosynthetic pathway...

Aerobic exercise enhances physiological performance by activating metabolic systems, including glucose oxidation and fat metabolism. Cellular glucose predominantly flows through glycolysis (95-98%), whereas a smaller fraction (2-5%) enters the hexosamine biosynthetic pathway (HBP), generating UDP-N-acetylglucosamine. Excessive carbohydrate intake and inactivity expedite glucose redirection toward the HBP via mass-action mechanisms, which impair glucose uptake and insulin signalling. Although exercise intensity is a key determinant of fuel selection, with lipid oxidation predominating at moderate intensities (25-40% ${\dot{V}}_{O_{2\max }}$ ) and glucose utilization becoming the primary energy source during vigorous activity (75-85% ${\dot{V}}_{O_{2\max }}$ ), the mechanisms by which exercise intensity regulates HBP activity remain poorly understood. To investigate this, 12 male C57BL/6 mice were randomly assigned to control (CG, n = 6) and aerobic exercise groups (AEG, n = 6), with AEG undergoing 8 weeks of ${\dot{V}}_{O_{2\max }}$ -based all-out aerobic exercise (∼65.72% ${\dot{V}}_{O_{2\max }}$ ). Untargeted plasma metabolomics was performed using ultra-high performance liquid chromatography-time-of-flight mass spectrometry and processed using apLCMS, xMSanalyzer, xMSannotator, MetaboAnalyst 6.0 and the KEGG database. A two-component partial least squares discriminant analysis model achieved 100% classification accuracy with strong explanatory and predictive performance (R² = 0.981, Q² = 0.673). Eight N-acetyl compounds were significantly decreased in AEG compared with CG (P = 0.0139; log₂ fold change = -1.17), corresponding to an approximate 0.445-fold decrease in AEG relative to CG, with amino sugar and nucleotide sugar metabolism being the sole significantly affected pathway (P = 0.0489). Four HBP intermediates (N-acetyl-d-glucosamine 6-phosphate, N-acetyl-α-d-glucosamine 1-phosphate, N-acetyl-d-mannosamine 6-phosphate, N-acetyl-α-d-galactosamine 1-phosphate) showed discriminatory performance (AUC = 0.833). All-out aerobic exercise suppresses HBP activity by lowering its key metabolic intermediates, suggesting a clear shift in glucose flux toward glycolysis. This metabolic redirection likely serves as a protective mechanism against the development of insulin resistance and diabetes.