DIA proteomics identifies time-of-day-dependent hippocampal proteomic signatures associated with exercise timing in type 2 diabetic mice

Type 2 diabetes mellitus (T2DM) is associated with neurological dysfunction, including alterations in emotional regulation, and exercise timing may influence physiological adaptation under metabolic stress. However, the molecular signatures associated with time-of-day-dependent exercise responses in the diabetic brain remain incompletely characterized. In this study, we investigated the effects of exercise timing on anxiety-like behaviors and hippocampal proteomic profiles in T2DM mice. Male C57BL/6J mice with T2DM were subjected to swimming exercise during either the early rest phase or early active phase (ZT15), followed by behavioral assessment and hippocampal proteomic profiling using data-independent acquisition (DIA) mass spectrometry. ZT15 exercise was associated with improved anxiety-like behavioral outcomes compared with diabetic controls. DIA-based proteomic analysis identified distinct hippocampal protein signatures associated with different exercise timings. Functional enrichment analyses identified timing-associated enrichment patterns involving transcriptional regulation, inflammation-related responses, and cytoskeletal-associated processes. These findings suggest that exercise performed at different times of day is accompanied by distinct hippocampal molecular responses under diabetic conditions. The identified proteomic signatures provide candidate molecular context and directions for future mechanistic studies of time-dependent exercise responses in metabolic disorders.