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.

The authors thank the animals that contributed to this research. The authors also acknowledge the use of Perplexity AI for assistance with English translation and language editing of this manuscript. This study was funded by the First-Class Discipline Team of Kunming Medical University (No. 2024XKTDPY19).

These authors contributed equally to this work: Jiyou Wu, Haojie Hu, Shunling Yuan and Weichang Guo. Faculty of Health Sciences and Sports, Macao Polytechnic University, Macao, 999078, China School of Physical Education, Yunnan Normal University, Kunming, 650500, China Key Laboratory of Exercise and Health Sciences of Fujian Province Universities, School of Physical Education and Sport Sciences, Fujian Normal University, Fuzhou, 350117, China Department of Physical Education, Kunming Medical University, Kunming, 650500, China The authors declare that they have no competing interests. Clinical trial number: not applicable.

All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the Animal Experiment Welfare and Ethics Review Committee of Kunming Medical University (Approval No. KMMUX2025070051; approval date: 2 July 2025). Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.