Objective: Based on the 4D label-free phosphoproteomic technique, we examined the differences in cognitive function and hippocampal phosphorylated protein expression in high-fat diet-induced obese mice after the intervention of semaglutide and empagliflozin, as well as the effects of both on protein activity and function in obese mice's hippocampal tissues and the signaling pathways involved. Methods: Thirty-two C57BL/6JC male mice were assigned to two groups randomly: A control group (group C, 10% of energy is from fat, n = 8) and a high-fat diet group (group H, 60% of energy is from fat, n = 24). The high-fat diet-induced obese mice were screened after 12 weeks of feeding based on the criterion that the bodyweight of mice in fat rich diet group was greater than or equal to 20% of the average body weight of the mice in the blank control group. Group H separate into group H (n = 8), group Semaglutide (group S, n = 8), and group empagliflozin (group E, n = 8). For a total of 12 weeks, group S received 30 nmol/kg/d bodyweight of semaglutide intraperitoneally, group E received 10 mg/kg/d bodyweight of empagliflozin via gavage, and groups C and H received equal amounts of saline by intraperitoneal injection and gavage. At the end of treatment, the mice were appraised for cognitive function employing the Morris water maze (MWM), and serum fasting glucose, lipids, and inflammatory parameters were measured. The 4D label-free phosphoproteomics method was employed to screen the differential phosphoproteins and loci in hippocampal tissues of mice in different treatment groups, and bioinformatics was used to analyze the biological processes, signaling pathways, and related protein-protein interaction (PPI) network analysis of these differentially phosphorylated proteins. Results: In comparison to normal controls, The escape latency of obese mice induced by high-fat diet was prolonged, the percentage of swimming time in the target quadrant was reduced, and the number of times of crossing the platform was reduced, whereas semaglutide and empagliflozin treatment reduced escape latency, increase the percentage of swim time in the target quadrant and increase the frequency of passing through the platform area, although there is little difference in the effect of the two drugs. The phosphoproteomic results showed 20,493 unique phosphorylated peptides, representing 21,239 phosphorylation sites and 4,290 phosphorylated proteins. Further analysis revealed that the proteins corresponding to these differentially phosphorylated sites are jointly distributed in signaling pathways such as dopaminergic synapses and axon guidance, and are involved in biological processes such as neuronal projection development, synaptic plasticity, and axonogenesis. Notably, the key factors voltage-dependent L-type calcium channel subunit alpha-1D (CACNA1D), voltage-dependent P/Q-type calcium channel subunit alpha-1A (CACNA1A), and voltage-dependent N-type calcium channel subunit alpha-1B (CACNA1B) were all found to be involved in the dopaminergic synapse pathway, and their expression was upregulated by semaglutide and empagliflozin. Conclusion: We found for the first time that a high-fat diet decreased CACNA1D, CACNA1A, and CACNA1B protein serine phosphorylation, which may affect neuronal development, synaptic plasticity, and cognitive function in mice. Notably, semaglutide and empagliflozin increased the phosphorylation of these proteins.

#	Concept	H-Index	Publications	Citations
1	Medicine	1	1	32
2	Cell Biology	1	1	32
3	Pathology	1	1	32
4	Cardiac Reprogramming	1	1	32
5	Cell Signaling	1	1	32

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