In this study, the mechanism by which GSK-3β regulates protein synthesis and lipid deposition was investigated in zebrafish (Danio rerio). The vector of pEGFP-N1-GSK-3β was constructed and injected into the muscle of zebrafish. It was found that the mRNA and protein expression of tuberous sclerosis complex 2 (TSC2) was significantly increased. However, the mRNA and protein expression of mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase 1 (S6K1), and 4E-binding protein 1 (4EBP1) was significantly decreased by the pEGFP-N1-GSK-3β vector in the muscle of zebrafish. In addition, the mRNA and protein expression of β-catenin, CCAAT/enhancer binding protein α (C/EBPα), and peroxisome proliferators-activated receptor γ (PPARγ) was significantly decreased, but the mRNA expression of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), ATP-citrate lyase (ACL), and HMG-CoA reductase (HMGCR) was significantly increased by the pEGFP-N1-GSK-3β vector. The activity of FAS, ACC, ACL, and HMGCR as well as the content of triglyceride (TG), total cholesterol (TC), and nonesterified fatty acids (NEFA) were significantly increased by the pEGFP-N1-GSK-3β vector in the muscle of zebrafish. The content of free amino acids Arg, Lys, His, Phe, Leu, Ile, Val, and Thr was significantly decreased by the pEGFP-N1-GSK-3β vector. The results indicate that GSK-3β may participate in regulating protein synthesis via TSC2/mTOR signaling and regulating lipid deposition via β-catenin in the muscle of zebrafish (Danio rerio).

ADCY9, a crucial member of the adenylate cyclase family, exerts neuroprotective and analgesic effects in the nervous system by modulating the activity of the cAMP/AMPK signaling pathway. However, the role of the ADCY9 gene in neural regeneration remains unreported. In this study, we utilized Dugesia japonica, a highly regenerative planarian species, as a model to systematically examine the spatiotemporal expression pattern of the ADCY9 gene during planarian brain regeneration and investigate its regulatory function in this process. The results demonstrated that the downregulation of ADCY9 resulted in abnormal brain regeneration in planarians, characterized by partial loss of the nerve cord, reduced numbers of collateral branches, and significant inhibition of the regeneration and differentiation of multiple neuron types. RNA sequencing revealed that the downregulation of ADCY9 led to 499 differentially expressed genes, with KEGG enrichment pathway analysis indicating significant associations with neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Double RNAi experiments demonstrated that simultaneous knockdown of ADCY9 and Mitofusin-1 significantly restored neural regeneration. Collectively, ADCY9 might promote the comprehensive reconstruction of neural structure by hierarchically regulating the regeneration intensity through negative regulation of the downstream inhibitory factor Mitofusin-1. This study discloses the function of ADCY9 in planarian neural regeneration, providing a theoretical foundation for its application in investigating neural regeneration mechanisms and neurodegenerative disease pathogenesis in higher vertebrates.