Identification, characterization and acute response to high starch diet of sirtuin family genes in Micropterus salmoides

The tolerance threshold for Micropterus salmoides regarding starch is below 10%, and above 20% will result in persistent hyperglycemia and the accumulation of glucose and lipids in hepatocytes. Studies on mammals have revealed the regulatory role of SIRT family genes in glucose and lipid metabolism. However, the structure, protein characteristics of the SIRT genes in M. salmoides and their response mechanisms to acute carbohydrate stress remain unclear, which hinders the in-depth analysis of this gene family and its application in targeted intervention studies. This study aimed to systematically identify the sirtuin family members in M. salmoides, characterize their gene structure and protein properties, and examine their response to acute high starch stress. Candidate genes were obtained based on the reference genome (GCF_014851395.1) HMM/BLASTP screening and CDD verification, and their physicochemical properties and evolutionary relationship were analyzed by bioinformatics. Two experimental treatment groups were established. Following a 24-hour fast, the fish were administered feed with 8% and 20% starch levels, respectively. Serum glucose level, liver pyruvate, NAD⁺ and NADH contents, and relative expression level of sirtuin were measured from 0 to 24 h. Seven non-clustered sirtuin genes were found across seven scaffolds. The CDS length, the quantity of exons and introns, and the protein's isoelectric point and stability exhibited substantial differences. All of them included Motif4, NAD⁺, and Zn²⁺ binding sites. The phylogenetic tree of SIRTs proteins was divided into four branches, closely associated with the sequences of Siniperca chuatsi and Oreochromis niloticus. sirt1-sirt7 were all highly expressed in the brain, and the expression level in muscle were generally the lowest. 24 hours post-feeding, the serum glucose levels in the 20% starch group was significantly elevated compared to the 8% starch group from 2 to 12 hours, peaking at 6 hours. Simultaneously, the concentration of pyruvate rose, the concentration of NAD⁺ diminished, and the concentration of NADH markedly increased at 6 hours. In comparison to the 8% starch group, the 20% starch group exhibited up-regulation of sirt1 at 2 hours, persisting until 12 hours, while sirt3 and sirt4 showed sequential increases between 8 and 24 hours; while the other members showed no significant temporal changes. In summary, this study identified 7 non-clustered and structurally diverse sirtuin genes in the genome of the M. salmoides, characterized by distinct physicochemical properties and conserved NAD⁺/Zn²⁺ catalytic domains. Under short-term high-starch nutritional stress, the temporal expression of sirt1, sirt3 and sirt4 was coupled with the changes in NAD⁺ and NADH contents, suggesting that they may be involved in regulating glycolipid metabolism. Among them, sirt1 responded earliest and could be used as the preferred target for precise nutritional regulation of fish. This study provides a theoretical basis for the subsequent verification of the function of the sirtuin family genes and the screening of the targets of precise nutritional regulation in fish.