Functional Analysis of Promoters from Three Subtypes of the PI3K Family and Their Roles in the Regulation of Lipid Metabolism by Insulin in Yellow Catfish Pelteobagrus fulvidraco.

In the present study, the length of 360, 1848 and 367 bp sequences of promoters from three subtypes of PI3K family (PI3KCa, PI3KC2b and PI3KC3) of yellow catfish were cloned and characterized. Bioinformatics analysis revealed that , and had different structures in their core promoter regions. The promoter regions of and had CpG islands but no CAAT and TATA box. In contrast, the promoter of had the canonical TATA and CAAT box but no CpG island. The binding sites of several transcription factors, such as HNF1, STAT and NF-κB, were predicted on promoter. The binding sites of transcription factors, such as FOXO1, PPAR-RXR, STAT, IK1, HNF6 and HNF3, were predicted on promoter and the binding sites of FOXO1 and STAT transcription factors were predicted on promoter. Deletion analysis indicated that these transcriptional factors were the potential regulators to mediate the activities of their promoters. Subsequent mutation analysis and electrophoretic mobility-shift assay (EMSA) demonstrated that HNF1 and IK1 directly bound with and promoters and negatively regulated the activities of and promoters, respectively. Conversely, FOXO1 directly bound with the and promoters and positively regulated their promoter activities. In addition, AS1842856 (AS, a potential FOXO1 inhibitor) incubation significantly reduced the relative luciferase activities of several plasmids of and but did not significantly influence the relative luciferase activities of the plasmids. Moreover, by using primary hepatocytes from yellow catfish, AS incubation significantly down-regulated the mRNA levels of , and and reduced triacylglyceride (TG) accumulation and insulin-induced TG accumulation, as well as the activities and the mRNA levels of several genes involved in lipid metabolism. Thus, the present study offers new insights into the mechanisms for transcriptional regulation of PI3Ks and for PI3Ks-mediated regulation of lipid metabolism by insulin in fish.