Characterization and mechanism of phosphoinositide 3-kinases (PI3Ks) members in insulin-induced changes of protein metabolism in yellow catfish Pelteobagrus fulvidraco.

In the present study, seven phosphoinositide 3-kinase (PI3K) members (PI3KCa, PI3KCb, PI3KCd, PI3KCg, PI3KC2a, PI3KC2b and PI3KC3, respectively) were isolated and characterized from yellow catfish Pelteobagrus fulvidraco, and their roles in insulin-induced changes of protein metabolism were determined. These seven PI3Ks can be divided into three classes, class I (including PI3KCa, PI3KCb, PI3KCd and PI3KCg), class II (including PI3KC2a and PI3KC2b) and class III (only including PI3KC3). Compared with mammals, all of these members share similar domain structure. Their mRNAs were widely expressed across ten tested tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, intestine, heart, kidney and ovary), but at variable levels. In the in vivo study, insulin treatment significantly increased hepatic protein content at 3h, accompanied with reduced plasma total amino acid contents and liver ALT activity, and with increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in liver. At 6h and 12h, insulin injection showed no significant effect on liver protein content and plasma total amino acid, but reduced liver ALT activity and increased liver total RNA and the mRNA levels of AKT2, mTORC1 and S6K1 in liver at 6h. In the in vitro study, insulin incubation also tended to increase protein content of hepatocytes, accompanied with reduced cell medium total amino acid contents and hepatocytes ALT activity, and increased total RNA content and the mRNA levels of PI3KCb, PI3KC2a, AKT2, mTORC1 and S6K1 in hepatocytes. However, insulin treatment showed no significant effect on GDH activity and mRNA expression of PI3KCa, PI3KCd, PI3KCg, PI3KC2b, PI3KC3 and eEF2 both in the in vivo and in vitro studies. Effects of insulin on the mRNA levels of eIF-4E and 4E-BP1 were different between the in vivo and in vitro studies, and also time-dependent. Compared to single insulin group, insulin+wortmannin group increased ALT activity at 6h but reduced T-RNA content at 6 and 12h. AKT2 and S6K1 mRNA levels at 6 and 12h, mRNA levels of mTORC1, 4E-BP1 and eEF2 at 3 and 6h, and EIF-4E mRNA levels at 3 and 12h, PI3KCb and PI3KC2a mRNA levels were significantly lower in insulin+wortmannin group than those in single insulin group. Thus, our study demonstrated that among seven PI3K members, PI3KCb and PI3KC2a were more sensitive to the insulin signaling pathway, and insulin stimulated hepatic protein synthesis in yellow catfish through PI3K signaling pathway.