Aquaculture Reports (Apr 2022)
Molecular cloning of AKT1 and AKT2 and their divergent responses to insulin and glucose at transcriptional level in the liver of Japanese flounder Paralichthys olivaceus
Abstract
The serine-threonine kinase AKT (protein kinase B) plays a vital role in insulin-mediated signaling transduction in mammals, but its role in carnivorous fish is still unclear. In the present study, AKT1 and AKT2 were cloned from Japanese flounder (Paralichthys olivaceus), and the full-length sequences of cDNA of AKT1 and AKT2 were 2335 bp and 3340 bp, encoding 473 and 482 amino acids, respectively. Tissue distribution results showed that AKT1 was most abundantly expressed in gill, intermediate in liver, spleen and brain. AKT2 was most highly expressed in liver, followed by gill, brain and eye. Compared with those intraperitoneally injected with phosphate buffer solution (PBS), after injected with 4 IU/kg of bovine insulin or 0.6 g/kg of glucose, the insulin-mediated insulin receptor (IR) / insulin receptor substrate (IRS) / phosphoinositide 3-kinase (PI3K) /AKT pathway and its downstream glucose metabolism pathways in the liver of Japanese flounder were significantly activated. AKT2 expression was significantly increased after insulin or glucose injection, but AKT1 not. Similar results were also found in the primary hepatocytes of Japanese flounder after 24-hour incubation with different doses of bovine insulin (50 nM, 100 nM and 200 nM) or PBS (Control). Knockdown of AKT2 in the hepatocytes of Japanese flounder inhibited glycolysis, glucose transport and glycogen synthesis. Meanwhile, knockdown of AKT2 promoted the phosphoenolpyruvate carboxykinase (PEPCK) expression and inhibited the glucose-6-phosphatase (G6P) expression in the gluconeogenesis pathway. It was confirmed in the present study that AKT2 was more sensitive to the insulin-mediated pathway than AKT1 in the liver of Japanese flounder after insulin or glucose administration. In addition, the glucose metabolism pathways could be mediated by AKT2 in the liver of Japanese flounder.
