Identification, Expression And Regulation Of Amphioxus G6Pase Gene With An Emphasis On Origin Of Liver

The cephalochordate amphioxus, which occupies a nodal position from invertebrates to vertebrates, has recently been repositioned at the base of the chordate phylum. Amphioxus is becoming an emerging model organism for insights into the origin and evolution of vertebrates. Liver is an organ characteriscit it of vertebrates, which plays a major role in glucose metabolism in vertebrate. Many studies suggested the presence of liver-like (hepatic caecum) tissues in amphioxus. However, if the hepatic caecum plays a similar regulatory function as the vertebrate liver remains an open question.Vertebrate glucose-6-phosphatase (G6Pase) consists of three isozymes:G6Pase-I, G6Pase-II and G6Pase-III. It is the enzymes that hydrolyze glucose-6-phosphate, resulting in the creation of a phosphate group and free glucose. This catalysis completes the final step in gluconeogenesis and glycogenolysis and therefore plays a key role in the homeostatic regulation of blood glucose levels. Despite extensive study on G6Pases in vertebrates, information regarding expression and regulation of G6Pase genes is rather limited in invertebrates.Here we report the identification of G6Pase gene in amphioxus Branchiostoma japonicum. The cDNA we obtained was 2053 bp long, which contains an open reading frame (ORF) of 1095 bp codes for a deduced protein of 364 amino acids. Sequence comparison revealed that the predicted protein was 29.8% to 39.1% identical with G6Pase-Ⅰ, G6Pase-Ⅱ and G6Pase-Ⅲ isolated from the vertebrates and 20.4% to 46.7% identical with G6Pases from the invertebrates. Comparison of the 3D structures of amphioxus and zebrafish G6Pases revealed a striking similarity although they both shared only 33.6% to 38.8% identity. These suggest that amphioxus G6Pase may play a similar role as zebrafish G6Pases do. The phylogenetic and genomic structure analyses reveals that amphioxus G6Pase bears close resemblance to vertebrate G6Pase-III and represents the archetype of vertebrate G6Pase from which the vertebrate G6Pase isoforms may be originated by 2 rounds of genome duplication during vertebrate evolution.The Real-time PCR revealed that it was abundantly expressed in the hepatic caecum and ovary in a tissue-specific manner. We also demonstrate that rzGH treatment for 8 h resulted in increase in the expression of G6Pase genes in both zebrafish and amphioxus in a dose-dependent manner. We then tested the time course effect of treatment with rzGH in both species that the G6Pase mRNA production in the organ cultures with rzGH remained markedly higher than that in the controls. Overall, GH treatment induces a closely similar expression pattern and trend of g6pases in both zebrafish and amphioxus.It was shown by Western blotting that rabbit anti-human glucose-6-phosphatase antibody G6Pase-a reacted with the extracts of amphioxus hepatic caecum extract as well as mouse liver extracts, both forming a single positive band with an apparent molecular mass of approximately 40.0 kDa, corresponding to the molecular weight we predicted. This indicated the presence of a native G6Pase in B. japonicum. We then determined the localization of G6Pase by means of measuring the activities of the enzyme in the hepatic caecum, hind gut and gut-free body in B. japonicum. It revealed that G6Pase activity in the hepatic caecum was 1.6 times higher than that in the hind-gut and the activity in gut-free body is extremely low, indicating that G6Pase mainly localized in the hepatic caecum. Next, G6Pase activity in hepatic caecum was analyzed under feeding and fasting conditions. Compared with control, the activity of G6Pase activity was significantly increased up to 182% by 48 h fasting, while the activity of G6Pase was decreased to 75% by 24 h feeding. These show that the enzymatic activity of G6Pase in amphioxus hepatic caecum is enhanced under fasting condition, but inhibited under feeding condition and that G6Pase activity in amphioxus hepatic caecum is subjected to regulation of feeding and fasting as observed in vertebrates.In conclusion, this study highlights that amphioxus G6Pase is structurally and functionally similar to G6Pases in the vertebrates. In addition, a similar expression pattern and trend of G6Pase genes following rzGH treatment in both zebrafish and amphioxus suggest that hormonal regulation of G6Pase genes might have evolved in amphioxus. All these provide functional evidences supporting the notion that the hepatic caecum is the liver homologue playing a key role in maintaining the glucose homeostasis in amphioxus.