SAT049 Goldfish LEAP2: Molecular Cloning, Functional Characterization, And Novel Regulation By IGF And Insulin At The Hepatic Level

Abstract Disclosure: F. Jin: None. X. Qin: None. C. Ye: None. M. He: None. A.O. Wong: None. Liver-expressed antimicrobial peptide 2 (LEAP2) is a peptide highly expressed in the liver and its secretion induced by bacterial infection constitutes an integral component of innate immunity in vertebrate species. Recently, LEAP2 has been confirmed to be the endogenous antagonist of the feeding regulator ghrelin, implying that the peptide may play a role in appetite control and energy homeostasis. However, whether LEAP2 can be regulated by metabolic/endocrine signals is still unclear and the signal transduction involved is totally unknown. Using goldfish as a model, three isoforms of LEAP2, namely LEAP2a, 2b & 2c, have been cloned and phylogenetic analysis confirmed that they could be clustered into the clade of fish LEAP2. Comparative synteny also revealed that the three isoforms were derived from separated genes with similar structural organization but located in different chromosomes of goldfish genome. The a.a. sequences and 3D protein structure of LEAP2a-c were found to be highly comparable with LEAP2 of other vertebrates and tissue expression profiling by RT-PCR also showed that they were expressed at high levels in the liver but to a lower extent in other tissues. As members of antimicrobial peptides, goldfish LEAP2a-c not only could exhibit antimicrobial activity against Staphylococcus epidermidis but also play important role in fish innate immunity, which is evidenced by their transcript expression induced by LPS & cytokines at hepatic level as well as in immune tissues in goldfish naturally infected with Aeromonas hydrophila. In goldfish liver cells, transcript expression of LEAP2a-c could be up-regulated in a time- and dose-dependent manner by IGF-I, IGF-II and insulin. Using a pharmacological approach, the stimulatory effects of IGF-I/-II and insulin on the three isoforms of LEAP2 were confirmed to be mediated by IGF-I receptor (IGF1R) but not insulin receptor activation. Furthermore, the PI3K/Akt, MEK/ERK and P38 MAPK pathways were shown to be involved in IGF-I induced LEAP2a-c gene expression. Our findings for the first time demonstrate that the endocrine signals targeting the liver (insulin) or produced locally within the liver (IGF-I & -II) can play a role in regulating LEAP2 expression at the hepatic level, which may shed light on a novel aspect of the functional crosstalk between innate immunity and feeding control/energy balance in fish model. Presentation: Saturday, June 17, 2023