Ghrelin induces clock gene expression in the liver of goldfish in vitro via protein kinase C and protein kinase A pathways

The liver is the most important link between the circadian system and metabolism. As a food entrainable oscillator, the hepatic clock needs to be entrained by food-related signals. The objective of the present study was to investigate the possible role of ghrelin (an orexigenic peptide mainly synthesized in the gastrointestinal tract) as an endogenous synchronizer of the liver oscillator in teleosts. To achieve this aim, we first examined the presence of ghrelin receptors in the liver of goldfish. Then, the ghrelin regulation of clock gene expression in the goldfish liver was studied. Finally, the possible involvement of the PLC/PKC and AC/PKA intracellular signaling pathways was investigated. Ghrelin receptor transcripts, ghs-r1a, are present in the majority of the goldfish hepatic cells. Ghrelin induces the mRNA expression of the positive (g bmal1a, gclock1a ) and negative (g per genes ) elements of the main loop of the molecular clock machinery, as well as of grev-erbα (auxiliary loop) in cultured liver. These effects are blocked, at least in part, by a ghrelin antagonist. Incubation of liver with a phospholipase-C inhibitor (U73122), a protein-kinase-C activator (phorbol-12-myristate-13–acetate) and a protein-kinase-C inhibitor (chelerythrine-chloride) demonstrates that the PLC-PKC pathway mediates such ghrelin actions. Studies with an adenylate cyclase activator (forskolin) and a protein-kinase-A inhibitor (H89) show that grev-erbα regulation could be due to an activation of protein-kinase-A. Taken together, present results show for the first time in vertebrates a direct action of ghrelin on hepatic clock genes and support a role for this hormone as a temporal messenger in the entrainment of liver circadian functions.