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

Abstract

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 phospholipase C/protein kinase C (PLC/ PKC) and adenylate cyclase/protein kinase A (AC/PKA) intracellular signalling pathways was investigated. Ghrelin receptor transcripts, ghs-r1a, are present in the majority of goldfish hepatic cells. Ghrelin induced the mRNA expression of the positive (gbmal1a, gclock1a) and negative (gper genes) elements of the main loop of the molecular clock machinery, as well as grev-erbα (auxiliary loop) in cultured liver. These effects were blocked, at least in part, by a ghrelin antagonist. Incubation of liver with a PLC inhibitor (U73122), a PKC activator ( phorbol 12-myristate 13-acetate) and a PKC inhibitor (chelerythrine chloride) demonstrated that the PLC/PKC pathway mediates such ghrelin actions. Experiments with an AC activator (forskolin) and a PKA inhibitor (H89) showed that grev-erbα regulation could be due to activation of PKA. Taken together, the 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.

Characterization of Ghrelin O-Acyltransferase (GOAT) in goldfish (Carassius auratus)

“…The existence of a rhythmic expression of all the components of the ghrelinergic system (ghrelin, GOAT and its main receptor) indicates that this system may be acting as an output of the circadian system in goldfish. Besides, the modulation of clock genes expression by ghrelin in goldfish tissues both in vivo [ 12 ] and in vitro [ 14 ] points to the ghrelinergic system as an input signal for the circadian system in fish.…”

“…This includes a role in a wide variety of physiological processes of all vertebrates, including fish, such as stimulation of growth hormone release from the pituitary, stimulation of food intake, carbohydrate utilization and adiposity and regulation of insulin secretion, among others [ 7 – 11 ]. In the last decade, ghrelin has also been involved in the circadian system of both mammals and fish, where it might act as both an input [ 12 – 14 ] and an output signal [ 15 ]. Although acylated ghrelin (acyl-GRL) is typically considered as the active form of the hormone, studies have recently shown that deacylated ghrelin (desacyl-GRL) also has some biological functions in vertebrates [ 16 – 18 ].…”

Insulin Controls Clock Gene Expression in the Liver of Goldfish Probably via Pi3k/Akt Pathway

“…Just like other crucial functions, such as feeding regulation, temporal homeostasis in the liver is probably regulated by multiple redundant signals. Specifically in goldfish, the orexigenic peptides orexin [26] and ghrelin [27,48], whose levels increase during fasting [49], can modulate the food anticipatory activity (FAA), which is the primary output of the food-entrainable oscillator (FEO) [26,27]. Unlike orexin and ghrelin, insulin levels increase directly in response to feeding [28,29]; thus, a stronger direct impact as a feeding input could be expected.…”

“…Unlike orexin and ghrelin, insulin levels increase directly in response to feeding [28,29]; thus, a stronger direct impact as a feeding input could be expected. Furthermore, several hormones apart from insulin directly affect clock gene expression in the fish liver, including ghrelin and also glucocorticoids, which will be further discussed [48,50]. Even more feeding-related signals have been implicated in liver clock entrainment in mammals, such as IGF-1 and oxyntomodulin (a hormone released by gastric filling), via induction of per1 and per2 [38,51].…”

“…Liver cultures were performed as previously described [48,50]. In short, on the day of the experiments, non-fed goldfish were sacrificed by anesthetic overdose with MS-222 (tricaine methanesulfonate, 0.3 g/L, Sigma-Aldrich) at 10:00 h. Then, the liver was quickly sampled, homogenized using a scalpel, and distributed in different wells (≈20 mg liver/well) of sterile culture 24-well multi-dish plates.…”