MELATONIN-SYNTHESIZING ENZYMES IN PINEAL, RETINA, LIVER, AND GUT OF THE GOLDFISH (CARASSIUS): mRNA EXPRESSION PATTERN AND REGULATION OF DAILY RHYTHMS BY LIGHTING CONDITIONS

Abstract

(MAX 350 WORDS)Melatonin is currently proposed to be synthesized in non-photosensitive organs of vertebrates, besides its well-known sites of synthesis, the pineal gland and the retina.However, very few studies have demonstrated gene expression of MEL synthesizing enzymes in extrapineal and extraretinal locations. In the present study, present study focuses on the circadian expression of the two key enzymes of the melatoninergic pathway, the AANAT and the HIOMT in central and peripheral locations of the goldfish we give report of the full-length cloning of the two enzymes catalyzing the final steps of melatonin biosynthesis, the arylalkylamine N-acetyltransferase (AANAT) and the hydroxyindole-Omethyltransferase (HIOMT), in a teleosts fish, the goldfish (Carassius auratus). Both enzymes showed high similarity with other teleost sequences, corresponding to the goldfish AANAT-2 and HIOMT-2. Two forms of AANATs were widely known to exist in teleosts, but this is the first time that two isoforms of HIOMT are deduced from a phylogenetic analysis.Both enzymes were detected in several peripheral locations, including liver and gut, being present results the first to find HIOMT in non-photosensitive structures of a fish species. No studies exist on transcriptional regulation of the expression of MEL biosynthesis enzymes in non photosensitive structures in fish The daily expression pattern of both genes in pineal gland, retina, liver and gut was investigated using quantitative real time RT-PCR and cosinor analysis. This is the first time that a rhythmic expression of AANAT and HIOMT are found in digestive tissues of a vertebrate species, supporting a functional melatonin synthesis pathway in liver and gut of the goldfish. Besides, the transcriptional regulation of Aanat-2 in pineal and peripheral locations of goldfish maintained under different lighting conditions was investigated expression of gAANAT-2 is analyzed under different lighting conditions including continuous light and darkness, revealing, as expected, light-dependent rhythms in pineal gland and retina, but also in liver. Nevertheless, the persistence in hindgut of these Aanat-2 rhythms in constant conditions suggests that the expression of this transcript is under circadian clock and entrained by non-photic cues. Our results reinforce the existence of melatonin synthesis in gut and liver of the goldfish, while the rhythmic expression profiles reported point to a regulation of both genes in gut and liver by peripheral oscillators entrainable to non-photic cues.

Interplay between the endocrine and circadian systems in fishes

“…Similarly, gut melatonin in goldfish also exhibits this dependence on scheduled feeding (Vera et al 2007). The close correlation between the acrophases of biosynthetic enzymes (aanat2 and hiomt2; Velarde et al 2010) and clock genes (per1a, per2a and cry3; Nisembaum et al 2012) in the goldfish hindgut supports the idea that gut melatonin rhythmicity is coupled to a gut molecular clock that may work independently of the pineal clock.…”

Light-dark cycle and feeding time differentially entrains the gut molecular clock of the goldfish (Carassius auratus).

“…Moreover, in zebrafish, Per3 rhythms damp down later in peripheral oscillators than retina (Kaneko et al, 2006). Thus, this tight feeding-time dependence of gPer3 expression could be involved in the greater sensitivity of peripheral oscillators to feeding cues reported in fish (Feliciano et al, 2011;Velarde et al, 2009Velarde et al, , 2010. Altogether, the available data on Per3 in fish suggest to us that the activation of the expression of this gene may be the input that entrains the FEO.…”

“…The gPer1a rhythm in the 24L group of goldfish could be due to the existence of a non-photic signal, probably food. Randomly supplied food has been previously proposed to drive Per1a in the goldfish liver (Feliciano et al, 2011), and also the rhythmic expression of the enzyme Aanat2 in the pineal, liver, and hindgut of this species (Velarde et al, 2010).…”

Molecular Evolution of Multiple Arylalkylamine N-Acetyltransferase (AANAT) in Fish

“…Consequently, current AANAT expression in the vertebrate retina may reflect its ancestral-conserved function as a detoxification enzyme. The expression of AANAT in extra-pineal and extra-retinal sites including the brain [37–40], raises the hypothesis that this enzyme is involved in detoxification functions in these tissues. Of special interest is the idea that AANAT acetylates dopamine in the brain.…”