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Abstract

Abstract The zebrafish (Danio rerio) has proven to be a useful genetic model for studying the circadian timing system. The canonical molecular mechanism underlying circadian rhythms is based upon auto-regulatory transcription-translation feedback loops (TTFLs). It is well established that zebrafish has a TTFLs-dependent light entrainable oscillator (LEO) present in most tissues and even cell lines however, like other vertebrates, they also possess a food entrainable oscillator (FEO). Recent studies have addressed how regular food availability can entrain circadian rhythms of increased locomotor activity that precedes the feeding time (so-called food-anticipatory activity, FAA). Unlike the extensively studied LEO, there is very little known about the molecular components and regulation of the FEO. My PhD programme focuses on the mechanism whereby food regulates the circadian clock in zebrafish. Using behavior recording, we have revealed that FEO exists in adult zebrafish by showing that scheduled, restricted food availability can entrain FAA independently of the timing of the light-dark (LD) cycle. By maintaining fish under a LD cycle and then feeding fish either in the middle of the light period (ML) or in the middle of the dark period (MD), we have performed a circadian metabolome analysis using hydrogen nuclear magnetic resonance (1H NMR) spectroscopy. We have revealed that most circadian metabolites peak in either the FAA or FAA anti-phase periods and thus there is a strong correlation between metabolite phases and mealtime. Furthermore, by comparing the circadian phases of metabolites between ML and MD, we have identified both light and feeding time-regulated rhythmic metabolites. Light-related circadian metabolites include nucleotides and non-essential amino acids while feeding regulated circadian metabolites include essential amino acids. The oxidized form of nicotinamide adenine dinucleotide (NAD+), which operates as a cofactor and occupies a central position connecting most circadian metabolic pathways, is under the control of both LEO and FEO in adult liver. The rhythmic expression of nampta, namptb and sirt1 which encode enzymes in NAD+ salvage pathways are also regulated by both LEO and FEO. Notably, the rhythmic transcripts which constitute elements of TTFLs are not influenced by the feeding time in the fish liver.

In order to explore the cellular metabolism under cell autonomous LEO control, we have performed metabolome measurements in cultured zebrafish primary hepatocytes which were exposed to light-dark (LD) and dark-light (DL) regimes. Unexpectedly, the cycling of nearly all the identified circadian metabolites in primary hepatocyte cultures is independent of the lighting conditions. In comparison, the mRNA expression of metabolism-related genes which oscillate strongly in adult livers are arhythmically expressed in hepatocytes. LEO-independent metabolic oscillations with a 24h period were also observed in zebrafish fibroblast cells (PAC2 and AB9), indicating that a metabolic oscillator could exist widely in zebrafish cells. Our subsequently studies have demonstrated that the phase of the metabolic oscillator is insensitive to serum refresh treatment. Finally, we have revealed that circadian metabolites in cavefish fibroblasts show aberrant rhythmicity with an infradian period of 40h-45h, which matches the extremely long period (~47h) of core circadian clock gene expression.

In conclusion, the zebrafish endogenous timekeeping system can be synchronized by diverse external signals and is under a multi-oscillatory control. The FEO is genetically and functionally independent of the classical core circadian clock TTFL of the LEO in zebrafish liver. Cellular metabolic oscillations could be driven by a non-transcriptional mechanism in both zebrafish and cavefish cell lines. Thus, the study of non-photic oscillators and non-transcriptional dependent circadian periodicity should provide us with a more accurate view of the entire circadian timing system.