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Abstract

Nutrient and energy metabolism in organisms oscillates in a time-of the-day-dependent manner under the control of an endogenous timing mechanism called the circadian clock. This is a cell autonomous, self-sustained molecular mechanism, which is synchronized by a key environmental signals, notably light and food availability. There is a wealth of evidence showing a bidirectional interaction between food-regulated clocks and the rhythmic expression of metabolic genes in peripheral tissues, notably the liver. For example, genetic or environmental disruption of the circadian clock is linked with metabolic disease, such as obesity and type 2 diabetes. Furthermore, cycling changes in cellular redox potential impact on the expression of circadian clock genes and influence energy metabolism. Therefore, it is vital to understand how animals integrate input from lighting conditions and food availability to ultimately coordinate their daily metabolic rhythms. In this regard, one key issue is whether there are genetically distinct light and food regulated circadian clock mechanisms. The Foulkes group has used zebrafish and blind cavefish models to demonstrate that certain metabolic pathways cycle according to the light dark cycle and are unaffected by the timing of feeding activity, while other pathways are predominantly feeding time regulated. Based on these preliminary data, this thesis project used fish models and fish-derived cell lines to explore the genetic mechanisms linking metabolism with light and food regulated circadian clocks.

The first part of this project aimed to explore at which stage during early zebrafish development a feeding-regulated clock first appears. Due to reduced feeding activity in constant darkness it was not feasible to examine the impact of feeding on clock gene expression. However, it was revealed that regular handling and disturbance of fish larvae, under otherwise constant environmental conditions results in the emergence of circadian clock rhythmicity. Several lines of evidence indicate that stress serves as a Zeitgeber and results in the emergence of rhythmicity in clock gene expression as well as clock outputs such as the cell cycle.

The second part of this thesis explored whether genetically distinct light and food regulated clocks coexist in fish cells and which transcriptional control mechanisms link food-regulated circadian clocks with metabolism. It was demonstrated that during restricted feeding in zebrafish, rhythmic expression of core clock genes in the liver is regulated according to the timing of light-dark cycles, whereas the expression of genes involved in the control of metabolism are influenced by feeding time. However, this study was unable to confirm previous data obtained using NMR, where it was shown that circadian rhythmicity in the levels of essential amino acids is regulated by the light-dark cycle while rhythmic non-essential amino acid levels are influenced by feeding time. Instead, by UPLC-MS/MS analysis, daily changes in the concentration of both essential and non-essential amino acids were shown to be set by the phase of regular timed feeding and not by the light dark cycle. Furthermore, the NAD+ biosynthesis pathway and autophagy were affected by a clock which is set by feeding time and not by light-dark cycles. In addition, regular nocturnal feeding resulted in an increase in obesity. These findings point to the presence of at least two distinct clock mechanism in the zebrafish liver.

In order to explore in more detail, the nature of the multiple clock mechanisms in zebrafish cells, the next part of this project employed multi-omics approaches and revealed infradian rhythmicity in amino acid concentrations in cultured fish cell lines. However, neither the expression of amino acid transporters nor autophagy exhibited infradian rhythmicity, instead showing circadian rhythmicity. In order to explore the involvement of the classical circadian clock mechanism in generating infradian rhythmicity, a cell line expressing a dominant negative form of clock1 gene was examined and shown to lack infradian rhythmicity in amino acid levels. Interestingly, the mRNA expression of Asparagine synthetase (asns) shows infradian rhythmicity, which are disrupted in Δclock1 cells. These data lead to the hypothesis that asns may be involved in the regulation of infradian rhythms in amino acid levels and point to a complex interplay between circadian and infradian rhythmicity.