The fruit fly Drosophila is a prime model in circadian research, but still little is known about its circadian regulation of metabolism. Daily rhythmicity in levels of several metabolites has been found, but knowledge about hydrophobic metabolites is limited. We here compared metabolite levels including lipids between period 01 (per 01) clock mutants and CantonS wildtype (WT CS) flies in an isogenic and non-isogenic background using LC-MS. In the non-isogenic background, metabolites with differing levels comprised essential amino acids, kynurenines, pterinates, glycero(phospho)lipids, and fatty acid esters. Notably, detectable diacylglycerols (DAG) and acylcarnitines (AC), involved in lipid metabolism, showed lower levels in per 01 mutants. Most of these differences disappeared in the isogenic background, yet the level differences for AC as well as DAG were consistent for fly bodies. AC levels were dependent on the time of day in WT CS in phase with food consumption under LD conditions, while DAGs showed weak daily oscillations. Two short-chain ACs continued to cycle even in constant darkness. per 01 mutants in LD showed no or very weak diel AC oscillations out of phase with feeding activity. The low levels of DAGs and ACs in per 01 did not correlate with lower total food consumption, body mass or weight. Clock mutant flies showed higher sensitivity to starvation independent of their background-dependent activity level. Our results suggest that neither feeding, energy storage nor mobilisation is significantly affected in per 01 mutants, but point towards impaired mitochondrial activity, supported by upregulation of the mitochondrial stress marker 4EBP in the clock mutants.

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Modern lifestyle often is at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a diminished or altered cycling of metabolites in the circulating tissue reflecting the current metabolic status.Drosophilais a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analysed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wildtype flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired inperiod01clock mutants. In wildtype flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of DGs, PEs and PCs peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. Rearing of wildtype flies on lipid-containing standard medium masked the rhythmic alterations of hemolymph lipid levels. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs and PCs in the hemolymph to the anabolic siesta phase, whith a strong influence of light on phase and modality. This finding opens the question whether and to what extent the circadian regulation of transport lipid levels in the hemolymph contributes to the health of the fly.