The Influence Of Time-restricted Feeding On The Circadian Clock Of Mouse Skin

The mammalian circadian clock is a hierarchical system with the central clock located in the suprachiasmatic nucleus and peripheral clocks ticking in almost all cells.The central clock is entrained by the day/night cycles and coordinates the peripheral clocks.It is believed that the circadian clock coordinates both physiological and cellular processes.Disruption of circadian clock accelerates aging and increases the risk of cancer,obesity and diabetes.The peripheral clocks,mainly studied in liver,could also be entrained by time-restricted feeding.We suppose that the clocks of different organs,even the circadian processes in the same tissue,may be entrained unequally and thus became desynchronized when feeding at abnormal time,which may reduce the fitness of the biological system.Here,C57BL/6J mice were grouped and fed respectively at 5 time windows:ZTO-24(the control,not restricted but mainly eating at night),ZT3-11,ZT5-9,ZTO-4 and ZT12-16.ZTO/24 and ZT12 correspond to lights-on and lights-off respectively.The circadian clock,transcriptome,circadian cell proliferation and sensitivity to UV of the telogen skin after time-restricted feeding was studied.Gene expression analysis by qPCR shows that the expression of Per2 in liver always peaks within 4 hours after the beginning of feeding.The peak time of Per2 in skin is shifted 4?8 hours forward in ZT5-9 feeding and unexpectedly 4-8 hours backward in ZTO-4 feeding.There is no substantial shift in ZT3-11 feeding and ZT12-16 feeding.The entrainment of Dbp is similar to Per2 in skin.We also found that the amplitude of Per2 and Dbp expression in skin is significantly lower in the day-time feeding groups.These results suggest that,besides the time-restricted feeding,there is another comparable time cue entraining the skin clock.When feeding at abnormal time,these time cues interfere with each other and the amplitude of the clock is dampened.Strand-specific total RNA sequencing was applied on ZT12-16,ZTO-4 and ZT5-9 feeding groups.In 22583 UCSC known canonical genes with reads mapped to exons,there is 32.41%.5.65%and 0.65%genes are circadian in at least one,two and three feeding groups respectively.These values are 20623,31.12%,5.11%,0.57%for introns and 22643,26.75%,3.15%,0.14%for antisense regions.These results indicates that only a small subset of circadian transcripts are dominantly regulated by the skin clock.The enriched Gene Ontology Biological Process terms of these three groups are similar and mainly related to cell cycle and metabolism,which suggests that the rhythmicity of some transcripts are regulated by the rhythmic processes or the signals that regulate the rhythmic processes.ANOVA was performed on the first three time points from the beginning of feeding.In exons,introns and antisense regions,there are 1028,913 and 254 genes up-regulated respectively and 998,977 and 408 genes down-regulated respectively.The up-regulated genes are enriched in lipid synthesis and translation-related processes,while the down-regulated genes are enriched in fatty acid oxidation and response to reactive oxygen species.These results suggest that the metabolism is tightly controlled by the feeding and may shift as much as the shift of feeding time.After feeding,the metabolism shifts from oxidative phase to reductive phase.BrdU labeling shows that the rhythm of cell proliferation is not obviously shifted.The basal epidermal cells go through S-phase mainly during the middle night under all the five feeding regimes.Unequal shift of cell cycle and metabolism may cause conflicts between DNA synthesis and oxidative metabolism,which may reduce the fitness of skin.Some mice undergone either ZT12-16 or ZT5-9 feeding were exposed to 500J/m2 UV at ZT9 and ZT21.Detection of photoproducts,CPDs and 6-4PPs in the skin DNA with ELISA shows that,at ZT9,the mice undergone ZT5-9 feeding get significantly higher DNA damage than the mice undergone ZT12-16 feeding.DNA damage at ZT9 is significantly lower than that at ZT21 in ZT12-16 feeding,while the difference becomes insignificant in ZT5-9 feeding.These results suggest that the fitness of skin is affected when the mice are fed at abnormal time.Therefore,the skin clock is entrained by at least two comparable cues,one is from feeding and the other is a combination of other system signals.These two cues,together with the cue from the skin clock,regulate the transcriptome and cellular processes.Each cue regulates different genes and cellular processes at unequal strength.Thus,when fed at different time,the combination of these cues vary from gene to gene and from process to process,and consequently the circadian genes and the circadian pattern of biological processes vary hugely.Under abnormal-time feeding,the coordination of different biological processes are disrupted,and the fitness of the skin is afffected.This study sheds light on the regulation of the circadian clock and the circadian rhythm of transcriptome and cellular processes in vivo.