Time of feeding and the intrinsic circadian clock drive rhythms in hepatic gene expression

Abstract

In mammals, the circadian oscillator generates approximately 24-h rhythms in feeding behavior, even under constant environmental conditions. Livers of mice held under constant darkness exhibit circadian rhythm in abundance in up to 15% of expressed transcripts. Therefore, oscillations in hepatic transcripts could be driven by rhythmic food intake or sustained by the hepatic circadian oscillator, or a combination of both. To address this question, we used distinct feeding and fasting paradigms on wild-type (WT) and circadian clock-deficient mice. We monitored temporal patterns of feeding and hepatic transcription. Both food availability and the temporal pattern of feeding determined the repertoire, phase, and amplitude of the circadian transcriptome in WT liver. In the absence of feeding, only a small subset of transcripts continued to express circadian patterns. Conversely, temporally restricted feeding restored rhythmic transcription of hundreds of genes in oscillator-deficient mouse liver. Our findings show that both temporal pattern of food intake and the circadian clock drive rhythmic transcription, thereby highlighting temporal regulation of hepatic transcription as an emergent property of the circadian system.

Fig. 1.

Restricted feeding restores feeding rhythms in cry1^−/−;cry2^−/− mice. The daily variation of the RER in mice is shown as an indicator of food intake. (A–D) Average (+SEM, n = 8 for WT, n = 4 for cry1^−/−;cry2^−/− mice under tRF, and n = 3 under ad lib conditions). (C) In addition to the average (red), the RER of the single mice is shown (gray). x-axis, white and black bars indicate day and night, respectively; red bars indicate food availability. ZT0 coincides with the onset of light, while ZT12 coincides with the onset of darkness.

Fig. 2.

Daily rhythms in AKT and CREB phosphorylation are driven by temporal feeding pattern. Total and phosphorylated protein levels of CREB (A and C) and AKT (B and D) in livers of WT (A and B), and cry1^−/−;cry2^−/− mice (C and D) under ad lib and tRF feeding conditions. Food availability is indicated in red.

Fig. 3.

Circadian and ultradian transcription is driven by feeding state controlled transcription factors CREB, FoxO1, SREBP-1/2, and ATF6. Median temporal expression pattern of transcriptional targets of CREB (A), FoxO1 (B), the SREBPs (C), and ATF6 (D) in the liver of WT and cry1^−/−;cry2^−/−.

Fig. 4.

Rhythmic food intake drives rhythmic gene expression. Heatmap rendering (A and C) and median expression changes (B and D) of food-induced and food-repressed transcripts in the indicated genotypes and feeding conditions. (A and B) Columns indicate time points, and rows indicate single transcripts. High levels are shown in yellow, whereas low levels are shown in blue. Data for WT are at 1-h resolution, while data for cry1^−/−;cry2^−/− mice are at 2-h resolution. Median expression values were smoothed by a 3-h moving average.

Fig. 5.

Temporally restricted feeding can partially restore approximately 24-h rhythm in gene expression in the livers of cry1^−/−;cry2^−/− mice. Heatmap rendering of temporal gene expression pattern of 617 probe sets scored rhythmic in cry1^−/−;cry2^−/− mouse liver under tRF.

Fig. 6.

Feeding schedule affects rhythmic transcription. (A) Transcripts that were scored as rhythmic in WT mice under both ad lib and tRF or fasting condition are shown in a Venn-Diagram. (B) Heatmap rendering of temporal gene expression pattern of the 1,743 “common cycler” transcripts is shown. (C) Venn-Diagram depicting only 368 of 2,997 transcripts rhythmic in ad lib-fed WT mice maintained a robust transcriptional rhythm under fasting conditions. (D) Heatmap rendering of these 368 transcripts, including most core clock components and known clock-controlled genes under ad lib and fasting conditions.

Fig. 7.

Feeding cue is essential for rhythmic transcription of a subset of transcripts and is integrated into the circadian clock through Per1 and Per2. (A and C) Mice were fasted for 24 h and re-fed at CT4 or left under fasted condition. mRNA levels of Per1 and Per2 in the livers of fasted (black) or re-fed (red) mice were determined by RT-qPCR at 2-h resolution. (B and D) mRNA levels of Per1 and Per2 in the liver of cry1^−/−;cry2^−/− mice under tRF conditions were determined by RT-qPCR at 4-h resolution.