Coordination of the transcriptome and metabolome by the circadian clock

Abstract

The circadian clock governs a large array of physiological functions through the transcriptional control of a significant fraction of the genome. Disruption of the clock leads to metabolic disorders, including obesity and diabetes. As food is a potent zeitgeber (ZT) for peripheral clocks, metabolites are implicated as cellular transducers of circadian time for tissues such as the liver. From a comprehensive dataset of over 500 metabolites identified by mass spectrometry, we reveal the coordinate clock-controlled oscillation of many metabolites, including those within the amino acid and carbohydrate metabolic pathways as well as the lipid, nucleotide, and xenobiotic metabolic pathways. Using computational modeling, we present evidence of synergistic nodes between the circadian transcriptome and specific metabolic pathways. Validation of these nodes reveals that diverse metabolic pathways, including the uracil salvage pathway, oscillate in a circadian fashion and in a CLOCK-dependent manner. This integrated map illustrates the coherence within the circadian metabolome, transcriptome, and proteome and how these are connected through specific nodes that operate in concert to achieve metabolic homeostasis.

Fig. 1.

Metabolic pathways of the liver containing diurnally regulated metabolites. Major metabolic pathways are represented in the liver by numerous metabolites that change in abundance throughout the 24-h cycle (orange lines, Clock^−/− metabolites; blue lines, WT metabolites). Metabolites that vary in abundance over time are shown. Pie charts depict the percentage of metabolites that changed over time (red) vs. those that did not (green). (N = 5 per genotype per time point.)

Fig. 2.

Metabolic pathway metabolites show disparate temporal peaks. Nucleotide and carbohydrate metabolites generally peaked at ZT9, whereas amino acid and xenobiotic metabolism-related biochemicals peaked at night (ZT15–ZT21).

Fig. 3.

Rhythmicity in metabolic subpathways. Metabolic pathway representation by subpathways and the percent of metabolites altered within each relative to genotype or time (only subpathways with ≥3 metabolites are graphed). The observed peak time (ZT hour) of metabolites within specific subpathways is shown on the right. ZT hour is denoted if all or the majority of metabolites identified within the category peak at one ZT. “Mult.” stands for “multiple” and refers to metabolite peaks within the subpathway that were distributed across multiple zeitgeber times. Dashes indicate that no metabolite within the subfamily showed altered abundance over time. Such subpathways include metabolites that were only altered in the Clock^−/− livers, and were not changed over time or oscillatory in expression patterns (where % greater than 0 is indicated) (*, glycolysis; **, gluconeogenesis).

Fig. 4.

Energy intake in clock knockout mice precedes the dark cycle. (A) Energy intake in 8-wk-old Clock-deficient (−/−) mice and WT littermate controls (+/+) (error ± SEM, *P < 0.05, Bonferroni posttest compared with WT). (B) Body weight in grams (g) of WT (+/+) and Clock-deficient (−/−) animals (error ± SEM, *P < 0.05, Mann-Whitney test compared with WT ZT12–24). (C) Average energy intake patterns in WT (+/+) and Clock-deficient (−/−) animals. (D and E) Respiratory exchange ratios (VCO₂/VO₂) of WT (+/+) and Clock-deficient (−/−) animals across a 24-h period (***P < 0.001, genotype; ***P < 0.001, time; two-way ANOVA) as well as during the hours of ZT8–12 and ZT12–24 (error ± SEM, ***P < 0.001 compared with WT). (F) Oxygen consumption in WT and Clock-deficient (−/−) animals during the resting (ZT0–ZT12) and active (ZT12–ZT24) periods. N = 7–8 animals per genotype (error ± SEM, ***P < 0.001, unpaired t test compared with WT).

Fig. 5.

Cohesiveness between computational networks and in vivo uracil metabolism. (A) The uracil network predicts an interaction with uridine phosphorylase 2 (UPP2). (B) UPP2 participates in the reversible reaction whereby uridine and orthophosphate are converted to uracil and ribose 1-phosphate. (C) Uridine and uracil oscillate in an antiphase pattern in WT livers but are nonoscillatory in Clock^−/− livers. (D and E) Semiquantitative PCR of Upp2 and quantification of Upp2 mRNA in WT (+/+) and Clock^−/− (−/−) livers (error ± SEM, *P < 0.05, genotype main effect; ***P < 0.001, time main effect; two-way ANOVA; *, P < 0.05 Bonferroni posttest). (F) UPP2 and Actin protein expression in WT and Clock^−/− livers (each band represents five pooled livers) (G) Diurnal binding of CLOCK to the Upp2 promoter. Immunoprecipitation of CLOCK from liver homogenates and quantification of CLOCK-bound target DNA by qPCR normalized to Upp2 input DNA. (For binding of CLOCK to the Dbp promoter in the same livers, see Fig. S9. N = 3–10 livers per data point.).