The human circadian metabolome

Abstract

The circadian clock orchestrates many aspects of human physiology, and disruption of this clock has been implicated in various pathologies, ranging from cancer to metabolic syndrome and diabetes. Although there is evidence that metabolism and the circadian clockwork are intimately linked on a transcriptional level, whether these effects are directly under clock control or are mediated by the rest-activity cycle and the timing of food intake is unclear. To answer this question, we conducted an unbiased screen in human subjects of the metabolome of blood plasma and saliva at different times of day. To minimize indirect effects, subjects were kept in a 40-h constant routine of enforced posture, constant dim light, hourly isocaloric meals, and sleep deprivation. Under these conditions, we found that ~15% of all identified metabolites in plasma and saliva were under circadian control, most notably fatty acids in plasma and amino acids in saliva. Our data suggest that there is a strong direct effect of the endogenous circadian clock on multiple human metabolic pathways that is independent of sleep or feeding. In addition, they identify multiple potential small-molecule biomarkers of human circadian phase and sleep pressure.

Fig. 1.

(A) Experimental design. Scheme of the 40-h extended wakefulness constant routine, sampling, and pooling of samples. For detailed explanations of the sampling and pooling procedures, see Methods. Black bars indicate sleep; open bars, extended wakefulness; red lines, sampling of blood and saliva; gray lines, isocaloric meals. Samples on either side of the circled numbers were pooled together for MS analysis. (B) Comparison of rhythmic cortisol levels measured by LC-MS (red) and ELISA (blue) from plasma and saliva, respectively, of the same subject pools. (C) Comparison of individuals with the pool in which they were used.

Fig. 2.

Heat plots for all identified metabolites in plasma (A) and saliva (B). The black bar indicates circadian metabolites, and the gray bar indicates monotonic increasing/decreasing metabolites. High levels of metabolites are shown in red (plasma) and yellow (saliva), and low levels are shown in green (plasma) and blue (saliva). (C–F) Pathway analyses (C and E) and time-of-day distribution (D and F) of peak phases of rhythmic metabolites in plasma (C and D) and saliva (E and F). Pathways are color-coded as follows: blue, lipids; orange, energy metabolism; gray, peptides; red, amino acids; green, carbohydrates; yellow, cofactors and vitamins.

Fig. 3.

Rhythmic metabolites in plasma and saliva. (A and B) Profiles of substances previously implicated in sleep–wake regulation. (C and D) Heat maps of all such substances. High levels of metabolites are shown in red (plasma) and yellow (saliva) whereas low levels are shown in green (plasma) and blue (saliva). The metabolites in blue are exemplified in A, and those in red are exemplified in B.