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. 2020 Oct 26;117(45):28402–28411. doi: 10.1073/pnas.2003524117

Fig. 1.

Fig. 1.

The circadian clock is regulated by integrated spatial and temporal mechanisms. (A) Circadian rhythms were shortened by autophagy-inducing AA starvation. Per2Luc MEFs were placed in different concentrations of AAs by varying the ratio between DMEM (100% AA) and EBSS (0% AA). After approximately 3 d, 2%, 5%, and 10% media were replaced with 100% medium and 100% medium was replaced with 5% medium while bioluminescence rhythms were measured. The low signal in 2% medium after replacement with the normal medium suggests that the starvation condition was not physiologically sustainable. n = 3 each. Data are representative of three experiments. (B) Circadian rhythms were lengthened and dampened by inhibition of autophagy in a dose-dependent manner. Note that robust rhythms were recovered after washout, indicating that the drugs did not affect cell viability at these doses. n = 3 each. Data are representative of three experiments. (C) Actograms of three mice heterozygous for Atg5 deletion. The black line indicates LD (12 h light:12 h dark) to DD (constant dark) transition, and the gray shading indicates TM treatment. No two mice showed a similar stable phase angle after TM treatment. Six more heterozygotes are shown in SI Appendix, Fig. S4. (D) Periods calculated from short intervals of stable phase angle (1 to 2 wk) were compared between WT (C57BL/6J) and Atg5 het mice. The average period of Atg5 het mice (Atg5fl/+; CAG-Cre-ER) was significantly longer than that of WT mice after TM treatment and significantly different before and after TM treatment (P < 0.01, two-tailed t test). The periods of Atg5 het mice before TM treatment were calculated from the actograms in the first 2 wk in DD. (E) Starvation-induced period shortening in WT mice. C57BL/6J mice were entrained in LD followed by DD for 2 wk, which served as baseline activity. A hypocalorie diet was provided at ZT12 of the previous LD for 12 d (indicated by the green line), followed by a return to ad libitum. The red line indicates the predicted continuous activity onset had the mice not been subjected to starvation. n = 8.