Mammalian Circadian Period, But Not Phase and Amplitude, Is Robust Against Redox and Metabolic Perturbations

Abstract

Aims: Circadian rhythms permeate all levels of biology to temporally regulate cell and whole-body physiology, although the cell-autonomous mechanism that confers ∼24-h periodicity is incompletely understood. Reports describing circadian oscillations of over-oxidized peroxiredoxin abundance have suggested that redox signaling plays an important role in the timekeeping mechanism. Here, we tested the functional contribution that redox state and primary metabolism make to mammalian cellular timekeeping.Results: We found a circadian rhythm in flux through primary glucose metabolic pathways, indicating rhythmic NAD(P)H production. Using pharmacological and genetic perturbations, however, we found that timekeeping was insensitive to changes in glycolytic flux, whereas oxidative pentose phosphate pathway (PPP) inhibition and other chronic redox stressors primarily affected circadian gene expression amplitude, not periodicity. Finally, acute changes in redox state decreased PER2 protein stability, phase dependently, to alter the subsequent phase of oscillation.Innovation: Circadian rhythms in primary cellular metabolism and redox state have been proposed to play a role in the cellular timekeeping mechanism. We present experimental data testing that hypothesis.Conclusion: Circadian flux through primary metabolism is cell autonomous, driving rhythmic NAD(P)+ redox cofactor turnover and maintaining a redox balance that is permissive for circadian gene expression cycles. Redox homeostasis and PPP flux, but not glycolysis, are necessary to maintain clock amplitude, but neither redox nor glucose metabolism determines circadian period. Furthermore, cellular rhythms are sensitive to acute changes in redox balance, at least partly through regulation of PER protein. Redox and metabolic state are, thus, both inputs and outputs, but not state variables, of cellular circadian timekeeping. Antioxid. Redox Signal. 28, 507–520.

Metabolic rhythms: A framework for coordinating cellular function

“…As the dilution rate and/or the glucose concentration decreases the period of oscillation becomes progressively longer and the fraction of unbudded cells increases (Beuse et al., ; Chin et al., ; Slavov & Botstein, ). This may be similar to glucose‐dependent effects on daily cycles that occur in fibroblasts, and U2OS cells and in strains of Neurospora that have lost metabolic compensation (Lamia et al., ; Putker et al., ; Sancar, Sancar, & Brunner, ). During LOC, glucose‐limited cells accumulate carbohydrates in the form of trehalose and glycogen (Ouyang, Xu, Mitsui, Motizuki, & Xu, ; Wang et al., ; Xu & Tsurugi, , ).…”

“…This process forms a critical part of the oscillation and populations deleted for glucose‐6‐phosphate dehydrogenase initiate cycling, but the oscillation stalls the first time it reaches LOC (Tu et al., , ). Genetic and pharmacological inhibition of the pentose phosphate pathway significantly affects circadian gene expression rhythms in mouse fibroblasts (Putker et al., ).…”

Circadian Clocks and Metabolism: Implications for Microbiome and Aging

“…Over- and hyper-peroxidation rhythms of peroxiredoxins have been found in human red blood cells [25], which have no nucleus that are found to be driven by hemoglobin auto-oxidation rhythms and are associated with the degradation of hyperoxidized peroxiredoxins [26]. Inhibiting the pentose phosphate pathway, a critical source for NADPH, was found to influence circadian rhythms in cells across species [27], with a recent study showing an effect on the amplitude and phase of the clock [28] This work further supports a feedback from cellular energy metabolism to the transcriptional/translational circadian clock.…”

Copper in the suprachiasmatic circadian clock: A possible link between multiple circadian oscillators

“…The redox state of the cell in turn can directly affect the function of some clock proteins (e.g., Rev‐Erbα) (Hirano et al., ; Rey et al., ; Wende et al., ). In addition, oxidative damage of clock proteins may enhance their degradation (e.g., PER2) or hinder their function, and oxidative stress has been shown to shift mammalian clocks through apoptosis signaling‐related kinase, a member of the MAPKKK (MAPKK kinase) family or the antioxidant transcription factor, NRF2 (nuclear factor E2‐like 2) (Imamura et al., ; Putker et al., ; Tamaru et al., ; Wible et al., ). It is still unclear how such effects on the core molecular clock are important for adjusting other processes to the metabolic state of the cell(s).…”

“…It is still unclear how such effects on the core molecular clock are important for adjusting other processes to the metabolic state of the cell(s). Nonetheless, one important clock function may be to coordinate enzymatic removal of ROS in anticipation of peak oxidative stress (Nagy & Reddy, ; Putker et al., ). Interestingly, the removal of ROS and cellular redox reactions involves redox recycling of antioxidant enzymes and molecules, (Kinoshita, Aoyama, & Nakaki, ; Nagy & Reddy, ), and studies using mature red blood cells have demonstrated circadian oscillations in the redox states of antioxidant enzymes in the absence of transcription/translation feedback loops (Cho, Yoon, Kim, Woo, & Rhee, ; Homma et al., ; O'Neill & Reddy, ).…”