Toward the Beginning of Time: Circadian Rhythms in Metabolism Precede Rhythms in Clock Gene Expression in Mouse Embryonic Stem Cells

Abstract

The appearance, progression, and potential role for circadian rhythms during early development have previously focused mainly on the suprachiasmatic nucleus (SCN) and peri- and postnatal expression of canonical clock genes. More recently, gene expression studies in embryonic stem cells have shown that some clock genes are expressed in undifferentiated cells; however rhythmicity was only established when cells are directed toward a neural fate. These studies also concluded that a functional clock is not present in ESCs, based solely on their gene expression. The null hypothesis underlying the present study is that embryonic stem cells become rhythmic in both clock gene expression and glucose utilization only when allowed to spontaneously differentiate. Undifferentiated stem cells (ESCs, n = 6 cultures/timepoint for all experiments) were either maintained in their pluripotent state or released into differentiation (dESCs, n = 6 cultures/timepoint for all experiments). Glucose utilization was assayed through 2-deoxyglucose uptake measurement, and clock gene and glucose transporter expression was assayed every 4 hours for 2 days in ESCs and dESCs by quantitative PCR (qPCR) in the same cell lysates. Undifferentiated stem cells expressed a self-sustained rhythm in glucose uptake that was not coincident with rhythmic expression of clock genes. This physiological rhythm was paralleled by glucose transporter mRNA expression. Upon differentiation, circadian patterns of some but not all clock genes were expressed, and the amplitude of the glucose utilization rhythm was enhanced in dESCs. These data provide the earliest evidence of a functional circadian clock, in addition to further challenging the idea that rhythmic transcription of clock genes are necessary for rhythmic physiological output and suggest a role for a clock-controlled physiology in the earliest stages of development.

Circadian clocks: from stem cells to tissue homeostasis and regeneration

“…This is in line with the emergence of circadian rhythmicity during differentiation of embryonic stem cells that do not possess a functional circadian clock 49, 53. Clock genes are expressed in ES cells, however, and the emergence of a functional clock system is closely linked to differentiation 49, 50, 51, 53, but these factors may have additional functions in those cells. The intimate link between clock proteins and the cell cycle was discussed above, and stem cell proliferation may be regulated by these factors.…”

“…In murine pluripotent stem cells, circadian rhythms were shown to be established when differentiation is induced upon withdrawal of leukemia inhibitor factor (LIF) (passive) or by the addition of retinoic acid (active) 49, 50, 51. When reversing differentiation through reprogramming 52, the clock is switched off again 49 (Fig 3A), which indicates that the (in)activation of the diurnal clock is a reversible process that is intensively linked with the differentiation state of a cell.…”

Circadian clock-mediated control of stem cell division and differentiation: beyond night and day

“…Thus, cell-intrinsic rhythms of circadian gene expression are probably present at the very earliest stages of development. It has even been suggested that circadian rhythms of energy consumption in ES cells might briefly precede the emergence of canonical circadian transcriptional oscillations (Paulose et al, 2012).…”

Circadian Redox and Metabolic Oscillations in Mammalian Systems

“…ESCs were previously considered not to possess any intrinsic timekeeping because although clock gene transcription factors such as BMAL1 are expressed, their levels do not exhibit circadian cycles until after differentiation. Paulose et al, however, report a self-sustained circadian rhythm in ESC uptake of 2-deoxyglucose prior to, and following, differentiation (102). Similar to the findings in erythrocytes, this suggests that circadian rhythms in metabolism (in this case glucose utilization) may persist in the absence of gene expression (cycling or otherwise), but does not satisfactorily address whether transcriptional cycles (when they are present) may run independently from metabolic oscillations, or are coupled with them.…”

“…Based on the examples above, however, it seems reasonable to expect an impending flood of observations detailing how metabolic cues can directly regulate the activity of circadian-relevant transcription factors and cellular rhythms in general. Moreover, since the stability and localization of identified clock proteins is regulated post-translationally, via phosphorylation/ubiquitination, through enzyme systems that appear to be conserved throughout the eukaryotes; it is not clear that the mRNA levels of the ''clock gene'' transcription factors actually need to oscillate for them to fulfill their role, merely that sufficient mRNA be present at the appropriate circadian phase to facilitate translation of nascent polypeptide chains (102). Clearly, however, as with cycles of transcriptional activation/repression, the degradation of these transcription factors and their mRNAs will increase the signal-to-noise ratio within this molecular circuit and thereby impart directionality to the cycle.…”