Toward the beginning of time: circadian rhythms in metabolism precede rhythms in clock gene expression in mouse embryonic stem cells

• Published in: PloS one 2012-11, Vol.7 (11), p.e49555-e49555
• Main Authors: Paulose, Jiffin K, Rucker, 3rd, Edmund B, Cassone, Vincent M
• Format: Article
• Language: English
• Subjects: Animals; Biological clocks; Biological Clocks - physiology; Biology; Circadian rhythm; Circadian Rhythm - physiology; Circadian rhythms; Clock gene; CLOCK Proteins - genetics; CLOCK Proteins - metabolism; Deoxyglucose; Developmental stages; Differentiation; Embryo cells; Embryonic stem cells; Embryonic Stem Cells - metabolism; Embryos; Energy Metabolism - physiology; Gene Expression; Genes; Glucose; Glucose metabolism; Glucose Transport Proteins, Facilitative - genetics; Glucose Transport Proteins, Facilitative - metabolism; Glucose transporter; House mouse; Kinases; Lysates; Metabolism; Mice; Null hypothesis; Period Circadian Proteins - genetics; Period Circadian Proteins - metabolism; Periodicity; Physiological aspects; Physiology; Pluripotency; Rhythm; RNA; RNA, Messenger - genetics; RNA, Messenger - metabolism; Stem cell transplantation; Stem cells; Suprachiasmatic nucleus; Transcription; Transcription (Genetics); Utilization
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0049555

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.