Metabolic drivers of co-expressed biological rhythms in vitro
Creators: name: Ting, Isaiah
Publisher: name: Zenodo
Year: 2021

description: Rapid biological rhythms with periods from milliseconds to hours (ultradian rhythms) have been uncovered in many physiological processes such as the feeding and activity behaviours in the common vole (Microtus arvalis) 1,2, human temperature regulation 3 and NREM-REM cycles 4. Van der Veen and Gerkema have unmasked widespread ultradian gene expression in vitro, that are co-expressed with daily, 24-hour rhythms 5. Gene Ontology analysis of the probe list that express ultradian gene expression in the mouse in vivo and in NIH 3T3 cells in vitro revealed a significant enrichment for metabolic process was observed which further provides evidence of the presence of ultradian rhythms within metabolic processes. One particularly ultradian gene is pdcd5, which exhibited robust ultradian rhythmicity, and is involved with programmed cell death 5.<br> A recent PubMed search with quoted search phrases “circadian rhythm” and “ultradian rhythm” yielded 32,265 and 788 articles, respectively, in the period between 2006 and 2021 showing “experimental neglect” of ultradian rhythms 6. Thus, more research is needed, specifically on the interactions of ultradian rhythms with metabolic processes and their contribution to maintenance of metabolic health. We therefore hypothesise that altering metabolic conditions can alter the balance between circadian and ultradian rhythms in several processes 7,8, including in pdcd5.<br> NIH 3T3 cells (mouse embryonic fibroblasts) that have been transduced with our custom-made Pdcd5::Luc reporter construct were used for in vitro tracking of ultradian rhythms in real-time. To address our hypothesis on metabolic challenge, cells were incubated with or without low glucose (0.5 mM) for 18- and 24-hours pre-measurement as well as low glucose during measurement. Bioluminescence recordings were undertaken at 10-minute intervals using a LumiCycle. The resulting data were analysed in MATLAB2019a© by detrending the data to a 12-h running average, a high-pass filter in order to remove unwanted slower rhythms, a sample autocorrelation step to validate the period found and cosinor-based rhythmometry used to find the dominant ultradian oscillatory period 9.<br> Using our approach, we were able to confirm circadian and ultradian rhythm in the expression of pdcd5. In control conditions (normal concentrations of glucose) cosinor analysis returned a straight line indicating no significant dominant ultradian oscillation. When incubated with low glucose for 18 hours pre-measurement, we were able to find a dominant ultradian period of 10.06h (± 0.14 h, p &lt; 0.0001, N=3); low glucose incubation for 24 hours pre-measurement revealed a period of 6.4 h (± 0.11 h, p = 0.0002, N=3). Using the analysis tools, we were able to remove the circadian signal, and unmask the higher frequency ultradian oscillations with a period of between 6 h and 10 h.<br> The findings in this project are the first cell culture-based model of ultradian rhythmicity in gene expression. Our analysis approach also successfully removes the long and overarching circadian rhythm that often masks the shorter frequency ultradian oscillation, often overlooked in measurements. The unmasking of ultradian signals from previously detected circadian rhythms using the spectral analysis pipeline (high-pass filter, sample autocorrelation) is novel work that advances our previous findings 5 where they have successfully unmasked ultradian rhythms in gene expression. Investigation of the effects of a metabolic challenge (represented by low glucose conditions) emphasised a switch in biological rhythm and timing in vitro that can be paralleled in arctic animals’ behavioural studies during the polar day and night where their activity and foraging behaviour reverts to shorter ultradian rhythms during a metabolic challenge 7,8. Altogether, these results indicate an interaction between ultradian rhythms and metabolic processes. Further research analysing the exact pathways that connect biological rhythms (ultradian rhythms) and metabolism is needed alongside the regulatory mechanisms underlying ultradian rhythms. This knowledge would lead to enhanced understanding of translational metabolic processes in healthy and pathological situations such as diabetes and the improved control of glucose metabolism through insulin release.