Synchronization between peripheral circadian clock and feeding-fasting cycles in microfluidic device sustains oscillatory pattern of transcriptome

Abstract

The circadian system cyclically regulates many physiological and behavioral processes within the day. Desynchronization between physiological and behavioral rhythms increases the risk of developing some, including metabolic, disorders. Here we investigate how the oscillatory nature of metabolic signals, resembling feeding-fasting cycles, sustains the cell-autonomous clock in peripheral tissues. By controlling the timing, period and frequency of glucose and insulin signals via microfluidics, we find a strong effect on Per2::Luc fibroblasts entrainment. We show that the circadian Per2 expression is better sustained via a 24 h period and 12 h:12 h frequency-encoded metabolic stimulation applied for 3 daily cycles, aligned to the cell-autonomous clock, entraining the expression of hundreds of genes mostly belonging to circadian rhythms and cell cycle pathways. On the contrary misaligned feeding-fasting cycles synchronize and amplify the expression of extracellular matrix-associated genes, aligned during the light phase. This study underlines the role of the synchronicity between life-style-associated metabolic signals and peripheral clocks on the circadian entrainment.

A role for gene expression and mRNA stability in nutritional compensation of the circadian clock

“…We used a U2OS Bmal1 - dLuc reporter cell line to compare rhythms in high versus low glucose medium and found a slight under-compensation phenotype (Supplementary Figure 6), similar to the single-cell rhythms report (Gagliano et al, 2021). Period length was 0.5 hours shorter in 25 mM (high) glucose compared to 5.56 mM (low) glucose, trending in the same direction as the ∼1.5-hour period shortening seen for the wild-type Neurospora clock across glucose concentrations (Figures 1 – 3).…”

“…We used a U2OS Bmal1-dLuc reporter cell line to compare rhythms in high versus low glucose medium and found a slight under-compensation phenotype (Supplementary Figure 6), similar to the single-cell rhythms report (Gagliano et al, 2021). Period length was 0. among the hits from a genome-wide screen for period length defects using U2OS cells (Zhang et al, 2009) or tested in a kinase/phosphatase siRNA screen (Maier et al, 2009); however, visual inspection of the genome-wide screen data confirmed that a subset of the pooled siRNAs did show period effects after CPSF6 or SETD2 knockdown (source: BioGPS database, "Circadian Genomics Screen" plugin).…”

The emergence of the circadian clock network in hiPSC-derived hepatocytes on chip

“…To reach this aim we take advantage of microfluidics technology applied in circadian studies as we recently reported. Microfluidic technology enables timely hepatic differentiation [21] combined with tight temporal control of the microenvironment [6]. This is an essential prerequisite for the synchronization of the cellautonomous circadian clock of differentiating cells.…”

“…Microfluidic devices were fabricated by standard softlithographic techniques as previously described [6]. The master mold was photo-lithographically patterned using a SU8-2100 (Microchem) negative photoresist to obtain a final thickness of 200 mm.…”

“…As previously described, the conventional technology (Petri dish cell culture) only offers limited possibilities in performing dynamic simulations. Microfluidics enables to accurately control of the temporal profile of biochemical composition over cell culture [6,21]. With this in mind, we developed a specific microfluidic platform that offers the unique possibilities to performed hepatic differentiation together with frequency-encoded stimulations and quantitative analysis of circadian gene expressions (Fig.…”