Circadian rhythm

3.1.1. Definition and concept

All photosensitive organisms from bacteria to humans are subjected to daily light/dark and temperature cycles due to the Earth rotation (Figure 1) (1). During evolution, an internal timekeeping mechanism called circadian clock, from the Latin “circa diem”, meaning about a day (24 hours (h)), has been acquired for a better adaptation by anticipating these environmental changes. Numerous biological processes are regulated by the circadian clock allowing proper gene expression and chemical reactions to occur at the right timing (2).

Four parameters characterize a circadian oscillation (Figure 2). Firstly, the period, which is defined as the time when an identical phenomenon occurs again. In case of circadian rhythm, the period length is around 24 h. Secondly, the amplitude that represents the difference between two successive points where the slope of the oscillation is equal to zero. Thirdly, the magnitude, defined as the absolute value of the amplitude. Lastly, the phase is the state of an oscillation at a time t.

Figure 2. Characteristics of an oscillation curve. The amplitude, the period and the phase are commonly used to define circadian oscillatory profiles.

0 0.5 1 1.5 2 2.5

0 24 48 72 96 120

Bioluminescence ratio

Time (hours)

Phase Period

Amplitude

Figure 1. (Adopted from (2)). Geophysical time drives circadian maintenance of homeostasis.

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3.1.2. Hierarchical organization of the circadian time-keeping system

In mammals, circadian control of physiology and behavior is coordinated by a central clock, located in the paired suprachiasmatic nuclei (SCN) of the hypothalamus (3), which synchronizes on a daily basis peripheral, or slave, oscillators found in most of the organs including liver (4), kidney (5), heart (6), adipose tissue (7), endocrine pancreas (8, 9), and skeletal muscle (10-12). It has been demonstrated that the clock is ticking in almost all body cells in a self-sustained autonomous manner (13-15).

Interestingly, it was recently discovered that inside the endocrine pancreas, the two main cell-types, i.e.

α- and β-cells, exhibit distinct clock properties, suggesting a very precise cell-type specific regulation (16). Interestingly, the peripheral cellular oscillators were found to be resilient to large variations in general transcription and temperature (17).

3.1.3. Synchronization of the clock

3.1.3.1. The circadian synchronization of central and peripheral clocks in vivo

The master pacemaker in the SCN is entrained by external Zeitgeber cues (from German, meaning

“time giver”), with the light intensity being the principal signal (18, 19), acting through the photosensitive retinal ganglion cells (ipRGCs) expressing the melanopsin photo pigment, and the optic nerve (20). This photo pigment is sensitive to the blue/green light spectrum, but less to the red light (21). SCN controls the synchrony of peripheral clocks directly, via sympathetic neural system and humoral signals, and indirectly by controlling rest-activity and temperature cycles (Figure 3) (18). Feeding/fasting cycle is an important Zeitgeber for peripheral oscillators, which was shown to be potent enough to entrain circadian peripheral clocks (22-29). Moreover, uncoupling between peripheral and SCN clocks has been demonstrated by inversion of feeding-fasting cycle (23, 30). In mouse liver with conditional clock disruption by hepatocyte-specific overexpression of Rev-Erbα in a doxycycline-dependent inducible manner, most of the transcripts became non-rhythmic (31). Importantly, 31 genes expressed in liver, including Per2, kept their circadian rhythmicity following liver clock disruption (31). Daytime-restricted feeding restored rhythmic gene expression in liver from Cry1^-/- ; Cry2^-/- double mutant mice (32). Taken together, these results indicate that rhythmicity in liver gene expression is entrained by systemic signals.

Moreover, variations of body temperature (33-38) and exercise (39-42) represent additional physiological synchronizers for peripheral clocks. Mimicked body temperature cycles, but not a serum shock, induce expression of the cold-inducible RNA-binding protein (CIRP/CIRBP) in culture fibroblasts, which is required for high amplitude circadian gene expression (43). Of note, change of 1°C of the body

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temperature was recently found to control rhythmic alternative splicing in mammals by regulating SR protein phosphorylation (44).

Figure 3. (Modified from (18)). Entrainment of peripheral clocks by the SCN. The central clock synchronizes the peripheral oscillators either directly, via nervous or humoral signals, or indirectly by changing behavior.

3.1.3.2. The in vitro synchronization

Field-breaking work by Ueli Schibler and colleagues demonstrated that a 50% horse serum pulse of 1 h is sufficient to synchronize the Rat1 fibroblast clock in vitro (13, 14), thus opening the new horizons for studying circadian oscillators in cells taken out of the body, cultured and synchronized in vitro. Later on, glucocorticoid hormone dexamethasone (25, 45-49), forskolin, and additional compounds (50, 51) were found to be potent in vitro synchronizers. Glucocorticoids bind the glucocorticoid receptor (GR), inducing its translocation to the nucleus, where it interacts with glucocorticoid response elements (GREs) to activate transcription of target genes (52-54). Forskolin acts by increasing cAMP levels using

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protein kinase A (PKA) signaling pathway. This results in the phosphorylation and activation of calcium/cAMP responsive element binding protein (CREB) (51). Synchronization of the circadian clock induces high expression of Per1, phenomenon called the immediate early response, required to initiate the cycle in Rat1 fibroblasts in vitro. Per2 was shown to be induced by certain synchronizers (horse serum), but its induction is not mandatory for cellular synchronization (50). Additional physiologically relevant stimuli including adrenaline, glucose or insulin are able to synchronize different cell types in vitro (16, 55). Employing cell cultures as model for studying circadian rhythm has been extensively developed since the pioneering work by Ueli Schibler. Moreover, application of this approach to the human primary cells, including primary human skin fibroblasts, which exhibited period length measured in vitro strongly correlating to the individual circadian phenotype in vivo (56, 57), offered a unique and powerful tool for studying human peripheral clocks in physiological condition and in diseases, which has been extensively utilized in this work.