I. The circadian system

The daily environmental changes imposed by the rotation of the earth drive circadian physiological processes in plants and mammals, including humans. While the molecular components defining circadian oscillations are conserved during evolution, several pathways—and even tissues—are specific to each species, which enables the fine-tuning and system plasticity necessary for the adaptation to environmental changes.

Circadian clocks enable species to adapt and anticipate daily and seasonal environmental changes. The circadian system is composed of: (i) an input pathway receiving and transmitting environmental cues to a central oscillator; (ii) a central oscillator that keeps circadian time and generates rhythm; and (iii) output mechanisms that relay this timing information to the systems, which regulate physiology and behavior.
Circadian clocks are uniquely characterized as entrainable (their period and phase can be changed by specific external cues, ie, Zeitgeber), self-sustained (their rhythmicity persists in constant conditions), and temperature-compensated (contrary to most metabolic activities, their period does not increase with body temperature).
In mammals, the circadian system is composed of a central pacemaker, which perceives and integrates light information and synchronizes peripheral oscillations to the daily rhythm. A core molecular clock expressed in all cells drives these oscillations. The light-input systems from plants to animals differ owing to their different
photoreceptive systems, but the molecular circadian machinery is evolutionary conserved.1

1. The clockwork machinery

In mammals, the core molecular clock is composed of interlocked positive and negative transcriptional/translational feedback loops. The components of the clock are expressed and active in all cells.
The bHLH-PAS activators CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) heterodimerize and promote transcription of target genes that present E-box cis-regulatory enhancer tandem in their promoter.
In this way, they induce the repressive arm of the clock, which includes CRY1/2 (cryptochromes 1 and 2) and PER1/2/3 (periods 1, 2, and 3), and the nuclear receptors Rev- Erb α and β (Figure 1). PER and CRY proteins form a complex able to translocate to the nucleus to inhibit the CLOCK:BMAL1 transactivating function, whereas Rev-Erb
α/β represses Bmal1 transcription through the recruitment of the complex formed by NCOR (nuclear repressor co-repressor 1) and HDAC3 (histone deacetylase 3). In parallel, CLOCK:BMAL1 induces the expression of another set of nuclear receptors, ROR α and β (RAR-orphan receptor), which function as transcriptional circadian activators to positively feedback on Bmal1 expression in competition with Rev-Erb α/β. Additional mechanisms control the stability of the repressor complex PER:CRY. CK 1α/β (casein kinase 1α and 1β) and AMPK (5’ AMP-activated protein kinase) induce the phosphorylation and destabilization of PER proteins, while FBXL3 (F-box and leucinerich
repeat protein 3) directs the E3 ubiquitin ligase-mediated degradation of CRY proteins.
This complete transcriptional system mandates fine-tuning of the clock and provides the plasticity needed to adjust intrinsic rhythms to external cues. Cytosolic loops involving cycling metabolites, such as NAD+ (nicotinamide adenine dinucleotide), are robustly coupled with the transcriptional loops and participate in the regulation of the clock (Figure 1).2

Deletion of individual clock genes in mice does not impact circadian rhythms when they are placed in a 12/12-hour light/dark schedule. The deletion of Bmal1 results in arrhythmia in constant darkness. Single deletion of the other clock genes generates a modest phenotype in free running conditions (ie, without external resetting cues), resulting in a clock with a short or long period length. However, the double knockouts of redundant clock components, such as Cry1 and Cry2, Per1 and Per2, and Clock and Npas2, also result in arrhythmia in constant darkness. The circadian network thus maintains genetic robustness to the loss of individual clock components.
Surprisingly, the deletion of individual components avoids cell autonomous circadian oscillations, suggesting that cellular rhythms are not as robustly controlled as locomotor activity behavior.3

2. The suprachiasmatic nucleus: central pacemaker of the clock machinery

The suprachiasmatic nucleus (SCN) is the master clock of the circadian system in mammals. The SCN, located in the hypothalamus, is composed of 15 000–20 000 neurons in rodents. Based on neuropeptide expression, the SCN is divided into a ventrolateral part (the core) and a dorsomedian part (the shell). The core of the SCN mainly expresses gastrin-releasing peptide (GRP) and vasoactive intestinal
polypeptide (VIP), whereas the shell contains the hormone vasopressin (AVP). The neurotransmitter γ-amino butyric acid (GABA), which is expressed throughout the SCN, is involved in the transmission of neuronal information between the core and the shell.
The molecular and cytosolic clock present in each neuron control ion conductances, leading to a rhythm in neuronal activity. Intrinsic rhythms of individual neurons are not entirely in phase. The combined electrical activity of individual SCN neurons integrates at the SCN network level to produce a sinusoidal-like waveform pattern, which oscillates with ∼24-h precision and peaks in phase with the active behavioral phase. Under the influence of environmental conditions, synchronization can either increase or decrease, leading to phase shifts in molecular clock oscillations.
Light is the strongest Zeitgeber for the SCN. Light information reaches the SCN through a monosynaptic pathway from the retina: the retinohypothalamic tract (RHT). RHT fibers end directly on the VIP-expressing neurons in the core of the SCN and primarily use the excitatory glutamate neurotransmitter. Light leads to the activation of immediate early transcription factors, such as CREB (cyclic AMP response element-binding protein), which induce sudden changes in clock gene expression, particularly the induction of Per1 and Per2 transcription. This change in
gene expression induces a phase shift in the electrical activity of the SCN, allowing a synchronization of the circadian rhythm to the daily light-dark cycle. Strikingly, the effect of light on the SCN varies depending on the time at which it is perceived. These different effects of light on the circadian phase of the SCN (delay or advance) depend on cellular signaling cascades and are an intrinsic property of the SCN.4

3. Output pathways from the mammalian circadian clock

Once the central pacemaker in the SCN has integrated the input signal, timing information has to be sent to peripheral oscillators. Then, the clock system coordinates behavior and metabolism with the external light-dark environment. The clock thus
controls different functions, according to both time-of-day and nutrient state. During sleep, clock enhances functions providing sufficient glucose to feed the brain; whereas during the postprandial state, clock stimulates processes required for nutrient storage.
To synchronize behavioral and physiological rhythms, signals are sent to the periphery via both neuronal pathways and diffusible molecules. SCN neurons project predominantly into the medial subparaventricular zone and the dorsomedial hypothalamus. The SCN communicates to the periphery in part via humoral signals released by other tissues of the central nervous system and via the sympathetic and parasympathetic nervous systems. These output signals regulate sleep, wakefulness, and behavioral activity and physiologically prepare internal organs and tissues for
food intake. For example, SCN targets the gallbladder, adrenal glands, liver, kidney, pancreas, white and brown adipose tissues, and muscles. Time cues sent by the SCN are thought to coordinate peripheral oscillators and impose cellular synchrony within a given tissue. However, additional cues—such as feeding or body temperature rhythms—strongly entrain peripheral oscillators independently of the central SCN pacemaker (Figure 2). Cycling metabolites secreted either by the brain or peripheral tissues might regulate circadian oscillations in the SCN. For example,
melatonin, which is released in a circadian fashion from the pineal gland, is involved in feedback regulation of the SCN. Circadian levels of ghrelin, leptin, insulin, or glucose might also affect circadian rhythmicity through the regulation of neuronal activity in the arcuate nucleus.5,6

“The Islet and Metabolism Keep Time”
I. The circadian system
II. Circadian regulation of the transcriptional network
III. Circadian rhythms and metabolism
IV. Clock and the islet
V. Conclusion