The circadian system is hierarchical and begins at the suprachiasmatic nucleus (SCN) in the anterior hypothalamus. The SCN is considered the master circadian pacemaker in mammals because it synchronizes all downstream peripheral clocks to the light/dark cycle (Liu et al., 2007b; Mohawk et al., 2012). Direct lesions to the SCN or downstream neurons resulted in the loss of this rhythmic ensemble, which led to the conclusion that the SCN is the master pacemaker (Moore and Eichler, 1972; Stephan and Zucker, 1972). Further, lesions along the light input pathway resulted in free-running rhythms that lacked synchrony to zeitgebers, indicating that, while rhythms can be generated by extra-SCN tissues, the SCN is necessary for entrainment to the light/dark cycle. Although the SCN responds to light, it also can work autonomously in its absence, as demonstrated by mice that maintain locomotor activity rhythms in constant darkness, a free-running condition that reveals the endogenous circadian clock function. These studies, especially those that are more recent and involved genetic perturbation and real-time in vivo bioluminescence recordings, led to the understanding that the SCN serves as the central oscillator and systemic circadian coordinator of the organism (Yoo et al., 2004; Sinturel et al., 2021).

Following the discovery of the master clock, SCN transplantation studies established that the donor SCN determines circadian parameters rather than the host. Tau mutant hamsters, which have a shortened free-running period, were key to this discovery. SCN transplantation studies involving homozygous tau mutant, heterozygous tau mutant, and wildtype hamsters with free-running periods of approximately 20, 22, and 24 h, respectively, determined that rhythmicity and period length were reflective of the donor SCN, regardless of the host (Ralph et al., 1990). Transplanted SCN were also shown to restore behavioral rhythms in arrhythmic and mutant mice through neural and diffusible signaling molecules; however, complete restoration of SCN requires intact neural projections, which cannot be maintained through transplantation.

The SCN regulates daily various behavioral and physiological rhythms through a complex neural circuitry within the hypothalamus. Internally, the SCN can be delineated into two subregions composed of various cell types expressing different neuropeptides: the ventral core and the dorsal shell. In the core region, vasoactive intestinal polypeptide (VIP) is the predominant neuropeptide (Yan et al., 2007). Mice deficient of the VIP receptor type 2 (VPAC2) show attenuation of locomotor activity rhythms and significantly impaired ability to phase shift the clock in response to jet lag (Hannibal et al., 2011). These data suggest that VIPergic signaling is crucial for generation of rhythms and phase shift in the SCN. In the SCN shell region, arginine vasopressin (AVP) is primarily expressed. A recent study using conditional Bmal1 deletion and reporter expression showed that circadian function in AVP neurons, not VIP neurons, is essential for SCN network synchrony and stability of circadian rhythmicity (Shan et al., 2020). The retinohypothalamic tract synapses onto core region neurons, where the light input first resets the ventral (VIP+) SCN and then, through internal coupling, synchronizes the dorsal (AVP+) SCN (Yan and Silver, 2002, 2004). While cell types expressing VIP and VIP receptor type 2 (VPAC2) are unequivocal, in unison they determine periodicity and phase in the SCN (Patton et al., 2020). These regions work together to generate rhythms within the SCN. However, in mediobasal hypothalamus (MBH)-lesioned mice, some locomotor activity rhythmicity and feeding-anticipatory activity was lost, indicating that there are other regions of the brain that play a role in maintenance and generation of circadian rhythms that have yet to be identified (Tahara et al., 2010).

The light input pathway of the SCN starts at the retina, which is essential for synchronizing the circadian clock to the local light/dark cycle (Liu et al., 2007a) (Figure 2). The retina contains intrinsically photosensitive retinal ganglion cells (ipRGCs), which respond to short-wave light through the G-coupled protein receptor (GPCR), melanopsin (Provencio et al., 1998). ipRGCs can be separated into six classes (M1-6), of which the M1 class contains the highest expression of melanopsin (Lazzerini Ospri et al., 2017). While melanopsin phototransduction appears to differ between classes, the photoisomerization reaction that occurs is the same (Do, 2019). Melanopsin readily interconverts between three states: two resting states, R and E, and an excited state, metamelanopsin (M), that is the intermediate between R and E (Matsuyama et al., 2012). Because short-wave light is more easily absorbed by resting states than the M state, it drives melanopsin to the M state (Matsuyama et al., 2012). When excited, the activated GPCR signals downstream effectors to depolarize cells by opening intracellular ion channels. However, the melanopsin-activated second-messengers have not clearly been identified and evidence suggests that they differ between ipRGC classes (Sonoda et al., 2018).

Regardless of the phototransduction signaling cascade, input signal is passed from the ipRGCs through the retinohypothalamic tract bilaterally to the ventral VIP + SCN, leading to increased PER1/2 expression via the Ca²⁺/cAMP-CREB pathway (Obrietan et al., 1998; Tischkau et al., 2003; Baek et al., 2018). Increased cellular Ca²⁺ levels activate calmodulin, which subsequently activates calmodulin-dependent kinase II (CaMKII) (Yokota et al., 2001). cAMP response element-binding protein (CREB) is then activated by phosphorylation and binds the cAMP response element in the PER1/2 promoter, driving PER1/2 transcription (McNulty et al., 1998; Obrietan et al., 1999; Tischkau et al., 2003) (Figure 3). Increased cellular levels of cAMP also activate CREB in a PKA-dependent manner (Obrietan et al., 1999; Schurov et al., 2002). Rhythmic cAMP-CREB signaling and control of PER1/2 expression drives SCN synchronization to the light/dark cycle.

It is possible that the SCN could be synchronized by cues other than light, such as meal times or exercise, but light is such a dominant cue that it is difficult to evaluate the strength of other zeitgebers. Despite that, phase shifts in the SCN have been observed in response to a hypocaloric diet under a 12:12 light/dark cycle (Mendoza et al., 2005). This challenges our widely-accepted assumption of light being the single zeitgeber for SCN entrainment. Notably, temperature changes can entrain the SCN ex vivo, but only under near-24-h cycles. This is illustrated in SCN explants cultured in 22-–26-h temperature cycles, with temperature drops from 37°C to 31°C (Bordyugov et al., 2015). However, the implications of these temperature entrainment results are unclear since daily changes in core body temperature typically fluctuate ± 0.5° (Barrett et al., 1993). The SCN appears to be impervious to entrainment to physiologically-relevant temperature changes (Buhr et al., 2010). Thus, light remains the most dominant and predictable zeitgeber for the SCN clock.

Circadian rhythms in the SCN clock are robust enough that they can be maintained for weeks or longer ex vivo (Evans et al., 2012). This robustness is generated in part by synchronization between SCN neurons (Liu et al., 2007b). Paracrine signaling between SCN neurons drives intercellular coupling (Maywood et al., 2011). Mathematical modeling of coupling between single-cell oscillators demonstrates that coupling strength is proportional to circadian amplitude (Finger et al., 2021). Thus, early efforts in these mathematical models showed that coupling between neurons improves circadian rhythms (Liu et al., 2007b; Mohawk et al., 2012). While coupling was originally thought to be a unique attribute to the SCN, it can also present in peripheral tissues (Sinturel et al., 2021). Recent studies suggest TGF-β signaling as a mechanism for coupling between the SCN and peripheral tissues (Finger et al., 2021). We discuss other major mechanisms of synchronization between the SCN and peripheral clocks in the following sections.

Peripheral clocks, which are found in virtually every tissue in the body, are kept in sync primarily through the SCN. A recent study used in vivo bioluminescence imaging to show the SCN is necessary for phase synchrony between organs and lesion of the SCN causes desynchrony of the cellular oscillators, leading to bioluminescence rhythms of reduced amplitude (Sinturel et al., 2021). There appear to be a multitude of pathways that the SCN uses to entrain and synchronize the peripheral oscillators. Concomitantly, there are many response elements in the PER1 and PER2 promoters, which provide mechanisms by which intra- and intercellular signals may reset the clock (Figure 3). First, sympathetic nerve activity through catecholamines, such as epinephrine and norepinephrine, is involved in the entrainment of peripheral organs by the SCN by regulating gene expression via a MAP kinase signaling cascade (Terazono et al., 2003). Downstream signals in this pathway lead to elevated levels of PER2, thereby resetting the clock. This pathway is such a reliable mechanism of clock resetting that forskolin, a known activator of this MAPK signaling cascade, is often used to synchronize cells in in vitro studies (Hu et al., 2021). Resetting through this pathway, however, requires expression of adrenergic receptors in peripheral tissues.

Rhythmic glucocorticoid (GC) secretion is also regulated by the SCN as a means of peripheral clock synchronization. The GC pathway is an ideal candidate for SCN synchronization of peripheral clocks as GC receptors (GR) are found in most peripheral tissues but not the SCN (Cheifetz, 1971; Irvine and Alexander, 1994; Lefcourt et al., 1995; Balsalobre et al., 2000; Cuesta et al., 2015; Boege et al., 2020). GR activation results in transcription of genes that contain glucocorticoid response elements (GREs) in their regulatory regions, including core clock genes PER1/2 (So et al., 2009) (Figure 3). Through this mechanism, the SCN is able to induce phase shifts in peripheral clocks and synchronize them to the light/dark cycle (Lamia et al., 2011; Cheon et al., 2013). Because the GR provides such a robust mechanism for synchronization, the GC analog dexamethasone, like forskolin, is commonly used to synchronize cells used in in vitro studies. However, while GC and adrenergic signaling is important for synchronization, adrenalectomized mice are still able to generate rhythms within peripheral tissues, which indicates that there are other signals and redundant mechanisms that take part in peripheral synchronization (Ikeda et al., 2015).

SCN-regulated daily fluctuations in core body temperature are used to entrain peripheral clocks (Buhr et al., 2010) (Figure 2). Core body temperature shows robust rhythms and typically fluctuates ± 0.5°C each day (Hanneman, 2001; Kräuchi, 2002; Sim et al., 2017). VPAC2-deficient mice showed severe attenuation of core body temperature rhythms, suggesting that the SCN is at least partially responsible for regulating body temperature. This provides a mechanism for synchronization of peripheral clocks through activation of heat shock factor 1, which directly binds to the regulatory sequences within the PER2 promoter to upregulate PER2 expression for phase resetting and synchronization (Buhr et al., 2010) (Figure 3).

While the SCN is necessary for phase synchrony between organs, hepatocytes in the liver, for example, can remain phase-coupled even in the absence of the SCN, suggesting there are redundant mechanisms of synchronization for peripheral clocks (Sinturel et al., 2021). Many of these mechanisms are activated by zeitgebers other than the light/dark cycle, and can cause phase shifts in peripheral tissues without signals from the SCN. Below we discuss how sleep, nutrition, and physical activity regulate circadian clocks throughout the body.

Sleep is thought to be regulated by two processes: circadian rhythms, also known as Process C, and homeostatic sleep drive, otherwise known as Process S. This is known as the Two-Process Model, which was first described by Borbely in 1982 (Borbély, 1982). The circadian sleep process is driven by the internal circadian clock and external time of day, whereas the homeostatic sleep process is driven by wake duration. While Process C is mainly controlled by the SCN, Process S is primarily regulated by the ventrolateral preoptic nuclei (VLPO) of the hypothalamus. Sleep debt increases progressively throughout wakefulness and is associated with accumulation of neuro-modulatory sleep factors, most notably adenosine (Peng et al., 2020). The mechanisms of these two processes converge through adenosine-mediated regulation of the clock: adenosine binds adenosine A1/A2 receptor, a GPCR expressed in several parts of the brain, and activates Ca²⁺-ERK-AP-1 and cAMP-CREB signaling, the same pathways activated by light (Jagannath et al., 2021). In the SCN, adenosine binds A1 to drive PER1/2 expression. Adenosine levels, and therefore sleep debt, gradually increase throughout wake time and decrease during sleep. While Process S is cumulative, Process C is time-of-day-dependent and regulated by core body temperature, glucocorticoids, and melatonin.

Melatonin, also known as the “darkness hormone,” is a regulator of the sleep/wake cycle (Figure 2). It is produced by the pineal gland, a neural substrate of the SCN, and is secreted with a circadian rhythm that peaks in the dark phase. Melatonin binds two GPCRs in the SCN: MT₁ and MT₂ (Dubocovich and Markowska, 2005). Activated MT₁ and MT₂ prevent CREB phosphorylation, thereby downregulating PER1/2 expression, counteracting the light effect (Kopp et al., 1997; von Gall et al., 1998, 2000; Jin et al., 2003). Melatonin has also been shown to mediate the PI3K/AKT pathway to increase BMAL1 expression in neuronal cells (Morishita et al., 2016; Beker et al., 2019). It is hypothesized that sleep entrains the SCN through melatonin secretion because MT₁ and MT₂ are expressed in the SCN. However, some evidence suggests that melatonin also regulates sleep through Process S by MT₂-mediated activation of the CaMK/CREB pathway in the VLPO (Wang et al., 2020). More studies are needed to better understand the various roles that MT₁ and MT₂ play in different parts of the brain.

Sleep is codependent on SCN-driven thermoregulation, which serves as a zeitgeber for peripheral tissues (Buhr et al., 2010) (Figure 2). Core body temperature (T_b) decreases as sleep approaches and can influence sleep quality, as demonstrated by the “warm bath effect.” Passive body heating before bed causes a phase delay in T_b rhythm and increases slow-wave sleep, also known as deep sleep, a restorative sleep phase which is known to be important for memory (Horne and Reid, 1985; Dorsey et al., 1999; Leong et al., 2019). These temperature changes can entrain peripheral tissues through activation of heat shock factor 1, which induces PER2 expression by binding to the heat shock element in its promoter (Kornmann et al., 2007; Buhr et al., 2010). Interestingly, a recent study suggests that this temperature synchronization in human-induced pluripotent stem cells occurs through DBP rather than PER2 expression and that HIF1α may be required (Kaneko et al., 2020). Therefore, the physiology of temperature synchronization requires more investigation to fully elucidate the mechanism and identify any differences between different cell types.

In summary, sleep is both an output of and a zeitgeber for the SCN. Sleep further regulates the clock through its impact on the timing of the feeding/fasting cycle and control of energy homeostasis. The sleep/wake rhythm has strong correlations with core body temperature rhythms, but it is unclear when sleep and temperature regulate the clock and when they are regulated by the clock.

In recent years, there has been a growing interest in chrononutrition, the study of interplay between nutrition and circadian timing. While meal times do not entrain the SCN, they are a strong zeitgeber for peripheral clocks and become the dominant zeitgeber in the absence of light cues or resetting cues emanating from the SCN, to coordinate circadian timing with feeding and fasting (Hamaguchi et al., 2015; Sinturel et al., 2021) (Figure 4). Elaborate peripheral clock synchronization mechanisms often involve multiple signaling pathways, some of which may be sufficient but not required for phase shifts, and can vary between different tissue types. Recent studies have led to a better understanding at the molecular level of how the nutritional cues affect the circadian clock function. It is likely that there are more synchronization mechanisms that have not yet been identified, but here we discuss what is established so far.

After feeding, a number of signaling pathways take part in nutrient storage and utilization, beginning with insulin release by the pancreas (Figure 4). This signals cells in metabolically active organs, including the liver and skeletal muscle, to take up glucose and upregulate catabolic pathways, such as glycolysis and the pentose phosphate pathway. When nutrients are depleted during times of fasting, glucocorticoid, and glucagon levels increase to initiate anabolic pathways, such as gluconeogenesis. The balance between catabolism and anabolism is critical for the maintenance of homeostasis, which has a strong circadian component. To maintain energy homeostasis, nutrient sensors Sirtuin 1 (SIRT1), poly (ADP-ribose) polymerase-1 (PARP-1), AMP-activated kinase (AMPK), and mechanistic target of rapamycin (mTOR) regulate metabolic pathways. These changes have been shown to modulate circadian clock function and cause clock resetting (Figure 4).

AMPK and mTOR are two master intracellular energy sensors that maintain energy homeostasis and proteostasis. They regulate the clock in response to feeding and fasting through several mechanisms (Figure 4). AMPK is an AMP/ADP-dependent protein kinase that is activated under glucose starvation and operates in a mTOR-dependent manner (Gwinn et al., 2008). AMPK was shown to target CK1ε and CRY1. CK1ε is activated by AMPK-mediated phosphorylation, resulting in enhanced phosphorylation and degradation of PER2. CRY1 is phosphorylated at S71 and S280 by AMPK, leading to its degradation (Um et al., 2007; Lamia et al., 2009). As AMPK activity is rhythmic, it functions in time-of-day-dependent resetting and synchronization of peripheral clocks (Lamia et al., 2009; Um et al., 2011).

mTOR regulates cell growth and proliferation through its activity as a serine/threonine kinase. It phosphorylates transcriptional repressors 4E-binding proteins (4E-BPs) and S6 kinase (S6K), which upregulate protein synthesis. Knock-down of mTOR in osteosarcoma cells, hepatocytes and adipocytes lengthens the cellular circadian period and reduces amplitude, while mTOR activation has the opposite effects (Ramanathan et al., 2018). mTOR affects clock function through posttranslational regulation of BMAL1 and CRY1 (Figure 4). First, S6K was shown to phosphorylate BMAL1 at Ser42, regulating the clock in two ways. BMAL1 phosphorylation destabilizes the protein-DNA interactions and causes its exclusion from the nucleus, thereby downregulating BMAL1/CLOCK transcriptional activity (Dang et al., 2016). Second, S6K was shown to modify BMAL1 in the cytoplasm to promote its involvement in global protein translation, thereby conferring circadian protein synthesis (Lipton et al., 2015). S6K activity on BMAL1 bridges the metabolic mTOR pathway and the circadian clock. Additionally, mTOR-mediated regulation of CRY1 degradation also regulates the clock. mTOR activation and subsequent autophagy inhibition lead to increased CRY1 protein levels, but not mRNA, indicating that that regulation likely takes place post-translationally (Ramanathan et al., 2018; Toledo et al., 2018). Therefore, mTOR and its downstream target take a multifaceted approach to regulating circadian transcription and translation.

In addition to being inhibited by AMPK, mTOR activity is also influenced by insulin and insulin-like growth factor-1 (IGF-1), which is elevated after meals (Levine et al., 2014) (Figure 4). Insulin and IGF-1 activate PI3K-catalyzed phosphorylation of PIP2–PIP3, which binds and activates AKT, an upstream effector and activator in the mTOR pathway (Crosby et al., 2019). Insulin and IGF-1 signaling was shown to upregulate PER2 protein synthesis through mTOR signaling (Crosby et al., 2019). Further, glucagon-CREB/CRTC2 signaling during fasting induces PER2 expression and PER2 acts to inhibit mTOR activity (Wu et al., 2019). The crosstalk between these signaling pathways provides mechanisms for fasting and feeding to reset the clock and ensure cell and tissue homeostasis.

The pentose phosphate pathway, which breaks down glucose to generate NADH and NADPH, creates another link between metabolism and timekeeping (Figure 4). Enzymes in this metabolic pathway help maintain redox homeostasis within cells. The pentose phosphate pathway regulates the clock through NADPH and the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) (Rey et al., 2016). The NRF2 gene contains an E-box element in the promoter region, and therefore is transcribed by BMAL1/CLOCK (Pekovic-Vaughan et al., 2014; Early et al., 2018). NRF2 in turn downregulates BMAL1/CLOCK by increasing NR1D1 and CRY2 expression (Fan et al., 2014; Wible et al., 2018). Inhibition of enzymes in the pentose phosphate pathway or glycolysis decreases cellular levels of NADH and NADPH and abolishes cellular redox rhythmicity (Ch et al., 2021). Furthermore, genetic perturbation of the folate pathway, which is critical for DNA synthesis and protein metabolism, has also been shown to alter the clock (Zhang et al., 2009). The interplay between circadian and metabolic pathways illustrate that the clock is deeply integrated with cell metabolism.

The NAD⁺-dependent protein deacetylase, sirtuin 1 (SIRT1), likewise plays an important role in circadian food-responsive entrainment in peripheral clocks (Figure 4). SIRT1 is upregulated in response to increasing NAD⁺ levels in times of energy stress and nutrient deprivation, and therefore is activated during times of fasting. Overexpression of SIRT1 has been shown to increase physical activity and lower total cholesterol, while SIRT1 deficiency increases blood glucose levels and ROS production, indicating that SIRT1 activation elicits phenotypes beneficial to overall health (Bordone et al., 2007; Wang et al., 2011).

In addition to regulation by cellular NAD⁺/NADH levels, SIRT1 expression and enzymatic activity are also clock-regulated and rhythmic (Nakahata et al., 2008; Zhou et al., 2014). Circadian regulation of SIRT1 in the liver and other peripheral tissues is important for rhythmic control of insulin sensitivity, a major marker for Type 2 Diabetes (Sun et al., 2007; Li et al., 2011; Zhou et al., 2014; Liu et al., 2016). BMAL1/CLOCK regulates SIRT1 expression by binding E-box elements in the promoter and activating transcription (Zhou et al., 2014; Liu et al., 2016). Thereafter, SIRT1 binds directly to BMAL1/CLOCK to drive expression of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme that catalyzes the rate-limiting step in NAD⁺ recycling (Nakahata et al., 2009). Interestingly, AMPK is necessary for NAMPT activation, suggesting that AMPK also modifies clock timing in part through regulation of cellular NAD⁺ levels (Um et al., 2011). Furthermore, SIRT1 was found to regulate AMPK and mTOR activation, highlighting the bidirectional relationship of many metabolic pathways and their integration with the clock mechanism (Wang et al., 2011; Price et al., 2012).

SIRT1 reciprocates this circadian regulation through its protein deacetylation activity and direct interactions with BMAL1, CLOCK, and PER2 (Asher et al., 2008; Nakahata et al., 2008; Wang et al., 2016) (Figure 4). SIRT1 binds and deacetylates BMAL1 and PER2, with PER2 deacetylation and subsequent degradation being the primary role of SIRT1 in regulating the circadian clock (Asher et al., 2008; Nakahata et al., 2008). This activity is required for oscillations in BMAL1, DBP, and PER2 expression (Asher et al., 2008; Nakahata et al., 2008; Foteinou et al., 2018). Nakahata et al. also found that SIRT1 is recruited to the DBP promoter E-box in a chromatin complex with BMAL1/CLOCK, indicating that it may regulate BMAL1/CLOCK-driven DBP expression through histone deacetylation in the DBP promoter (Nakahata et al., 2008). These data suggest that SIRT1 regulates the clock not only at the post-translational level but also through histone modification and epigenetic control.

SIRT1 can also indirectly modify clock gene expression through deacetylation and activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) (Nemoto et al., 2005) (Figure 4). The primary function of PGC-1α is to drive mitochondrial biogenesis in response to low cellular energy levels (Than et al., 2011). In myotubes and hepatocytes, PGC-1α activation has also been shown to elevate BMAL1 and CLOCK mRNA and protein levels through activation of RORα/γ (Liu C. et al., 2007). PGC-1α knock-out in the liver and skeletal muscle attenuates rhythmic expression of CLOCK and PER1, as well as genes, such as pyruvate dehydrogenase kinase 4, that are essential for metabolism (Liu C. et al., 2007). This suggests that PGC-1α is required for circadian regulation of metabolism and SIRT1 appears to be an important regulator in peripheral clocks (Chang and Guarente, 2013).

Similar to SIRT1, poly (ADP-ribose) polymerase-1 (PARP-1) is another NAD⁺ sensor that modulates circadian clock protein activity in response to nutrient depletion. PARP-1 is a versatile enzyme that post-translationally modifies other proteins to facilitate DNA damage repair (Ray Chaudhuri and Nussenzweig, 2017). Its activity in the liver oscillates with a circadian rhythm and can respond to daily changes in cellular NAD⁺ levels. PARP-1 activity peaks at the beginning of the light phase between ZT0 and ZT4 in mice. It binds and poly (ADP-ribosyl)ates CLOCK, disrupting interactions between BMAL1/CLOCK and DNA, subsequently inhibiting BMAL1/CLOCK transcriptional activity and downregulating target gene expression (Asher et al., 2010) (Figure 4).

Hormonal factors such as insulin, IGF-1, glucagon and GCs, and these intracellular nutrient sensors work together to facilitate entrainment of peripheral clocks and align them to the feeding/fasting cycle. Because feeding and fasting are able to entrain peripheral clocks but not the SCN, it is important to be mindful of mealtimes in order to keep peripheral clocks in phase with the SCN (Hamaguchi et al., 2015). Generally, fasting upregulates PER/CRY transcription through increased BMAL1/CLOCK activity, but the regulatory mechanisms are complicated and many are intertwined. Moreover, mouse studies have shown that clock changes in response to feeding cues are tissue-specific and can vary drastically (Manella et al., 2021). Clock regulation by metabolic pathways occurs at the transcriptional, translational, and post-translational levels. Likewise, clock regulation also occurs at the intracellular, intercellular, and inter-tissue levels. It is likely that there are more biochemical pathways that alter the clock than what is discussed here, and future studies should explore these mechanisms.

Physical activity and other types of physiological stress serve as zeitgebers for peripheral clocks and employ many of the same signaling pathways as fasting (Figure 5). Notably, exercise is able to restore dampened rhythms in peripheral clocks caused by SCN lesion, highlighting its strength in resetting the clock (Sasaki et al., 2016a). Expression of core clock genes BMAL1, CLOCK, PER1/2, and CRY1/2 is activity-dependent in skeletal muscle and can be modulated through several pathways (Dyar et al., 2015).

Like fasting, physical activity uses GC signaling to synchronize peripheral tissues (Figure 5). In mice, forced exercise has been shown to cause phase shifts in the kidney, liver, and submandibular glands by activating the sympathetic nervous system and the hypothalamus-pituitary-adrenal gland axis (Sasaki et al., 2016a). This exercise-induced phase shift was associated with increases in GCs, epinephrine, and norepinephrine levels, emphasizing the importance of stress response in entrainment of peripheral clocks. In fact, even without exercise, increasing levels of epinephrine alone is sufficient to induce Per1 expression in the liver and restore damped oscillations of Bmal1 and Per2 in SCN-lesioned mice (Terazono et al., 2003).

While the SCN utilizes adrenergic signaling to synchronize peripheral clocks through activation of p38, this pathway can also be activated by exercise and cause phase shifts (Goldsmith and Bell-Pedersen, 2013). Exercise induces secretion of epinephrine and norepinephrine, which activates p38 in a β2AR-dependent manner (Proll et al., 1993; Zheng et al., 2000) (Figure 5). Epinephrine and norepinephrine bind to adrenergic receptors, including the β₂ adrenergic receptor (β₂AR; Proll et al., 1993; Zheng et al., 2000). These receptors activate p38, a MAP kinase, in a PKA-dependent manner (Hu et al., 2021). p38 has been shown to activate MEF, which drives transcription of PGC-1α, initiating a positive feedback loop that continues to upregulate PGC-1α expression (Baar et al., 2002; Pilegaard et al., 2003; Akimoto et al., 2005; Miura et al., 2007). PGC-1α is normally used to stimulate mitochondrial biogenesis through CRTC3 in times of energy stress (Than et al., 2011). Upon activation, PGC-1α also elevates BMAL1 and CLOCK expression through RORα/γ, as discussed earlier (Figure 4).

Additionally, the p38 signaling cascade is codependent on nuclear factor of activated T-cells (NFAT) proteins, a family of transcription factors that act as nerve activity sensors in skeletal muscle (McCullagh et al., 2004; Tothova et al., 2006). NFATc1, which is normally phosphorylated and located in the cytosol, is dephosphorylated in response to electrical stimulation and subsequently translocated to the nucleus (Liu et al., 2001; Tothova et al., 2006). Pathway enrichment analysis has shown that NFAT transcription factors upregulate circadian pathways in muscle tissue through CREB and p38 (Dyar et al., 2015). Perturbation of NFAT signaling was shown to affect cell- and tissue-autonomous circadian clock function (Lee et al., 2019). These pathways provide a mechanism by which physical activity resets the clock through GC and epinephrine signaling and electrical stimulation.

Muscle contraction that occurs during exercise resets the circadian clock in muscle tissue in a manner similar to light-resetting in the SCN (Figure 5). Exercise increases intracellular levels of cAMP and stimulates activation of IP3, which binds to calcium channels in the ER to trigger calcium release into the cytosol (Dunbar and Kalinski, 1994; Berdeaux and Stewart, 2012). The calcium-dependent CaMK/CREB pathway is activated in skeletal muscle, resulting in increased PER2 expression (Small et al., 2020). This pathway is also activated in fibroblasts in response to depleted cAMP following activation of p38 and PKA (Delghandi et al., 2005). In cardiomyocytes, the exercise-induced increase in cytosolic Ca²⁺ levels leads to activation of CAMKII, which then activates MEF2 (Duran et al., 2017; Rodrigues et al., 2018). MEF2 can also be activated by p38, so it is possible that these pathways work synergistically in various tissues to reset peripheral clocks (Yang et al., 1999). While CaMK/CREB signaling has been shown to contribute significantly to clock resetting in skeletal and cardiac muscle, future studies should investigate the role of these pathways in other tissues. For example, exercise-induced parathyroid hormone (PTH) secretion can activate PKA by increasing intracellular cAMP and Ca²⁺ levels through binding of PTH receptor 1, which is highly expressed in the bone and kidney (Abou-Samra et al., 1992; Aghaloo et al., 2006; Tong et al., 2017). Activation of this pathway has been shown to induce Per1/2 transcription activation in chondrocytes, but future studies should determine whether this is sufficient to cause phase shifts in clock gene expression (Hinoi et al., 2006). Additionally, it would be interesting to determine whether the same effect is seen in other tissues, such as kidney and bone.

In addition to inducing epinephrine and GC secretion, exercise also increases cellular energy demand. Lower cellular levels of ATP and NADH activate AMPK and SIRT1 pathways, as discussed (Figure 4). Energy demand is also compensated by upregulation of cellular respiration. Oxidative phosphorylation elevates production of reactive oxygen species (ROS), which cause oxidative stress in the cell and are eliminated following activation of NRF2 (Turrens, 2003; Shadel and Horvath, 2015; Done et al., 2017). NRF2 is a transcription factor that represses inflammation by upregulating expression of genes containing antioxidant response elements. A major transcriptional target of NRF2 is glutathione S-transferase (GSTA4), an enzyme that disposes of ROS by catalyzing their conjugation to reduced glutathione. Besides serving its cytoprotective role, NRF2 also modifies clock gene expression and rhythmicity by upregulating NR1D1 and CRY2 expression (Wible et al., 2018) (Figure 5). Interestingly, NRF2 expression and activity are regulated by BMAL1 and CK2, respectively, and antioxidant gene peroxiredoxin-6 expression is upregulated by BMAL1, so the clock plays an important part in maintenance of ROS homeostasis in tissues (Kondratov et al., 2006; Early et al., 2018; Chhunchha et al., 2020; Jang et al., 2020).

Besides causing irreversible damage to DNA, at high levels ROS can reset the clock independently of NRF2. The transcription factor heat shock factor 1 (HSF1) is recruited to BMAL/CLOCK. Hyperphosphorylated HSF1 accumulates in the nucleus and binds promoters of heat-shock genes in a circadian manner (Reinke et al., 2008). The BMAL1/CLOCK/HSF1 complex is subsequently phosphorylated by CK2 at BMAL1-S90 and HSF1-T142 in order to promote transcription of PER2 (Kornmann et al., 2007; Tamaru et al., 2013) (Figure 3). Redox conditions also alter BMAL1/CLOCK activity through its transcriptional coactivator p300, which acetylates CCG loci and activates BMAL1/CLOCK in a redox-dependent manner (Rey et al., 2016). Therefore, generation of ROS ties exercise and oxidative phosphorylation to the circadian clock.

The increase in oxidative phosphorylation to keep up with the energy demand in response to physical activity also creates hypoxic conditions, which induce expression of hypoxia-inducible factor 1α (HIF1α). HIF1α activates AMPK, an inhibitor of mTOR, thereby slowing cellular growth and proliferation, similar to the response to fasting conditions. Inhibition of mTOR prevents phosphorylation of BMAL1, causing it to remain localized in the nucleus for a longer period of time and ultimately resulting in lengthening of the circadian period and amplitude reduction (Ramanathan et al., 2018). HIF1α also binds directly to E-boxes in a BMAL1-dependent manner to drive transcription of clock genes including PER2 (Peek et al., 2017). HIF1α is targeted by BMAL1 and, conversely, PER2 is also targeted by HIF1α, which directly binds the HRE-containing PER2 promoter (Wu et al., 2017) (Figure 3). Stabilizing HIF1α/2α lengthens the period through induction of circadian gene expression, creating a bidirectional relationship between circadian and hypoxic signaling pathways (Peek et al., 2017). Taken together with the p38/NFAT pathways, exercise is able to reset the clock in many peripheral tissues through nutrient starvation/low energy conditions and glucocorticoid signaling.

The implications of clock reset are important to consider when scheduling exercise activities, but when is the best time of day to exercise? There is evidence to support the late afternoon (between 16:00 and 20:00) to be the time for optimal time to maximize performance (Chtourou and Souissi, 2012; Mirizio et al., 2020). In agreement with that, muscle strength appears to peak between 16:00 and 20:00 (Douglas et al., 2021). However, higher performance does not necessarily correlate with increased output of other benefits of exercise. Therefore, whether the afternoon is the best time of day to exercise is unclear. Regular exercise can improve sleep quality, decrease daytime fatigue, and improve cardiovascular and muscular endurance. Surprisingly, studies show that there is no difference in these improvements between people who exercise in the morning and those who exercise in the evening (Alley et al., 2015; Saidi et al., 2021). Notably, there is contrasting evidence regarding the best exercise schedule for weight loss and lowering blood pressure, which could be due to a number of factors discussed hereafter (Brito et al., 2015, 2019, 2021; Blankenship et al., 2021).

Many variables have brought about controversy in studies investigating the effects of exercise on the clock. First, some authors report that forced exercise causes phase shifts but voluntary exercise does not (Sasaki et al., 2016a). Therefore, the type of exercise must be justified. Second, duration and intensity vary between studies. While exercise duration can be easily controlled for, intensity is more difficult, especially in humans, because physical fitness can vary drastically between individuals (Lewis et al., 2018). Other variables that should be considered in animal studies to ensure reproducibility include room temperature and humidity, number of animals per cage, food availability, and training load progression (Garrigos et al., 2021).

Because the light/dark cycle is the dominant zeitgeber in SCN entrainment and setting of the sleep/wake cycle, light/dark disruption, as experienced in jet lag and shift work, and artificial light at night (LAN) can disrupt circadian rhythms by altering clock gene expression. A rise in use of electronics has caused an increase in humans’ exposure to LAN, which has been shown to cause phase shifts in the SCN of many mammals, including mice and humans, in a duration- and intensity-dependent manner (Kronauer et al., 2019). Mouse studies have shown that exposure to even dim light during the dark phase can alter Per and Cry expression in the SCN (Shuboni and Yan, 2010). Furthermore, as little as a single day of sleep disruption can alter chromatin accessibility and attenuate rhythmic clock gene expression in the cerebral cortex of mice (Hor et al., 2019). The behavioral phenotype suggested the mice had recovered within 48 h. However, gene expression perturbation persisted for up to 7 days later, demonstrating the long-term effects of acute sleep disruption.

While short-term exposure to LAN is sufficient to cause phase shifts and attenuate rhythms, chronic exposure can increase risk of serious diseases such as cancer, heart attack, and stroke (Brown et al., 2009; Lee S. et al., 2010; Arendt, 2010). This is especially problematic for night shift workers and those who experience chronic jet lag, such as flight attendants. Studies have shown that melatonin suppression, which occurs in response to LAN, promotes carcinogenesis, while melatonin treatment can attenuate tumor growth (Davis and Mirick, 2006). Shift work is classified as a carcinogen, and the risk of cancer, as well as heart disease, further increases with the time spent carrying out shift work (Schernhammer et al., 2001; Megdal et al., 2005; Davis and Mirick, 2006; Vetter et al., 2016; Yousef et al., 2020). When sleeping out of phase, many clock genes lose rhythmicity (Archer et al., 2014). This loss of clock gene expression is associated with poorer cancer prognosis, likely due to loss of PER2 rhythmicity specifically (Cadenas et al., 2014). PER2 is thought to act as a tumor suppressor, in part by stabilizing p53, but also through regulation of the cell cycle (Wood et al., 2008; Xiang et al., 2008; Gotoh et al., 2014; Farshadi et al., 2020). Downregulation of PER2 increases Cyclin D and Cyclin E levels and increases the rate of tumor growth in breast cancer, disrupts DNA damage repair pathways and results in increased tumor growth (Fu et al., 2002; Xiang et al., 2008; Yang et al., 2009). It is also possible that internal circadian misalignment causes cancer via genomic perturbation. This is supported by the fact that genomes of night shift workers are significantly hypomethylated compared to those of day shift workers (Bhatti et al., 2015). Therefore, shift work, light at night, and chronic jet lag may increase the risk of cancer and other diseases through several mechanisms, including rhythmic expression attenuation, downregulation of clock genes, and covalent modifications to DNA. Given that shift work comprises many essential jobs that cannot end with the typical workday, solutions should be identified to mitigate the effects and reduce the risks of chronic jet lag. Additionally, the association between shift work and cancer requires further investigation because there is also evidence that shift work does not significantly contribute to carcinogenesis (Yang et al., 2021).

In addition to environmental factors, heritable mutations can also disrupt sleep. This is illustrated by familial advanced sleep phase syndrome (FASPS), which is characterized by a significantly advanced shift in the daily sleep/wake cycle (Jones et al., 1999). Sleep duration is normal in FASPS, however, a PER2 mutation leading to decreased phosphorylation by CKIε causes a phase advance (Toh et al., 2001). Conversely, in familial delayed sleep phase syndrome (DSPS, a CKIε substitution mutation at Ser408 can increase the phosphorylation rate by 1.8-fold compared to wild type and delay the phase of sleep onset/offset (Takano et al., 2004). DSPS can also be attributed to a single point mutation that disrupts a splice site in CRY1. This leads to deletion of exon 11 (CRY1Δ11) and gives rise to a significantly longer circadian period (Patke et al., 2017). In addition to causing insomnia, CRY1Δ11 is also associated with attention-deficit/hyperactivity disorder (ADHD), major depressive disorder, and anxiety (Onat et al., 2020). Because DSPS is characterized by the resulting phenotype and not the underlying genetic perturbation, it can also be caused by a variation in the PER3 gene. The frequency of a shorter polymorphic repeat region in PER3 was found to be significantly higher in a population of DSPS subject group compared to controls (Archer et al., 2003). However, this association weakens with age (Jones et al., 2007). Despite the potential underlying differences behind DSPS, the preference for later wake time can affect quality of life, so it may be useful identifying genetic markers that predispose individuals to DSPS.

Mental health disorders can also have a significant impact on sleep homeostasis, and therefore clock function (Liberman et al., 2018). For example, sleep disruption is an early symptom of psychosis (Lynch et al., 2018). This may be due in part to N-methyl-D-aspartate receptor (NMDAR) dysfunction, which is associated with psychiatric disorders and suspected to impact sleep regulation (Wang et al., 2020). NMDA increases intracellular Ca²⁺ levels in the SCN, resetting the clock through activation of the Ca²⁺/cAMP-CREB signaling pathway (Schurov et al., 2002) (Figure 2). NMDAR antagonism has been shown to lengthen the circadian period, but that effect is reversed with melatonin treatment (Wang et al., 2020). Attention deficit hyperactivity disorder (ADHD) is also strongly associated with disruption of sleep and circadian rhythms, specifically BMAL1 and PER2 rhythms (Baird et al., 2012). Surprisingly however, rather than reversing these phenotypes, stimulants used to treat ADHD can worsen sleep disturbance and cause further phase shifts (Coogan et al., 2019). ADHD is thought to be caused by a reduction in dopaminergic signaling (Volkow et al., 2009). Likewise, depression, which is also associated with insomnia, has been attributed to dysregulation of dopaminergic signaling (Grace, 2016; Li et al., 2016). Therefore, ADHD, depression, and other mental health disorders may cause circadian disruptions due to decreased activation of the Ca²⁺/cAMP-CREB pathway, as dopamine has been shown to activate this signaling cascade (McNulty et al., 1998; Schurov et al., 2002). More research is needed to fully understand the interplay between psychological disorders and the sleep/wake cycle.

Unfortunately, aging is also associated with lower sleep quality. Specifically, sleep fragmentation and also impaired cognitive function have been attributed to aging (Yoon et al., 2003; Kaneshwaran et al., 2019). This sleep fragmentation is thought to alter circadian rhythms via Ca²⁺/cAMP-CREB signaling, as prevention of CREB phosphorylation by melatonin was shown to be attenuated in the SCN of aged mice (von Gall and Weaver, 2008). Nevertheless, more research is needed to fully understand sleep dysregulation and attenuation of circadian rhythms with aging. Notably, there is a bidirectional relationship between aging and sleep disruption. Chronic sleep deprivation has proven to be detrimental to long-term memory and to impair overall cognitive function, eventually leading to dementia (Lo et al., 2016; Cousins and Fernández, 2019; Zhang et al., 2019; Garcia-Aviles et al., 2021).

Bettering our understanding of how aging and other disease states affect the clock will be useful in identifying interventions that will lessen the detrimental effects on sleep and clock function. There are many factors, environmental or otherwise, that affect both sleep quality and quantity. However, it is important to minimize sleep disruption whenever possible to enhance circadian clock function and improve overall health.

Under normal conditions, the feeding/fasting cycle is aligned with the sleep/wake and light/dark cycles. However, mistimed meals can cause phase shifts in peripheral tissues that can have serious consequences on overall health. Asynchrony between the SCN and peripheral tissues, otherwise known as internal circadian misalignment, has been shown to lower glucose tolerance, cause a phase shift in cortisol secretion, increase the risk of obesity, and increase the overall levels of triglycerides (Marcheva et al., 2010; Morris et al., 2016; Grant et al., 2021). These physiological changes consequently increase the risk of obesity, diabetes, cardiovascular diseases, cancer, and other age-linked diseases (Davis and Mirick, 2006; Scheer et al., 2009; Haus and Smolensky, 2013; Shi et al., 2013; Gan et al., 2015).

Meals are a dominant zeitgeber for peripheral clocks, and mistimed meals can cause drastic phase shifts of up to 12 h or more (Damiola et al., 2000; Le Minh et al., 2001; Sujino et al., 2012; Wolff and Esser, 2012; Salgado-Delgado et al., 2013; Wehrens et al., 2017). Peripheral clocks are sensitive enough to changes that skipping breakfast for even 1 day can significantly alter clock gene expression (Jakubowicz et al., 2017). This is especially relevant to shift workers who are more likely to regularly have mistimed meals. The negative effects of restricted feeding (RF) during the sleep phase, also known as the inactive phase, have been well documented in rodent models. Disrupting the clock through RF can lower insulin sensitivity, reverse core body temperature cycling, and increase total caloric intake (Satoh et al., 2006; Bray et al., 2013; Zhou et al., 2014; Liu et al., 2016). In humans, eating at night has been shown to attenuate rhythms and decrease total sleep time (Morris et al., 2016; Grant et al., 2021). Even delaying meals by just a few hours can cause phase shifts in peripheral tissues (Wehrens et al., 2017).

Intermittent fasting (IF) is a habit that restricts meals to roughly 6–12 h per day. It has been found to provide numerous benefits in weight loss and anti-aging efforts, including increased insulin sensitivity, thereby reducing the risk of diabetes (Schenk et al., 2011). Further, in mice, time-restricted feeding was shown to lower fat accumulation, improve glucose tolerance, increase insulin sensitivity, and reduce inflammation (Chaix et al., 2014, 2019). However, in human studies, many participants restrict meals to the afternoons and evenings, delaying time cues that would normally reset peripheral clocks in the morning. Rather than improving health, eating later dinners can increase the risk of obesity (Tahara and Shibata, 2013). Shifting the time of restricted feeding to start at the beginning of the active phase could allow participants to continue reaping the benefits of fasting without disrupting the circadian clock (Regmi et al., 2021). A recent clinical trial showed that early time-restricted feeding, a form of IF, improved insulin sensitivity, blood pressure, and oxidative stress levels without causing weight loss (Sutton et al., 2018). However, a study involving 24-h fasts after breakfast resulted in decreased expression of antioxidant enzymes, indicating that there can be adverse effects to IF, possibly due to fasting for too long (Liu et al., 2021). Finally, there is contradictory evidence regarding the effects of weight loss in response to time-restricted feeding in humans (Adafer et al., 2020). This may be due, at least in part, to the differences in populations that were evaluated, the length of time throughout the day when eating was permitted, and the duration of the studies. Therefore, more research is needed to optimize the timing and nutritional value of diets that provide the best outcomes in disease prevention and anti-aging. There are also many factors that prevent participation in IF, including cost, perceived health benefits, and wake/sleep time, that must be addressed before IF can be implemented on a larger scale (Jefcoate et al., 2021; Veronda and Irish, 2021).

In addition to timing, it’s also important to be mindful of the nutritional value of meals. Increasingly popular diets often require participants to severely restrict particular food groups. High-fat diets, such as the ketogenic diet, have proven to have many benefits. For example, the ketogenic diet was effective in treating epilepsy and Type 2 Diabetes in young and middle-aged adults (Napoleão et al., 2021). While ketosis-inducing diets can improve metabolic and other syndromes, they can have negative effects on the circadian clock. For example, a high-fat diet has been shown to lengthen the free-running period in mice as well as attenuate rhythms of plasma insulin, in part through inhibition of AMPK and pentose phosphate pathway enzymes (Kohsaka et al., 2007; Stucchi et al., 2012). Further, obesity induced by a long-term high-fat diet was shown to increase the expression levels of core clock genes Bmal1, Dbp, and CK1ε in mouse livers and Bmal1, Clock, Per1/2/3, Cry1/2, Dbp, and CK1ε in the kidneys (Hsieh et al., 2010). On the other hand, a balanced diet containing carbs, proteins, lipids, and vitamins/minerals was shown to be important for liver entrainment and sufficient to cause phase shifts (Hirao et al., 2009). Furthermore, caloric restriction was shown to activate SIRT1, which is known to have many benefits, including lower cholesterol, lower plasma glucose levels, and increased insulin sensitivity (Bordone et al., 2007; Schenk et al., 2011). Therefore, it is important to balance nutritional intake with physiological requirements.

Scheduling meals and drug administration for appropriate times can help peripheral clocks stay synchronized to the SCN. Preventing circadian misalignment is important for maintaining overall health and maximizing physiological outputs such as sleep and muscle strength (Leatherwood and Dragoo, 2013; Morris et al., 2016; Song et al., 2017; Aoyama et al., 2021). Circadian misalignment has also been shown to accelerate the progression of various diseases and therefore should be minimized whenever possible (Tahara and Shibata, 2016). Because of the crosstalk between metabolic pathways and the circadian clock, chrononutrition shows high potential for prevention and treatment of disease states (Barrea et al., 2021).

Timing of physical activity is important in regulating the circadian clock, as the direction in which exercise-induced phase shift occurs is time-of-day-dependent. Exercise sessions in the early evening (before melatonin onset) have been shown to cause significant phase advance, while bed rest and exercise late at night can cause phase delays in healthy adult men (Buxton et al., 2003). Timing of exercise has also been shown to affect which metabolic pathways are activated in response to activity. For example, forced acute exercise in the early rest phase was shown to stimulate carbohydrate metabolism in the skeletal muscle of mice. In contrast, forced exercise in the early active phase stimulated metabolism of lipids, amino acids, and ketones, as well as activated the HIF-1a signaling cascade (Sato et al., 2019). However, while exercise can be a strong zeitgeber for some peripheral clocks, it does not appear to be as effective for entrainment as meal times. In fact, exercise-induced phase shifts in peripheral tissue can be dependent on meal times (Sasaki et al., 2016b). Because physical activity is a less dominant zeitgeber than feeding, scheduled exercise cannot effectively entrain peripheral clocks if it is not in phase with meal times. In summary, physical activity can either couple peripheral clocks to the SCN or cause desynchronization. This affects not only shift workers, but also anyone who exercises late at night.