Circadian clock system : the perturbation of pancreatic cellular clocks promote the onset of diabetes


The circadian clock system (from the Latin “circa diem,” about a day) allows organisms to anticipate periodic changes of geophysical time and to adjust to those changes.

Nearly all cells comprise molecular clocks that regulate and synchronize metabolic functions to a 24-hour cycle of day-night changes.

Today, increasing evidence show that disturbances in internal clocks stemming from frequent time zone changes, irregular work schedules and aging have a significant impact on the development of metabolic diseases in human beings, including type-2 diabetes.

Such disturbances seem to prevent the proper functioning of the cells in the pancreatic islet that secrete insulin and glucagon, the hormones that regulate blood sugar levels.

By comparing the pancreatic cells of type 2 diabetic human donors with those of healthy people, researchers at the University of Geneva (UNIGE) and at the University Hospitals of Geneva (HUG), Switzerland, were able to demonstrate, for the first time, that the pancreatic islet cells derived from the Type 2 Diabetic human donors bear compromised circadian oscillators.

The disruption of the circadian clocks was concomitant with the perturbation of hormone secretion. Moreover, using clock modulator molecule dubbed Nobiletin, extracted from lemon peel, the researchers succeeded in “repairing” the disrupted cellular clocks and in partial restoring of the islet cell function.

These results, published in the Proceedings of the National Academy of Sciences, provide a first insight into innovative approach for diabetes care.

Two years ago, the team led by Charna Dibner, principal investigator at UNIGE Faculty of Medicine, has already shown that in rodents, the perturbation of pancreatic cellular clocks led to disrupted insulin and glucagon secretion, thus promoting the onset of diabetes.

But what is the situation in human beings?

Volodymyr Petrenko, a researcher in Dr. Dibner’s lab and the first author of these publications, says, “We had also previously observed that if the clocks of human pancreatic cells were artificially disrupted in the cellular culture in vitro, secretion of the key islet hormones – insulin and glucagon – was compromised.

Hence, our next step, that we report here, was to unravel whether the circadian rhythms were perturbed in human pancreatic islets in type 2 diabetes, and, if so, how would this perturbation affect the islet function.”

Using combined bioluminescence-fluorescence time-lapse microscopy, a technology that allows tracking the molecular clock activity in living cells very precisely over time, the scientists compared the behavior of pancreatic cell of type 2 diabetic donors and those of healthy subjects throughout the day.

“The verdict is indisputable,” says Charna Dibner. The biological rhythms of the islet cells in type-2 diabetes exhibit both reduced amplitudes of circadian oscillations and poor synchronization capacity.

“As a result, hormone secretion is no longer coordinated. Moreover, the defects in temporal coordination of insulin and glucagon secretion observed in patients with type-2 diabetes were comparable to those measured in healthy islet cells with artificially-disrupted circadian clock.”

It’s all in the timing

Circadian clocks represent the daily cycles governing the various cellular functions. There are several interlocking levels of synchronization of these clocks, the main one being light, which in particular regulates the central clock located in the cerebral hypothalamus.

Like a conductor in the orchestra, it regulates peripheral clocks present in organs and cells. The latter are therefore partly centrally regulated, but function differently in each organ, and even in each cell, depending on their functions.

“Pancreatic cells are also subject to the rhythm of fasting and food intake, and to a tight hormonal regulation,” says Charna Dibner.

“Coordinating all levels of regulation therefore allows the optimization of metabolic functions. Clock deregulation in pancreatic islet leads to a compromised function: They stop anticipating food-derived signals.

Indeed, if you eat the same food, but at night rather than during the day, you may gain weight much faster due to a suboptimal response of your metabolism.”

Setting the right time again

Step two of their research: the Geneva scientists used Nobiletin, a small clock modulator molecule and a natural ingredient of lemon peel, in order to resynchronize the clocks.

“By acting on one of the core-clock components, it efficiently resets the amplitude of the oscillations in the human islets” says Volodymyr Petrenko.

“And as soon as we got the clocks back in sync, we also observed an improvement in insulin secretion.”

“This is the first proof of principle that repairing compromised circadian clocks may help improve the function of pancreatic islet hormone secretion,” says Charna Dibner.

“We will continue by exploring this repair mechanism in vivo, first in animal models. Our society has experienced epidemic growth in metabolic diseases, concomitant with shifted working and eating schedules, and lack of sleep.

By re-synchronizing the perturbed molecular clocks, either by personalized eating and exercise schedules or with the help of clock modulator molecules, we hope to ultimately be able to provide an innovative solution to an epidemic metabolic problem affecting an ever-increasing proportion of the world’s population.”

Circadian rhythms (CR) are endogenous autonomous oscillators of physiological activities resulting 24-h day/night cycles, which allow organisms to adapt to a fluctuating environment (Reppert and Weaver, 2002Dibner et al., 2010).

The core pacemaker of CR lies in the SCN, which plays crucial roles in maintenance of systemic CR and regulates peripheral tissue clocks through secretion of endogenous regulatory factors (Dibner et al., 2010).

The molecular clock of the CR system, which is present in all cells, is made up of oscillating clock-related proteins that compose TTFLs (Gekakis et al., 1998).

The core TTFL is composed of the transcriptional activator proteins CLOCK and BMAL1, and the repressor proteins Period-1 (PER1), PER2, PER3, Cryptochrome-1 (CRY1) and CRY2 (Gekakis et al., 1998).

Other loops are coupled to the core TTFL to maintain oscillation. The first sub-loop is composed of RORs and nuclear REV-ERB receptors.

The second sub-loop comprises D-box-related genes, which include DBP, TEF, and HLF (Figure 1Preitner et al., 2002Sato et al., 2004).

Moreover, recent studies have suggested that circadian regulation is heavily involved in gene expression. A considerable portion (approximately 10%) of genes expressed in cells or tissues have been found to display circadian oscillations, resulting in identification of these genes as “CCGs” (Duffield, 2003Zhang et al., 2014).

Figure 1. The molecular mechanism of circadian rhythms. CLOCK and BMAL1 activate the cis-acting element E-box to initiate the transcription of downstream genes such as Pers and Crys, while the accumulated PER and CRY proteins, in turn, bind to CLOCK/BMAL1 and switch them from an activated state to an inhibited state, suppressing the transcriptional activation of downstream genes. ROR/REV-ERB and DBP (TEF, HLF)/E4BP4, acting on other cis-acting elements such as RORE and D-box, participating in the regulation of the core feedback loop. CCGs refer to the clock-controlled genes. The circles represent proteins, the squares represent genes or clock-related elements, the red arrows represent transcriptional activation, and the orange lines with horizontal bars represent transcriptional

Mammalian tissues and cells have an autonomous circadian oscillator with a period of roughly 24 h.

External stimuli are essential for maintaining the appropriate circadian oscillations (Bass and Takahashi, 2010Roenneberg and Merrow, 2016Tahara et al., 2017). 

In vivo, CR is mainly entrained by environmental signals such as light, food, and arousal stimuli. In the SCN, the circadian clock mainly responds to the LD cycle.

In peripheral tissues, CR can be synchronized by food or temperature (Damiola et al., 2000Hara et al., 2001Buhr et al., 2010).

Moreover, internal signals such as circulating hormones, cytokines, metabolites, sympathetic nervous activation, and body temperature are significant timing cues that regulate peripheral clocks (Albrecht, 2012Menaker et al., 2013). I

n vitro, CR are difficult to observe due to lack of SCN signals. As such, external stimuli should be applied to induce CR in cultured cells or explants (Figure 2).

Figure 2. Schematic summary of in vivo and in vitro circadian synchronization. In vivo, the photic zeitgeber mainly entrains the central clock, which regulates the peripheral clocks through the internal timing cues including autonomic innervations, endocrine signaling and body temperature; the non-photic zeitgebers including arousal stimuli, temperature and food mainly entrain the peripheral clocks. In vitro, the circadian oscillations of cells or explants can be synchronized by temperature cycles, chemical factors (such as Dex, Fsk, or horse serum) and mechanical stimuli.

The field of chronobiology requires detection of features of CR in tissues or cells. Through chronological collection or luminescence monitoring, the basic data resulting from circadian fluctuations in vivo or in vitro can be accurately recorded. Approaches such as FFT (Moore et al., 2014), JTK-Cycle (Hughes et al., 2010), or autocorrelation (Levine et al., 2002) can be used to analyze the rhythmic features of CR. Subsequently, we introduced several circadian databases established in recent years.

Finally, we summarized in vivo and in vitro factors that can alter CR. In vivo factors include entrainments such as light, food, and temperature, while the in vitro factors include cell density (Noguchi et al., 2013), osmotic pressure, PH, mechanical stimulus, temperature, oxygen concentration, and microorganisms (Haspel et al., 2014). These factors can lead to circadian disruptions, and result in various diseases such as cancer, dysplasia, and metabolic and behavioral disorders (Takahashi et al., 2008).

The Mammalian Circadian Clock System

The circadian clock system is the totality of all oscillators in organisms coupled to various physiological processes. This system generally consists of three parts in mammals including the input pathway, the core circadian clock, and the output pathway.

The input pathway senses external timing signals, for example, light/dark, and sends information to the core circadian clock.

The core circadian clock forms endogenous CR according to external time cues to allow for adaptation to the environment. Based on changes in the core circadian clock, the output pathway adjusts the physiological activities in various tissues and organs through neurohumoral regulation.

In mammals, the core pacemaker of the CR system exists in the SCN, which exhibits endogenous rhythmic oscillations both at the tissue and cell levels, plays a vital role in maintenance and alterations of CR, and provides outputs to peripheral tissues after synchronization by external time cues (Dibner et al., 2010).

The SCN can be distinguished into two main areas: the VL core area and the DM shell area (Welsh et al., 2010). The core area mainly expresses the neuropeptide VIP, while the shell area expresses AVP.

The VL-SCN, which controls essential physiological activities such as exercise, body temperature, heart rate, and hormone synthesis, serves to couple the circadian system. VIP is released periodically from the VL core region, binding to VPAC2 on the neuronal surface, resulting in cell depolarization and induction of PER1 and PER2 (Stepanyuk et al., 2014).

VIP deficiency impairs synchronization of the cells, leading to weakening of whole-body rhythms (Loh et al., 2011). AVP-deficient rats exhibit weakened rhythms, but do not experience changes in circadian pacemaking.

As such, the AVP-SCN is considered to function as the output (Mieda, 2019). In addition to VIP and AVP, other neurotransmitters such as glutamate and GABA, which are present in the SCN, conduce to regulate areas upstream and downstream of the circadian clock system (Morin et al., 2006).

The SCN has historically been considered to be the only endogenous circadian clock with autonomous oscillations, and peripheral clocks were not believed to spontaneously oscillate, but could oscillate under pacing by the SCN.

However, studies have demonstrated that the peripheral clocks also have the ability to oscillate autonomously and keep their internal rhythms (Balsalobre et al., 1998Yoo et al., 2004). The predominant theory is that the SCN, as the master pacemaker, can drive peripheral clock rhythms (Dibner et al., 2010).

Peripheral clocks are under the regulation of endogenous regulatory factors from the SCN. In an organized CR system, the connection between the SCN and peripheral tissues is a complex network, and the gene expression patterns of peripheral tissues are under the control of various complex factors including autonomic innervations, endocrine signaling, temperature, and local signals (Figure 2Mohawk et al., 2012).

Within cells, the CR system regulates cell biological behaviors either directly through the TTFLs or indirectly (Figure 1).

The core TTFL contains two transcription activators, CLOCK and BMAL1, which generate a heterodimer, bind to the cis-acting element E-box (5′-CACGTG-3′), and activates transcription of the PER and CRY genes at the beginning of the cycle (Gekakis et al., 1998).

After several hours, PER and CRY proteins accumulate, dimerize, and generate a complex which translocates to the nucleus and inhibits the CLOCK/BMAL1 heterodimer (Xu et al., 2015).

Other loops are coupled to the core TTFL to complete the oscillation. The first sub-loop comprises RORs and REV-ERB, which directly targets at CLOCK/BMAL1 or ROREs (Preitner et al., 2002Sato et al., 2004).

The second CLOCK/BMAL1-driven sub-loop contains the PAR-bZip factors DBP, TEF, and HLF. The repressor NFIL3 (or E4BP4), driven by the REV-ERB/ROR loop, interacts with these proteins at sites containing D-boxes (Mitsui et al., 2001Gachon et al., 2004Takahashi, 2017).

These three interlocking TTFLs form 24-h cycles of transcription with diverse expression phases, resting with the combination of cis-elements (E-box, RORE, D-box) in the promoters and enhancers of specific CCGs.

Circadian Rhythm Synchronizers

Nearly every eukaryote has a cell-autonomous circadian clock which exhibits 24-h physiological oscillations and can be influenced by external timing cues.

These external timing cues, also called “synchronizers,” “zeitgeber,” or ”entraining agents” can reset the body’s circadian clock and place all cells at the same phase of circadian oscillation, a process called circadian rhythm synchronization.

The word “ZT” is used to describe external cues that synchronize CR such as LD cycles or temperature cycles, and the word “CT” is utilized to describe timing without external signals (Li et al., 2017).

In the field of chronobiology research, whether in vivo or in vitro, researchers use these entraining agents to synchronize CR of the experimental animals or cells. A variety of factors which act as synchronizers are summarized in Figure 2.


Most organisms acquire the time information through changing light intensity throughout the day to reset their own clock, referred to as “photic entrainment” (Roenneberg and Foster, 1997).

In chronobiology experiments, light is often served as a stimulus to induce responses from the circadian clock. The light signaling cascades that entrain the circadian clock are fairly complex. 

In vivo, pRGCs receive light information, which is transmitted to the SCN directly through the RHT (Hattar et al., 2002Ishida et al., 2005Kalsbeek et al., 2006van Diepen et al., 2015Pilorz et al., 2016Mouland et al., 2017Astiz et al., 2019), resulting in regulation of the peripheral clocks by the SCN via secreting neurohumoral factors (Mohawk et al., 2012).

The light-sensitive photopigment melanopsin (Provencio et al., 2000), is expressed in the pRGCs which mediate a series of responses to light (Hankins et al., 2008Do and Yau, 2010Schmidt et al., 2011).

Stimulation of melanopsin causes activation of G protein coupled signaling cascades and the PLC pathway, leading to opening of TRPCs such as TRPC6 and TRPC7, Ca2+ influx, and cellular depolarization (Poletini et al., 2015).

The input pathway to the SCN mainly depends on the monosynaptic RHT. The primary neurotransmitters in the RHT are glutamate, PACAP, SP, and aspartate (Ebling, 1996Chen et al., 1999Hannibal, 2002Golombek et al., 2003Hannibal and Fahrenkrug, 2004Fahrenkrug, 2006).

Glutamate, which binds to glutamatergic receptors such as NMDA or AMPA receptors, is the main signal for photic entrainment (Mikkelsen et al., 1993Golombek and Ralph, 1996Chambille, 1999Peytevin et al., 2000Michel and Colwell, 2001Paul et al., 2005), and induces increased intracellular Ca2+ concentrations (Kim et al., 2005).

PACAP, which activates PAC1 receptor, is also responsible for mediating synchronization to light (Hannibal et al., 19982008Shen et al., 2000Colwell et al., 2004Hashimoto et al., 2006). Ca2+ influx activates a range of kinases including PKA, MAPK, CaMK, PKCA, and PKG (Meijer and Schwartz, 2003).

These kinases are involved in phosphorylation of CREB, which binds to cAMP response elements in promoters, resulting in transcription of clock genes such as PER1 and PER2 (Meijer and Schwartz, 2003).

Moreover, transcription factors such as c-FOS (Travnickova-Bendova et al., 2002) and EGR1 (Riedel et al., 2018) also participate in regulation of the SCN clock through optical signals.

Based on RHT innervation and the neurochemical natures of the cells, the SCN is distinguished into two main subdivisions: the VL area (core area) and the DM area (shell area) (Moore et al., 2002).

The VL-SCN, which contains VIP and gastrin-releasing peptide, is located above the optic chiasm, and receives most of its photic input from RHT innervation. In contrast, the DM-SCN region, where neurons contain AVP and calretinin, receives mostly neural signals from the hypothalamus, limbic areas, and the VL-SCN (Golombek and Rosenstein, 2010).

VL-SCN neurons respond to photic stimuli during the subjective night and communicate with DM-SCN neurons through several neurotransmitters such as VIP, GRP, and SP (Best et al., 1999Berson et al., 2002), resulting in synchronization of the DM-SCN to the expression of these proteins.

Therefore, DM-SCN neurons depend on neuropeptide release from the VL-SCN, although they show stronger autonomous oscillations (Moga and Moore, 1997Meijer and Schwartz, 2003Antle and Silver, 2005).

Arousal Stimuli

Other stimuli can synchronize CR, resulting in “non-photic entrainment.” Arousal stimuli are non-photic entrainments which include social interactions, exercise, restraint stress, and caffeine-induced arousal (Mistlberger and Skene, 2004).

Different species respond differently to arousal stimuli. For example, during the daytime, wheel running or gentle handling cause a robust phase advance in locomotor activity rhythms of Syrian hamsters, whereas these stimuli do not arouse a similar phase advance in the behavioral rhythms of mice or rats (Antle and Mistlberger, 2000Mistlberger et al., 2002).

However, phase advance in peripheral clocks such as liver and kidney can be induced by restraint stress or running wheel exercise during the intermediate of the light phase in mice (Tahara et al., 2015Sasaki et al., 2016).

These studies suggested that under LD cycles, peripheral tissues directly respond to behavioral entrainment without influencing the SCN. However, in constant darkness during projected daytime, behavioral rhythms of mice can also be observed under several-hour daily wheel stimulation (Mistlberger, 1991).

These results imply that the master clock in mice is primarily impacted by light rather than arousal stimuli.

A review by Golombek and Rosenstein (2010) showed that arousal stimuli transmit to the SCN through two major pathways. The GHT from the thalamic IGL, employs NPY, GABA, and endorphins as neurotransmitters, and plays a role in activation of the Y2 receptor/PKC pathway.

The other is the serotonergic median raphe nucleus projection to the SCN, where serotonergic effects are mediated by 5-HT1A/7 receptors and PKA activation (Golombek and Rosenstein, 2010). The mechanisms responsible for behavioral entrainment of peripheral clocks include physiological factors such as glucocorticoids, sympathetic nerves, oxidative stress, hypoxia, PH, cytokines, and temperature (Tahara and Shibata, 2018).


The CR of mammals can be synchronized by light, and thus the central clock for light response, the SCN, is considered a light-entrainable oscillator.

Food is a non-photic stimulus that can reset the circadian rhythm.

Many studies have confirmed the existence of a FEO, but the anatomical location(s) and molecular timekeeping mechanisms of the FEO have not been determined.

In the case of mammalian research, food is provided and consumed within a few hours. This condition is called time-restricted feeding, or temporal food restriction, and is frequently used to study FAA (Challet et al., 1998).

FAA refers to the output of the FEO, which appears under time-restricted feeding, but disappears under ad libitum access, and reoccurs during the following fasting (Pendergast and Yamazaki, 2018).

Mice allowed access to a standard diet ad libitum typically ingest 60–80% of their daily food intake at night.

Feeding-fasting signals reset the circadian clocks in the peripheral tissues by causing periodic availability of many circulating macronutrients (Woods, 2005).

For example, ingestion causes release of insulin into the blood and induces clock gene expression in insulin-sensitive tissues (such as liver, adipose, and muscle) (Oike et al., 2014). Feeding also enhances blood glucose levels, and high glucose concentrations can down-regulate the expression of PER1 and PER2 in fibroblasts (Hirota et al., 2010), and indirectly regulate AMPK, which controls the stability of CRYs (Lamia et al., 2009).

Recent evidence showed that the liver may be a communication center of the FAA synchronization signal. Under time-restricted feeding, the liver increased βOHB production through regulation of Cpt1a and Hmgcs2 by Per2, which was then used as a signal to cause animals to anticipate feeding time (Chavan et al., 2016).

In addition, food restriction may alter the anabolic/catabolic cycles of tissues, which may affect the cellular redox state (Rutter et al., 2002) and further influence the circadian feedback loop (Mendoza, 2007).

For instance, NADH and NADPH, classical cofactors for intracellular redox reactions, can promote binding of CLOCK/BMAL1 and NPAS2/BMAL1 dimers to DNA, while their oxidized forms, NAD(P)+, inhibit this binding (Rutter et al., 20012002Schibler and Naef, 2005). Furthermore, restricted feeding may influence body temperature cycles, which have been confirmed to entrain peripheral clocks (Brown et al., 2002).

Although the central oscillators are not affected by temporal food restriction during the light phase of the day (Damiola et al., 2000Stokkan et al., 2001), SCN-derived physiological rhythms such as locomotor activity and body temperature can be entrained by caloric restriction (also called hypocaloric diet, characterized by caloric intake reduction to 60% of the animal’s normal daily food intake) under LD cycles (Challet et al., 1997) or time-restricted feeding in constant darkness (Holmes and Mistlberger, 2000).

Although the mechanisms by which caloric restriction affects the SCN clock has not been characterized, previous studies suggested that receptors of metabolism-related hormones such as insulin, leptin, and ghrelin, which exist in SCN cells, may involve in synchronizing the SCN (Guan et al., 1997Zigman et al., 2006).

Moreover, feeding regulates brain structures that project straightly to the SCN such as the DM hypothalamus and the arcuate nucleus through orexin and ghrelin (Akiyama et al., 2004Mieda et al., 2004LeSauter et al., 2009Moriya et al., 2009Acosta-Galvan et al., 2011).

Moreover, glucose influences the neural activity phase in vitro SCN slices (Hall et al., 1997). Further researches are required to evaluate the involvement of these factors and their probable impacts on the SCN.


Temperature is a non-photic synchronizer with a weaker synchronization effect than that of light.

Roberto Refinetti found that only 60–80% mice can be synchronized by ambient temperature cycles, and stable entrainment takes longer in response to temperature cycles than LD cycles (Refinetti, 2010).

Though multiple physiological processes rely on ambient temperature, the CR system has a significant feature called temperature compensation, in which circadian oscillations remain resistant to temperature changes, resulting in the period length still maintaining approximately 24 h despite ambient temperature changes (Isojima et al., 2009Narasimamurthy and Virshup, 2017).

In addition, the SCN clock does not respond to temperature stimuli, whereas cells and tissues outside of the SCN can be synchronized by temperature fluctuations (Brown et al., 2002Buhr et al., 2010Ohnishi et al., 2014).

The reason why the SCN is resistant to temperature changes could be that the circadian clocks in the SCN cells have robust couplings. After uncoupling of SCN cells using tetrodotoxin or nimodipine, temperature sensitivity was detected (Buhr et al., 2010).

Because temperature is a weak synchronizer, it is not typically used as a zeitgeber in animal experiments.

The phase shift of the clock in response to simulated body temperature fluctuations has been demonstrated in vitro (Brown et al., 2002Buhr et al., 2010Saini et al., 2012Dudek et al., 2017), and the amplitude of circadian gene expression was enhanced by temperature cycles (Dibner et al., 2009Sporl et al., 2011Dudek et al., 2017).

Mammalian cells sense temperature fluctuations through a series of temperature-stimulated TRP channel subfamily members called thermo-TRPs, each of which is activated in a narrow thermal range (Poletini et al., 2015).

The intrinsic mechanism of temperature effects on CR may be mediated by HSF1 and CIRBPs (Ki et al., 2015). HSF1 is a circadian transcription factor which binds the heat shock element sequence, causing circadian activation of downstream promoters such as Per2 (Kornmann et al., 2007Reinke et al., 2008Tamaru et al., 2011). CIRBP-mediated post-transcriptional regulation allows high-amplitude clock genes express, including that of the core clock gene CLOCK (Ki et al., 2015).

Chemical Factors

In vitro experiments lack the humoral and neuronal factors that can act as entrainments in vivo. As such, cultured cells or explants are desynchronized and circadian oscillations are absent. Thus, to observe endogenous oscillations of cells or explants in vitro, chemical factors with similar effects to in vivo entrainments are required.

Many chemical factors can function as synchronizers. It has been reported that glucocorticoid, a kind of anti-inflammatory hormones released by the adrenal cortex, serves as a vigorous synchronizer of peripheral tissues (Balsalobre et al., 2000abYamamoto et al., 2005Segall et al., 2006So et al., 2009Cheon et al., 2013).

Dex, an artificial glucocorticoid, exposure for 1 h can be used to restart the oscillations of circadian clock genes (Balsalobre et al., 2000bWu et al., 2008). Glucocorticoids activate GRs, which bind to the GREs on the promoters of core clock genes such as Per1, Per2, and E4bp4, thereby directly activating the core clock cycle, or by activating the transcription factor HNF4alpha, which targets downstream rhythmic genes without GRE elements (Reddy et al., 2007Cheon et al., 2013). Dex cannot be used for SCN synchronization, because the SCN does not express GRs.

A study performed in 1998 indicated that serum shock could induce the rhythmic expression of PerlPer2Reverb-α, Dbp, and Tef in cultured rat fibroblasts (Balsalobre et al., 1998).

Thereafter, serum shock was used to synchronize the circadian oscillations of various tissues, immortalized cells, and fibroblast cells. This study suggested that various factors in the blood could stimulate rhythmic oscillations.

Previous reports showed that various factors such as such as EGF (Izumo et al., 2006), calcium (Balsalobre et al., 2000b), glucose (Hirota et al., 2002), PGE2 (Tsuchiya et al., 2005), 1α,25-dihydroxyvitamin D3 (Gutierrez-Monreal et al., 2014), and atomoxetine (O’Keeffe et al., 2012) can synchronize clock genes.

Moreover, serum shock can induce Ser/Thr phosphorylation of CLOCK through the Ca2+-dependent PKC pathway (Shim et al., 2007), or activate the p42/44 MAPK pathway in a manner similar to that observed in response to light pulses (Ginty et al., 1993Yagita and Okamura, 2000), which may cause resetting of the clock. In addition, a previous report showed that blood-borne signals activate GTPase RhoA, which promotes G-actin polymerization into F-actin, resulting in release of MRTFs into the nucleus, thus regulating the transcription of clock genes (Esnault et al., 2014).

Another common chemical synchronizing agent, Fsk, has a similar mechanism to that of serum. Fsk directly activates AC, which activates CREB through phosphorylation by promoting the synthesis of cAMP and activating PKA indirectly (Yagita and Okamura, 2000).

Mechanical Stimuli and Oxidative/ Hypoxia Stress

A recent study showed that intermittent uniaxial stretching of bone marrow-derived mesenchymal stem cells, DPSCs, and adipose tissue-derived mesenchymal stem cells can reset their CR, resulting in a synchronization effect similar to that observed in response to Dex treatment (Rogers et al., 2017).

Changing media also could reset cellular CR (Yeom et al., 2010Guenthner et al., 2014). Mechanical stimuli provide researchers with alternate mechanisms to reset the circadian clocks of cells, such as DPSCs, which cannot be synchronized by other methods. However, the mechanisms by which mechanical stimuli induced synchronization are still unclear.

It might be involved the RhoA pathway, by which short-duration fluid shear force can induce changes expression levels of clock genes in osteoblasts (Hamamura et al., 2012).

Oxidative or hypoxic stimuli may lead to circadian clock entrainment. In vitro, oxidative stimulation via hydrogen peroxide synchronizes cellular circadian oscillations in dose- and time-dependent manners (Tamaru et al., 2013). 

In vivo, phase shifts in peripheral clocks such as those in the kidney and liver are observed after hydrogen peroxide injection (Geerdink et al., 2016). However, whether the master clock responds to oxidative stress is unknown. Oxygen cycles (12-h 5%:12-h 8%) synchronize cellular clocks via a key transcription factor in cultured fibroblasts, HIF1α, which has similar a similar PAS domain to CLOCK and BMAL1 (Adamovich et al., 2017).

Furthermore, at the onset of a 6-h-shifted dark period, hypoxic stimulation (14% O2) for 2 h advanced phases of locomotor activities in response to a new LD cycle (Adamovich et al., 2017). These results suggested that both central and peripheral oscillations can be reset by hypoxic stimulation. Taken together, oxygen signals may induce circadian synchronization in vitro.

More information: Volodymyr Petrenko et al, In pancreatic islets from type 2 diabetes patients, the dampened circadian oscillators lead to reduced insulin and glucagon exocytosis, Proceedings of the National Academy of Sciences (2020). DOI: 10.1073/pnas.1916539117


Please enter your comment!
Please enter your name here

Questo sito usa Akismet per ridurre lo spam. Scopri come i tuoi dati vengono elaborati.