Review

doi: 10.1111/joim.12347

Circadian timing of metabolism in animal models and

humans
C. Dibner1 & U. Schibler2
From the 1Department of Endocrinology, Diabetes, Nutrition and Hypertension, University Hospital of Geneva; and 2Department of Molecular
Biology, Sciences III, University of Geneva, Geneva, Switzerland

Abstract. Dibner C, Schibler U (University Hospital of the hypothalamus, is synchronized every day to the
Geneva; and University of Geneva, Geneva, photoperiod. In turn, the SCN determines the
Switzerland). Circadian timing of metabolism in phase of the cellular clocks in peripheral organs
animal models and humans (Review). J Intern Med through a wide variety of signalling pathways
2015; 277: 513–527. dependent on feeding cycles, body temperature
rhythms, oscillating bloodborne signals and, in
Most living beings, including humans, must adapt some organs, inputs of the peripheral nervous
to rhythmically occurring daily changes in their system. A major purpose of circadian clocks in
environment that are generated by the Earth’s peripheral tissues is the temporal orchestration of
rotation. In the course of evolution, these organ- key metabolic processes, including food processing
isms have acquired an internal circadian timing (metabolism and xenobiotic detoxification). Here,
system that can anticipate environmental oscilla- we review some recent findings regarding the
tions and thereby govern their rhythmic physiology molecular and cellular composition of the circadian
in a proactive manner. In mammals, the circadian timing system and discuss its implications for the
timing system coordinates virtually all physiologi- temporal coordination of metabolism in health and
cal processes encompassing vigilance states, disease. We focus primarily on metabolic disorders
metabolism, endocrine functions and cardiovascu- such as obesity and type 2 diabetes, although
lar activity. Research performed during the past circadian misalignments (shiftwork or ‘social jet
two decades has established that almost every cell lag’) have also been associated with the aetiology of
in the body possesses its own circadian time- human malignancies.
keeper. The resulting clock network is organized
in a hierarchical manner. A master pacemaker, Keywords: circadian oscillator, glucose homeostasis,
located in the suprachiasmatic nucleus (SCN) of human peripheral clocks, metabolic diseases.

I do not understand’. According to this notion, we

Introduction: Molecular composition of circadian oscillators
are close to understanding the clockwork circuitry
Organisms belonging to phyla of all three domains in only one system, cyanobacteria. By simply
of life, Archaea, Bacteria and Eukaryota, harbour mixing the three clock proteins KaiA, KaiB and
endogenous timekeepers known as circadian KaiC with ATP, Takao Kondo and et al. succeeded
clocks. These time-measuring devices enable the in reassembling a molecular oscillator generating
organisms to adapt their behaviour and physiology 24-h rhythms of protein phosphorylation/dephos-
in a proactive manner to daily cycles of light phorylation during extended time periods in the
intensity and temperature generated by the Earth’s test tube [1]. However, none of the three Kai
rotation around its own axis. During the past two proteins of cyanobacteria bears any amino acid
decades, impressive progress has been made in the sequence similarity to the clock proteins of the
molecular and cellular understanding of circadian other above-mentioned biological systems, and it is
clocks in several biological systems, including therefore unlikely that a similar reductionist
cyanobacteria, fungi, green plants, insects and approach will be successful in the next few years.
mammals. Genetic and biochemical approaches In fact, even the circadian clocks of different
have revealed essential clock genes in all of these eukaryotic systems (fungi, plants and metazoans)
systems, and the number of these genes is cur- share little if any sequence homology and may thus
rently increasing. The physicist and Nobel Laure- have been generated independently by convergent
ate Richard Feynman stated: ‘What I cannot create, evolution. The only commonality between the

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 C. Dibner & U. Schibler Review: Circadian clocks in health and disease

known circadian oscillators of fungi, plants and TRAP150 [12] and SRC-2 [13]. The mRNA and
metazoans is the involvement of negative feedback protein levels of CRY and PER increase, and these
loops in gene expression. Those operative in mam- corepressors assemble into large heterotypic pro-
malian cells are shown schematically in Fig. 1. tein complexes that bind to the CLOCK–BMAL1
heterodimers. This results in the removal and
However, according to an alternative hypothesis, inactivation of the CLOCK–BMAL1 activator com-
there may be a common basic oscillator relying on plexes from the promoters and enhancers of the
metabolic feedback loops. Evidence for such an CRY (Cry) and PER (Per) genes and thereby in the
oscillator driving daily redox cycles of antioxidant repression of Cry and Per transcription. As a
peroxiredoxin proteins has recently been provided. consequence, the concentrations of CRY1/2 and
Circadian peroxiredoxin oxidation/reduction PER1/2 decrease until they can no longer inhibit
cycles that do not rely on canonical clock genes their own expression, and a new daily accumula-
have been observed in cyanobacteria, archaea, tion cycle of CRY–PER complexes can ensue. In a
algae, fungi, plants, roundworms, insects and secondary feedback loop, which involves the
mammals [2]. Remarkably, these circadian cycles nuclear orphan receptors REV-ERBa, REV-ERBb,
even operate in human and mouse red blood cells, RORa, RORb (only expressed in neurons) and
which are devoid of nuclei and ribosomes and thus RORc, BMAL1 and CLOCK control their own tem-
incapable of transcription and translation (Fig. 2) poral expression. Thus, the heterodimer CLOCK–
[3, 4]. It remains to be investigated whether or to BMAL1 binds to E-box promoter and enhancer
what extent the known circadian oscillators are elements with the Rev-erba and Rev-erbb genes,
coupled to the metabolic clock, the molecular and its cyclic activity leads to the circadian expres-
‘cogwheels’ of which have not yet been identified sion of REV-ERBa and REV-ERBb. The REV-ERB
(Fig. 1). repressor proteins compete with ROR-PGC-1a acti-
vator/coactivator complexes for the binding to
As mentioned above, the canonical molecular RORE elements within the Bmal1 and Clock genes
oscillator of mammals relies on feedback loops in and win this competition when they reach their
clock gene expression. These engage a complex highest circadian concentration. Once bound to
array of transcriptional and post-transcriptional RORE elements, they recruit the NCoR1–HDAC3
mechanisms that have been extensively reviewed corepressor complex and repress transcription of
elsewhere [5–7]. Therefore, here we will describe the Bmal1 and Clock genes [14]. The transcrip-
these mechanisms only to the degree required in tional mechanisms operative in the primary and
order to understand the following sections. First, it secondary feedback loops are assisted by a large
is worth noting that the major purpose of a number of post-translational mechanisms, includ-
molecular clock is to generate oscillations in the ing protein phosphorylation/dephosphorylation,
expression of clock-controlled output genes, which ubiquitination, sumoylation, acetylation/deacety-
eventually drive overt rhythms in physiology and lation and poly ADP-ribosylation. Some of these
behaviour. This has been accomplished by meta- post-translational protein modifications provide a
zoan evolution through the selection of complex link between circadian and metabolic cycles.
molecular interactions modulating high-amplitude Again, the complex interlocked gene expression
fluctuations in the activity of some key transcrip- circuitries outlined above have evolved for one
tion factors. The heterodimeric transcription fac- major purpose, namely the circadian expression of
tors CLOCK and BMAL1, which bind E-box motifs genes leading to overt cycles in physiology and
of hundreds of clock-controlled genes in a highly behaviour. As illustrated in Fig. 1, the transcrip-
rhythmic fashion, are the central drivers of the tion cycle depending on CLOCK–BMAL1/E-box
molecular oscillator [8, 9]. These transcriptional interactions (i.e. the primary feedback loop) runs
activator proteins regulate their cyclic activity and in antiphase with the transcription cycle driven by
balanced accumulation through two intercon- ROR/RORE interactions (i.e. the secondary feed-
nected feedback loops (Fig. 1). In the primary back loop). Therefore, the transcription cycles of
feedback loop, they stimulate the transcription of output genes controlled by CLOCK–BMAL1 and
the genes encoding the four corepressor proteins RORs display phase differences of ~180° (~12 h).
period 1 (PER1), period 2 (PER2), cryptochrome 1 Additional phases can be generated by clock out-
(CRY1) and cryptochrome 2 (CRY2) through the put regulators, such as PAR bZIP transcription
binding of E-box motifs and the recruitment of factors and E4BP4/NFIL3, whose rhythmic expres-
various coactivators, including CBP/p300 [10, 11], sion is governed by the primary and secondary

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Fig. 1 Mammalian core clock machinery. In mammals, the molecular oscillator circuitry is composed of two interlocked
negative feedback loops of gene expression. This canonical oscillator is known as the transcriptional translational feedback
loop (TTFL). The two transcription factors CLOCK (C) and BMAL1 (B) bind to E-box DNA motifs and recruit the coactivator
proteins CBP/p300 and a few other polypeptides (shown in light grey). This leads to the activation of the genes Per1, Per2,
Cry1 and Cry2 in the primary feedback loop, and Rev-erba and Rev-erbb in the secondary feedback loop. PER1, PER2,
CRY1 and CRY2 assemble into large repressor complexes containing many additional polypeptides. These repressor
complexes attenuate the transactivation potential of CLOCK and BMAL1, after having reached a threshold level of activity.
As a consequence, the level of PER and CRY repressor complexes diminishes until they no longer inhibit the activity of
CLOCK and BMAL1, and a new 24-h PER and CRY production cycle can be initiated. In a second feedback loop, CLOCK and
BMAL1 activate and PER/CRY complexes repress the transcription of the genes encoding the two negatively acting nuclear
orphan receptors REV-ERBa and REV-ERBb (REV). Circadian REV expression then results in the cyclic repression of the
Clock and Bmal1 genes. When REV concentrations are low, the positively acting nuclear orphan ROR receptors bind to
RORE elements within promoter and enhancer regions of these two genes and activate transcription by PGC1 coactivator
complexes. When REV concentrations are high, REVs compete with RORs for the binding to RORE elements and recruit
NCoR/HDAC3 corepressor complexes. Per1 and Per2 also serve as immediate early genes in the synchronization of the
molecular clockwork circuitry. Thus, their transcription can be stimulated by immediate early transcription factors whose
activity is controlled by systemic cues, such as hormones, second messengers, temperature and neurotransmitters. cAMP
responsive element (CRE)-binding protein (CREB), HSF1 binding to heat shock elements (HSEs), serum response factor (SRF)
binding to serum response elements (SREs) and glucocorticoid receptor (GR) binding to glucocorticoid responsive elements
(GREs) are amongst these immediate early transcription factors. The activation of Per1 and Per2 expression by these
systemically regulated transcription factors plays an important role in the phase resetting of circadian clocks. A redox
oscillator of unknown molecular composition drives circadian cycles of peroxiredoxin (PRX) oxidations. The schematic
diagram suggests that this redox oscillator is coupled to the TTFL, but this remains to be determined, adapted with changes
from [103] (Swiss Med Wkly. 2014 Jul 24;144:w13984. www.smw.ch.). Arrows and bars at the end of the connecting solid
and dashed lines indicate activation and repression of transcription, respectively.

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Fig. 2 Peripheral clocks and redox oscillators in humans. In humans, as in other mammals, virtually all cells harbour cell-
autonomous and self-sustained molecular oscillators (Fig. 1). The central clock in the suprachiasmatic nucleus (SCN)
synchronizes these peripheral oscillators by a plethora of neural and humoral signals (for details, see ref. [6] and references
therein). Circadian gene expression, in the form of luminescence cycles produced by luciferase reporter genes, has been
monitored in several human tissues, including the thyroid gland [150], pancreatic islets [20] and skin fibroblasts [145].
Moreover, circadian peroxiredoxin redox oscillations have been described in human erythrocytes, which are devoid of nuclei
and ribosomes and thus incapable of transcription and translation [4]. It is not yet clear whether these redox oscillations are
also synchronized by the SCN master pacemaker.

feedback loops, respectively, or by combinations of oscillator. Using circadian reporter genes encod-
transcription factors with different phases in activ- ing fluorescent proteins or firefly luciferase in
ity and/or accumulation [15, 16]. The generation of conjunction with highly sensitive fluorescence or
different phases in circadian gene expression is luminescence microscopy techniques, respectively,
essential, as different metabolic reactions and rhythmic gene expression can now be easily
other biological processes must be guided to dis- recorded in real time and in individual cells in
tinct time windows in order to optimize cellular and tissue culture [18–21]. It has been determined
organismic physiology. Indeed, one of the functions using these approaches that the period lengths
of biological clocks is to temporally isolate essential for individual cells can vary by several hours.
but chemically incompatible reactions. For exam- With such large variations, the phase coherence
ple, it makes sense to separate glycogen synthesis between cells and organs would be expected to
and glycogen utilization (phosphorolysis) in time, degenerate rapidly in the body if the network of
and the circadian clock helps to coordinate these cellular circadian oscillators was not periodically
antagonistic processes (ref. [17] and references synchronized. These phase adjustments are
therein). accomplished by rhythmic signals controlled by
the suprachiasmatic nucleus (SCN) located in the
ventral hypothalamus (Fig. 2). The SCN, also
Architecture of the circadian timing system: central and peripheral
known as the circadian master pacemaker, con-
oscillators
sists of two tiny clusters of cells, comprising about
As is clear from the above discussion, the molec- 100 000 neurons in humans and about 10 000
ular clockwork circuitry functions in a cell-auton- cells in mice [22]. It is commonly subdivided into
omous manner. In mammals, virtually every cell ventrolateral and dorsomedian areas, known as
harbours such a self-sustained and cell-autonomous the core and the shell, respectively. The SCN is

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composed of various cell types that can be charac- immediate early transcription factor CREB. The
terized by their expression of different neuropep- latter binds to promoter and enhancer sequences
tides, including vasopressin, vasoactive intestinal of Per genes and thereby elicits the rapid accumu-
peptide, gastrin-releasing peptide and neuromedin lation of PER1 and PER2. These corepressor pro-
S [23]. The molecular oscillators of individual SCN teins attenuate the transactivation potential of
neurons, like those of individual fibroblasts, pro- CLOCK–BMAL1 and thus delay or advance the
gressively desynchronize when kept in tissue cul- phase of circadian gene expression, depending on
ture. However, in organotypic brain slice cultures, whether the light has been provided during the first
in which the SCN tissue organization is conserved, or second half of the night, respectively. During
the cellular oscillators remain synchronized for subjective daytime, that is during the resting phase
weeks, due to strong intercellular coupling through of (nocturnal) laboratory rodents in constant dark-
synaptic and paracrine mechanisms [23]. This ness, the phase of the SCN pacemaker is not
explains why animals housed in the absence of susceptible to light [26, 27].
external timing cues (constant darkness and tem-
perature) display strongly rhythmic behaviour The SCN synchronizes the countless cellular clocks
throughout their lifespan. Under normal condi- in peripheral organs by employing multiple signal
tions, the SCN is synchronized daily to geophysical transduction pathways depending on feeding
time by the photoperiod (24-h light–dark cycle), cycles, oscillating hormones (e.g. glucocorticoids)
and also by social cues in humans and some other and body temperature rhythms (Fig. 2). Feeding
species [24]. Although how the SCN is affected by rhythms, which are driven by rest–activity cycles,
social cues is poorly understood, some of the are clearly the dominant zeitgebers (time givers) for
pathways involved in light-dependent phase most peripheral organs [28, 29]. If nocturnal mice
entrainment of the SCN have been elucidated. Of or rats are offered food exclusively during the day
note, a very small percentage of ganglion cells of (i.e. during their resting phase), the phase of
the inner retinal layer, known as intrinsically circadian gene expression in peripheral tissues
photosensitive ganglion cells (ipRGCs), are both progressively changes until it is completely
necessary and sufficient for photic phase entrain- inverted after 1 or 2 weeks. Because the SCN
ment of the SCN. These ganglion cells are directly master pacemaker is fairly resilient to feeding
connected to SCN neurons via the retinohypotha- rhythms and maintains its original phase, the
lamic tract. ipRGCs convey photic signals that are peripheral clocks become uncoupled from the
perceived by two completely different but function- SCN by inverted feeding rhythms. If food is again
ally redundant mechanisms to SCN neurons. First, provided ad libitum, the peripheral oscillators
ipRGCs are themselves photosensitive, due to the resume their original phase within 2 or 3 days. A
expression of the nonvisual photoreceptor mela- possible explanation for the slow phase inversion
nopsin. Secondly, they serve as a relay station following daytime feeding and the rapid phase re-
between a fraction of classical rod and cone pho- inversion after food is again freely available is as
toreceptors in the outer retinal layer and the SCN. follows. Under normal conditions (i.e. if food is
Due to this functional redundancy, melanopsin provided ad libitum or exclusively during the noc-
knockout mice with normal vision or blind mice turnal activity phase), the SCN synchronizes
lacking all classical rod and cone photoreceptors peripheral clocks indirectly via behaviour (i.e.
can synchronize the circadian clock to 24-h light– feeding rhythms depending on rest–activity cycles)
dark cycles. However, following genetic ablation of and more directly via hormones, the cyclic secre-
ipRGCs by the expression of the diphtheria toxin A tion of which remains roughly in phase with the
subunit from the melanopsin gene locus, mice are SCN. If conflicting feeding cycles are imposed (i.e.
no longer capable of synchronizing the SCN to daily daytime feeding for nocturnal animals), the SCN
light–dark cycles. These animals possess normal uses the more direct signals to counteract the
vision but free-run with an intrinsic period length indirect but dominant signals that depend on
of 23.8 h, irrespective of whether they are housed metabolic cycles. As soon as the conflicting feeding
in constant darkness or under 24-h light–dark rhythms are terminated, the SCN immediately
cycles [25]. The photic signals transmitted from resumes its hierarchical role as a master pace-
ipRGCs to the SCN via the retinohypothalamic maker and adjusts the phase of the peripheral
tract elicit an increase in Ca2+ in postsynaptic oscillators to normal within 2 or 3 days. Because of
neurons. This in turn triggers the activation of a recently developed in vivo luminescence record-
various kinases and, as a consequence, of the ing device, the RT-Biolumicorder, circadian gene

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expression in peripheral organs can now be mon- core clock components (for reviews see [51–54]).
itored in real time in freely moving mice. By For example, NADs, indicators of energy metabo-
determining the phase-shifting kinetics of circa- lism, can affect the activity of clock transcription
dian liver gene expression in intact and SCN- factors directly or via NAD-dependent enzymes.
lesioned mice, the synchronization of liver clocks Rutter et al. [55] were the first to provide compel-
by indirect feeding-dependent signals and more ling evidence for an NAD–clock connection by
direct SCN-dependent signals could be experimen- studying the impact of NAD cofactors on the
tally confirmed [30]. Glucocorticoids, the daily binding of CLOCK/NPAS2–BMAL1 heterodimers
plasma levels of which oscillate at least 10-fold in to their cognate E-box DNA motifs. In these
humans and laboratory rodents [31, 32], are biochemical experiments, the DNA binding of these
amongst the SCN-dependent signals involved in transcription factors strongly depended on the
the synchronization of peripheral clocks [33, 34]. ratio of reduced to oxidized NAD [NAD(P)H/NAD
By contrast, nicotinamide adenine dinucleotide (P)+]. The activation of PGC-1a by SIRT1-mediated
(NAD+)-sensing enzymes such as the poly (ADP- deacetylation boosts Bmal1 and Clock transcrip-
ribose) polymerase 1 (PARP-1) [35] and the protein tion in the SCN, and this enhances the amplitude
deacetylase sirtuin (SIRT)1 [36, 37] may serve as of circadian gene transcription in SCN neurons [56]
regulators for adjusting the phase of peripheral and, perhaps, in peripheral cell types [36]. As
clocks (i.e. hepatocyte clocks) to metabolic cycles. SIRT1 levels decrease with ageing, the amplitude of
Body temperature rhythms are perhaps the most circadian gene expression is also reduced [56]. This
unexpected zeitgebers for peripheral clocks [38– may offer a mechanistic explanation for the dete-
40]. The amplitude of body temperature fluctua- rioration of circadian behaviour and physiology in
tions is <1% in mice (minimum 308 °K to maximum old animals and humans. PARP-1, an NAD-depen-
311 °K) and humans (minimum 309 °K to maxi- dent enzyme that adds poly (ADP-ribose) residues
mum 310 °K). However, simulated murine body onto its substrate proteins, displays robust diurnal
temperature cycles imposed on cultured fibro- activity cycles, and CLOCK was identified as a
blasts efficiently synchronize their circadian PARP-1 target protein. The results of genetic loss-
clocks, and even human body temperature cycles of-function studies suggest that PARP-1 partici-
phase-entrain the clocks of about 20% of the pates in the synchronization of liver clocks by
cultured cells. Heat shock transcription factor 1 feeding cycles [35]. Hepatic NAD+ levels indeed
(HSF1) [39, 40] and, possibly, the cold-inducible oscillate during the day, and at least in part, this
RNA-binding protein CIRP have a role in the rhythm is driven by the circadian expression of
synchronization of circadian gene expression by nicotinamide phosphoribosyltransferase (NAMPT)
body temperature cycles. Nonetheless, few molec- [57]. In fact, NAMPT expression and intracellular
ular details are known about the signalling path- NAD+ concentrations follow a circadian rhythm
ways relevant for the synchronization of peripheral even in cultured fibroblasts maintained under
clocks in vivo (see ref. [41]). constant conditions, suggesting that the circadian
clock drives these oscillations [58]. In liver, NAD+
Circadian coordination of metabolism fluctuations, through rhythmically activating
SIRT3, also govern daily rhythms of oxidative
Interactions between circadian and metabolic cycles
phosphorylation in mitochondria [59].
The results of genomewide transcriptome profiling
studies and the observation that feeding–fasting Mutations in the essential clock genes Bmal1 and
cycles are dominant zeitgebers for the synchroni- Clock [60, 61] were found to cause various meta-
zation of peripheral oscillators indicate that a bolic disorders. Conversely, perturbations of met-
major purpose of circadian clocks in peripheral abolic pathways in mice fed a high-fat diet dampen
tissues is the temporal orchestration of food pro- the amplitude of circadian oscillations and
cessing (metabolism and xenobiotic detoxification). lengthen their period [62]. Moreover, the expres-
This conjecture is further supported by extensive sion of circadian output genes dramatically
metabolomic and lipidomic studies in laboratory changes in such animals, partly due to inhibition
rodents and humans, in which the levels of large of CLOCK–BMAL1 recruitment to chromatin and
numbers of metabolites were found to oscillate in activation of PPARc [63].
tissues, plasma and saliva [42–50]. Several recent
studies have demonstrated a strong relationship The results of genomewide transcriptome profiling
between metabolites and the activities of circadian studies performed in various tissues suggest that

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the clock orchestrates a large diversity of metabolic nisms in the control of clock output pathways and
processes in the body. In rodent models, the emphasize the critical role of feeding cycles in these
presence of peripheral circadian oscillators and processes. Quantitative mass spectrometric analy-
their impact on gene expression and organ func- sis of the liver circadian acetylome revealed the
tion have been demonstrated in liver (e.g. ref. [64, possible roles of lysine acetylation in tuning
65]), pancreatic islets [61, 66–68], skeletal muscle metabolism in the cytosol and mitochondria [93].
[69–71] and adipose tissue ([72–74]. A large num- Thus, enzymes involved in gluconeogenesis, gly-
ber of key metabolic functions are subject to daily colysis, citric acid cycle, amino acid metabolism
oscillations. These include carbohydrate and lipid and fatty acid metabolism exhibited strong circa-
metabolism, and xenobiotic detoxification by the dian accumulation patterns according to this
liver, kidney and small intestine [70, 75–77]. Of study. Aforementioned metabolomic studies con-
note, functional liver clocks contribute to glucose ducted during the past few years in tissues directly
homeostasis by driving a daily rhythm of hepatic verified the pervasiveness of diurnal metabolism.
glucose export [78]. Further evidence for the inter-
connection between circadian oscillators and
Circadian clockwork perturbations and metabolic defects
metabolism comes from several recent studies of
the nuclear receptor and core clock gene Rev-erba, Under homeostatic conditions, the clock acts as a
which was found to be crucial for proper lipid and driver of metabolic physiology. Perhaps not sur-
carbohydrate metabolism [76, 79–82]. Of interest, prisingly, perturbations of either the circadian or
Rev-erba has recently been shown to represent an metabolic system (e.g. behaviour misalignment,
important link between circadian clock and therm- shiftwork or high-fat diet) can result in the disrup-
ogenic networks, through the regulation of brown tion of metabolic pathways and circadian clock
adipose tissue function. Low levels (during the function (such as dampening of the amplitude and
early morning hours) or deletion of Rev-erba ren- increasing the period length under free-running
dered mice cold resistant, due to Rev-erba-induced conditions [62, 94]). Mice homozygous for domi-
repression of uncoupling protein 1 in brown adi- nant negative Clock alleles develop hyperphagia,
pose tissue in a cell-autonomous manner [83]. obesity and hyperglycaemia [62]. Similarly, mice
Moreover, nuclear receptors play a critical role in with an adipocyte-specific Bmal1 knockout become
controlling major metabolic networks in the liver, obese [95], and those with an islet-specific Bmal1
skeletal muscle and adipose tissue, allowing fine- ablation develop type 2 diabetes (T2D) [61].
tuning of the metabolic processes to feast and
famine cycles [79, 81, 84]. Of note, FGF family There is robust evidence for a strong reciprocal
proteins represent an important regulatory path association between a number of metabolic disor-
through which nuclear receptors exert control on ders, including obesity and diabetes, and the
nutritional homeostasis [85–87]. Although most of circadian clockwork [51, 60, 94, 96, 97]. In an
these findings emphasize the role of peripheral elegant study, Joseph Bass and colleagues dem-
clocks in the temporal orchestration of metabo- onstrated a direct connection between clockwork
lism, other studies have shown that cyclic systemic function in endocrine pancreatic beta cells and
signals, directly or indirectly depending on the SCN T2D [61]. To investigate the effect of the endocrine
master clock, also contribute to daily oscillations of pancreatic clock on islet function, the authors
metabolic activities [88, 89]. assessed circadian expression of the functional
islet-specific genes, as well as insulin secretion and
During the past decade, research on circadian gene glucose-stimulated insulin response at different
expression in mammals has largely focused on phases of the circadian cycle. In addition to mRNAs
transcriptome analysis. However, recent proteomic specified by core clock genes, including mRNAs
studies have revealed that a large fraction of encoding proteins involved in glucose metabolism,
oscillating liver proteins is encoded by mRNAs, insulin signalling and cellular proliferation, islet
the accumulation of which does not vary through- transcripts were found to exhibit circadian oscilla-
out the day [90–92]. Moreover, the cyclic expres- tions. The oscillations of these transcripts were
sion of numerous proteins persisted in genetically attenuated or abolished in the islets isolated from
arrhythmic mice maintained on a daily feeding ClockD19 mutant mice, consistent with the atten-
rhythm [90]. These results, in combination with uation of PER2::luciferase bioluminescence cycles
mathematical modelling, highlight the importance recorded from these islets. ClockD19 mutant mice
of translational and/or post-translational mecha- exhibited hyperglycaemia, significantly elevated

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fasting glucose levels and impaired glucose toler- shiftwork, might engender serious health problems
ance and glucose-stimulated insulin secretion. including cardiovascular disease, metabolic disor-
Moreover, these conditions deteriorated with age. ders (see ref. [54] and references therein) and
The decrease in insulin secretion in these mice was cancer [99]. The connection between malignant
most probably due to a defect in insulin exocytosis. transformations and perturbed circadian clock
In keeping with the transcriptome profiling results, function may be reciprocal. Thus, in a vicious
ClockD19 mice had smaller islets with reduced feed-forward loop, circadian rhythms can be dis-
proliferation rates. A similar phenotype was rupted in patients with cancer [100] and tumour
observed in Bmal1 knockout mice, which also do growth may be accelerated by circadian disruption
not possess a functional CLOCK–BMAL1 heterodi- [101, 102]. However, because of a number of
mer [61]. In line with these findings, a different possible confounding effects, the association
pancreas-specific Bmal1 knockout mouse model between circadian disruption and health deterio-
showed impaired insulin secretion and glucose ration must be interpreted with caution. Indeed, it
homeostasis [68]. Of note, the adverse effects of may be difficult to discriminate between the effects
islet-specific Bmal1 deficiency on glucose levels, caused by the circadian misalignment per see and
impaired glucose tolerance and insulin secretion those elicited by changes in lifestyle generally
were more pronounced than in either ClockD19 associated with rotating shiftwork (for review, see
mutant or global Bmal1 knockout mice. As sug- ref. [103]).
gested by Charles Weitz and colleagues [78] on the
basis of observations in mice with a hepatocyte-
Circadian clock and the aetiology of obesity and T2D
specific Bmal1 knockout, the rest–activity and
associated fasting–feeding rhythms governed by Insulin is released from pancreatic islets in a
the SCN must be counterbalanced by metabolic strongly circadian manner, and its levels in the
rhythms controlled by clocks in peripheral organs, blood exhibit a circadian pattern with a nocturnal
such as the liver and the endocrine pancreas. dip in humans [104]. Of note, the sensitivity of
skeletal muscle to insulin is also controlled by the
An additional link between circadian clock compo- clock [105]. Thus, glucose homeostasis is tightly
nents and glucose homeostasis has been provided controlled by the circadian system not only in
by Zhang et al. [98]. They revealed that the core laboratory rodents but also in humans [106].
clock proteins CRY1 and CRY2 modulate fasting Genetic linkage analyses in human subjects have
glucose levels through the inhibition of glucagon- indicated that hPER2 and hCRY2 polymorphisms
induced gluconeogenesis. This is accomplished by might be associated with blood glucose levels [107,
dampening cAMP accumulation and thus CREB 108]. Polymorphisms in hCLOCK were linked to
activity. Moreover, hepatic overexpression of Cry1 predisposition to obesity whereas two hBMAL1
lowered blood glucose levels and improved insulin haplotypes were associated with T2D and hyper-
sensitivity in the setting of insulin resistance (db/ tension (for review, see ref. [109]). Although the
db mice), emphasizing the critical role of the clock rs2287161 polymorphism of hCRY1 per se does not
proteins for glucose homeostasis [98]. Collectively, show a significant impact on glucose homeostasis,
these findings indicate that obesity and high-fat in combination with a high dietary carbohydrate
feeding reciprocally affect the circadian system in intake, it is strongly associated with the develop-
rodents, highlighting the tight connection between ment of elevated insulin resistance and diabetes in
metabolic and circadian rhythms. homozygous subjects [110].

Circadian clock and human metabolic diseases Melatonin is a circulating neurohormone, which is
predominantly secreted by the pineal gland during
Synchrony between the internal clock and the environment: circadian
the night. It regulates circadian rhythms by assist-
misalignment studies
ing the translation of photoperiodic information in
A modern lifestyle, with exposure to artificial light, the brain. Melatonin signalling is mainly mediated
irregular meal times and short sleep duration, by two receptors, MT1 and MT2, encoded by the
provokes chronic desynchrony between internal MTNR1A and MTNR1B genes, respectively. The
clocks and environmental cues; this is known as strongest MT2 expression levels are detected in
‘social jet lag’. Numerous epidemiological studies the retina and in the SCN (for review, see ref. [104,
have suggested that such circadian misalign- 111, 112]). MT2-mediated melatonin signalling
ment, in particular prolonged periods of rotating may indirectly regulate glucose level and insulin

520 ª 2015 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2015, 277; 513–527
 C. Dibner & U. Schibler Review: Circadian clocks in health and disease

secretion through the SCN [113]. As mentioned than during sleep. Functional cell-autonomous
above, in healthy individuals, insulin secretion clocks were characterized in human pancreatic
follows a circadian rhythm governed by the islets islets (Fig. 2) and in beta cells synchronized in
themselves, and the phase of this rhythm might be culture by a dexamethasone pulse and by cyclic
modulated by melatonin [114]. MTNR1A is in changes in temperature [20]. These clocks drive
general more highly expressed than MTNR1B in rhythmic islet gene expression [20] and impact on
pancreatic islets. However, the presence of MT2 the basal and induced insulin secretion in cultured
has been confirmed both in islets and beta cells human islet cells (C. Saini and C. Dibner, unpub-
analysed by fluorescence-activated cell sorting, lished results). The analysis of clock gene expres-
suggesting a possible role of this less-abundant sion in human islets suggested that the
melatonin receptor isoform in the regulation of accumulation of the mRNAs encoding PER2,
insulin secretion [115]. Genomewide association PER3 and CRY2 is downregulated in patients with
studies in humans have demonstrated a connec- T2D in comparison with their healthy counterparts
tion between MTNR1B variants and hyperglyca- [124].
emia, impaired early phase insulin secretion and
beta cell function [115–117]. Moreover, it has been When interpreting the impact of core clock tran-
shown [115–117] that MTNR1B haplotypes predict scription factors on insulin secretion and diabetes
T2D development. Thus, MT2, expressed in (e.g. [ref. 61]) or blood glucose levels and insulin
human pancreatic beta cells, might represent an sensitivity (e.g. ref. [98]), it should be acknowl-
important link between the circadian clock and edged that these transcriptional regulatory pro-
glucose homeostasis and provide new opportuni- teins may implement functions not related to
ties for T2D diagnostics and possible treatments circadian rhythmicity. In other words, when study-
[118]. ing organisms with mutations in core clock genes,
we must discriminate between clock gene and
Patients with circadian misalignments show pro- clock phenotypes. For example, the susceptibility
found perturbations in plasma glucose and insulin of different clock mutant mice to oncogenic trans-
levels (for review, see ref. [119]). There is accumu- formations, metabolic disorders or bone defects
lating evidence that insufficient sleep, which is [125, 126] may have no causal relationship with
widespread in modern society, represents a major circadian rhythmicity. Bmal1 knockout mice exhi-
risk factor for a number of conditions, in particular bit an early ageing phenotype and a number of age-
metabolic diseases [75, 109, 120]. Due to technical related pathologies [127]. Yet, these phenotypes
and ethical limitations associated with studies in are not observed in ClockD19 mutant mice or Per1/
human subjects, molecular investigations of the Per2 and Cry1/Cry2 double-knockout mice. Fur-
influence of circadian clocks on human metabo- thermore, BMAL1 acts as a negative regulator of
lism have been initiated recently but are still the mammalian target of rapamycin complex 1
relatively limited. As mentioned above, it has been (mTORC1) pathway [128]. As a consequence,
demonstrated that obesity is tightly and recipro- mTORC1 activity is increased in Bmal1 knockout
cally linked to circadian oscillator function in mice, and this may account for the accelerated
rodents [60, 62]. In humans, however, the situa- ageing observed in these animals. Similarly, it has
tion is somewhat more complicated. In a study been reported that REV-ERBa modulates inflam-
using biopsies of subcutaneous white adipose matory responses by macrophages [129] by atten-
tissue harvested at 6-h intervals from lean and uating the production of inflammatory cytokines
obese subjects and patients with T2D (n = 8–11 such as interleukin 6 [130] and Ccl2 [131, 132]. In
subjects per group), no major changes in circadian this case, the high-amplitude expression of REV-
clock gene expression were detected between the ERBa may account for the strongly circadian
groups [121]. The same group later reported a gating of inflammatory symptoms, although
highly significant reduction in the amplitude of what the value of this gating might be remains
circadian melatonin production in a different unclear.
cohort of patients with T2D [122]. In a study
focused on obesity, Markwald et al. [123] found Recent studies in rodent, as well as prospective
that sleep time restriction resulted in an increase human studies, have provided increasing evi-
in calorie consumption and weight gain, and a dence of the link between sleep disturbances,
phase shift in circadian melatonin production. Yet, circadian system disruption and metabolic dis-
more calories are burned during waking hours eases [133]. Of note, obstructive sleep apnoea

ª 2015 The Association for the Publication of the Journal of Internal Medicine 521
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 C. Dibner & U. Schibler Review: Circadian clocks in health and disease

(OSA), which combines sleep fragmentation and author) were housed for several weeks under
hypoxaemia, represents a major risk factor for conditions in which they did not receive any
the development of obesity, insulin resistance external timing cues. The period lengths of body
and possibly diabetes [133]. The association temperature cycles and urine production rhythms
between OSA and the development of obesity were considerably longer than 24 h in 85% of the
and insulin resistance was observed in adult and examined individuals (average: ~25 h).
paediatric populations [134]. A plausible expla-
nation for the link between OSA, the circadian Subsequent studies on human circadian rhythms
system and metabolic disturbances could involve have employed additional daytime-dependent
changes in circadian patterns of metabolic hor- parameters, such as saliva and blood levels of
mones caused by sleep apnoea. However, in a melatonin [122], plasma cortisol levels [32] and
recent study, no changes in circadian variations thoracic skin surface temperature [100, 139].
of ghrelin, leptin, resistin and adiponectin were Moreover, several relatively noninvasive sampling
revealed in patients with OSA compared to con- procedures, including repetitive oral mucosal
trol subjects [135]. OSA is also strongly associ- biopsy [140] and hair follicle analysis [141], have
ated with cardiovascular diseases, including been developed. However, none of these methods
stroke and acute coronary syndromes. For exam- provides reliable assessments of free-running cir-
ple, plasminogen activator inhibitor-1 (PAI-1), a cadian rhythms.
key circulating prothrombotic factor that inhibits
fibrinolysis and exhibits a robust circadian In view of these limitations, substantial efforts
rhythm in human subjects, is elevated in both have been directed towards establishing tech-
cardiovascular diseases and OSA and might thus niques for examining human circadian oscillators
represent an important link between the two in in vitro synchronized human primary explants/
conditions. The sharp early morning peak in cells from tissue biopsies. Indeed, the results of a
PAI-1 expression is caused by the circadian number of studies in cultured fibroblasts sug-
system and might explain the morning peak in gested that these cellular clocks represent an
adverse cardiovascular events [136]. The pres- excellent experimental system to study mamma-
ence of OSA has been shown to adversely affect lian circadian oscillator functions [142–144].
circadian fibrinolytic balance, with higher mean Moreover, fibroblasts expressing the luciferase
PAI-1 activity and lower tissue-type plasminogen reporter from various circadian promoters consti-
activator activity in OSA patients compared with tute an ideal model system for the molecular
control subjects. This perturbation might account dissection of basic oscillator properties in living
for the increased rate of cardiovascular events in cells with high temporal resolution and precision
patients with OSA, with alterations of circadian [6, 19]. Brown and colleagues have demonstrated
clock function these patients being responsible that cultured primary human skin fibroblasts
[137]. These findings suggest an important rela- represent an excellent experimental system for
tion between OSA, the circadian system and the dissection of human clock properties (Fig. 2);
metabolic disease aetiology. However, whether perhaps not surprisingly, circadian clock param-
glycaemic control in patients with T2D can be eters measured by the continuous recording of
improved by treating OSA remains controversial circadian bioluminescence cycles produced by
[133]. human skin fibroblasts varied widely amongst
the cells harvested from different donors [145].
Remarkably, circadian oscillator characteristics
Circadian rhythm in synchronized primary cells and tissue explants of
measured in cultured skin fibroblasts correlate
human subjects
relatively well with rhythmic human behaviour, as
The characterization of circadian rhythms in demonstrated in human subjects in whom circa-
humans is an extremely challenging and costly dian physiology was examined under laboratory
endeavour, as it requires prolonged subject obser- conditions [146] or individuals who completed the
vation under controlled laboratory conditions. The Munich chronotype questionnaire [147, 148]. The
‘bunker experiments’ conducted by Aschoff [138] findings of these studies suggested that long and
were the first and perhaps best-known examples of short periods of fibroblast clock gene expression
studies in which the free-running clocks of human were frequently associated with late (‘owl-like’)
subjects were investigated. In these studies, vol- and early (‘lark-like’) chronotypes, respectively
unteers (mostly university students as well as the [149]. Accordingly, studies of peripheral organs

522 ª 2015 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2015, 277; 513–527
 C. Dibner & U. Schibler Review: Circadian clocks in health and disease

are promising for increasing understanding of the between the circadian clock and these metabolic
entire circadian timing system in humans. In the disorders, modulation of the oscillator could
meantime, robust oscillations in gene expression provide a potential novel therapeutic option.
have also been investigated in human pancreatic Here, we have summarized some recent studies
islets maintained in organotypic cultures [20], in performed in laboratory rodents and human
human primary thyrocytes (Fig. 2, ref. [150]) and subjects that have made significant contributions
in in vitro differentiated skeletal myotubes (Lau- towards understanding the molecular link
rent Perrin and C. Dibner, unpublished results). between circadian gene expression and metabolic
An exciting observation regarding the impact of dysfunctions, such as obesity, the metabolic
human bloodborne factors on the period length of syndrome and T2D. These studies highlight the
circadian gene expression in cultured cells was importance of circadian clock function – or at
recently reported. Surprisingly, cultured fibro- least circadian ‘clock gene’ function – in energy
blasts exposed to serum collected from elderly homeostasis and metabolism. However, many
subjects had shorter periods than fibroblasts other clinically relevant physiological processes
exposed to serum harvested from young individ- are driven by the circadian timing system and are
uals [151]. This suggests that circadian molecular therefore daytime dependent. Particularly well-
oscillators are more plastic than hitherto antici- known examples are bronchial asthma with
pated in that they can change their properties around-the-clock changes in dyspnoea frequency,
according to their environment. The recording of symptoms of rheumatoid arthritis, oscillations in
circadian gene expression in cultured cells may blood pressure (i.e. a morning rise preceded by
even be useful for diagnostic purposes. It will thus night time dipping) (for review, see ref. [152]) and
be exciting to examine circadian clock properties xenobiotic detoxification (for review, see ref.
in cells treated with serum obtained from human [102]). The enzymes involved in the latter process
subjects with various disorders, such as meta- also metabolize therapeutic drugs and thus are
bolic diseases, chronic inflammatory conditions relevant in determining pharmacokinetic param-
and cancer [150]. eters. Hence, the tolerability and efficacy of
cancer chemotherapeutic agents can vary signif-
icantly as a function of delivery time [101, 153,
Conclusions
154]. In the clinic, a significant improvement in
Based on several observations, the temporal tolerability has been shown in randomized trials
orchestration of metabolism seems to be one of in which patients with cancer received the same
the primary functions of the mammalian circa- sinusoidal chronotherapy schedule over 24 h,
dian timing system. Indeed, most if not all compared to constant-rate infusion or chrono-
metabolic processes are subject to diurnal oscil- therapy in which the timing was not optimized
lations, and conversely, daily feeding–fasting [100, 155]. Moreover, the quality of life of
rhythms are the dominant timing cues in the patients with regard to fatigue and weight loss
synchronization of circadian clocks in peripheral was significantly improved in those receiving a
organs. The disruption of circadian physiology daytime-adapted drug-delivery regimen [139].
imposed by social constraints on many individu- Even though chronotherapeutics is still in its
als (chronic ‘social jet lag’) can have severe infancy, it has great potential for improving the
consequences on their health. In addition to efficacy of treatments and reducing the burden of
increasing sleep deficit and irregular meal times, unwanted side effects. In the modern era of
there is a concomitant worldwide rise in the personalized medicine, knowledge of individual
incidence of metabolic disorders and T2D. Both chronotypes may be critical for treatment and
biological and epidemiological studies suggest a may have to be included as an important com-
direct link between lifestyle and these metabolic ponent in diagnostic procedures. We hope that
disorders. However, the genetic and biochemical the number of clinicians willing to take into
associations between human circadian clocks account this rhythmic physiology when treating
and metabolic disorders are still poorly under- patients will dramatically increase in the near
stood. It is therefore of utmost scientific and future.
clinical importance to further investigate the
emerging connection between circadian oscillator
Conflict of interest statement
function and the aetiology of obesity and T2D. If
we succeed in establishing a molecular link No conflicts of interest to declared.

ª 2015 The Association for the Publication of the Journal of Internal Medicine 523
Journal of Internal Medicine, 2015, 277; 513–527
 C. Dibner & U. Schibler Review: Circadian clocks in health and disease

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