(2018) - Froy, O., & Garaulet, M.The Circadian Clock in White and Brown Adipose Tissue Mechanistic, Endocrine, and Clinical Aspects PDF

The circadian clock in white and brown adipose tissue: mechanistic,
endocrine and clinical aspects
Oren Froy, Marta Garaulet

ADVANCE ARTICLE: Endocrine Reviews
Endocrine Reviews
Endocrine Society
Submitted: July 24, 2017

Accepted: February 22, 2018
First Online: February 27, 2018
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Endocrine Reviews; Copyright 2018 DOI: 10.1210/er.2017-00193
The circadian clock in adipose tissue
The circadian clock in white and brown adipose tissue: mechanistic,

endocrine and clinical aspects
Oren Froy1, Marta Garaulet2
1
Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and
Environment, The Hebrew University of Jerusalem, Rehovot, Israel.
2
Department of Physiology, University of Murcia, Murcia Spain; IMIB-Arrixaca, Murcia, Spain
Received 24 July 2017. Accepted 22 February 2018.
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Obesity is a major risk factor for the development of illnesses, such as insulin resistance and
hypertension and has become a serious public health problem. Mammals have developed a
circadian clock located in the hypothalamic suprachiasmatic nuclei (SCN) that responds to
the environmental light-dark cycle. Clocks similar to the one located in the SCN are found in
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peripheral tissues, such as the kidney, liver and adipose tissue. The circadian clock regulates
metabolism and energy homeostasis in peripheral tissues by mediating activity and/or
expression of key metabolic enzymes and transport systems. Knockouts or mutations in clock
genes that lead to disruption of cellular rhythmicity have provided evidence to the tight link
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between the circadian clock and metabolism. In addition, key proteins play a dual role in
regulating the core clock mechanism as well as adipose tissue metabolism and link circadian
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rhythms with lipogenesis and lipolysis. Adipose tissues are distinguished as white, brown and
beige (or brite), each with unique metabolic characteristics. Recently, the role of the circadian
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clock in regulating the differentiation into the different adipose tissues has been investigated.
In this review, the role of clock proteins and the downstream signaling pathways in white,
brown and brite adipose tissue function and differentiation will be reviewed. In addition,
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chronodisruption and metabolic disorders and clinical aspects of circadian adiposity will be
addressed.
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The role of the circadian clock in regulating adipose tissue function and differentiation and key
proteins that play a dual role in regulating clock mechanism and adipose tissue metabolism.
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Essential Points:
• The circadian clock controls energy homeostasis by regulating circadian expression and/or
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activity of enzymes, hormones, and transport systems involved in metabolism.

• Disruption of circadian rhythms leads to obesity and metabolic disorders.
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• The circadian clock regulates the function of and differentiation into white and brown adipose
tissue.
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• Key proteins play a dual role in regulating the core clock mechanism as well as adipose tissue
metabolism and link circadian rhythms with lipogenesis, lipolysis and differentiation.
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1. Circadian rhythms and the circadian clock

Organisms on our planet developed an endogenous circadian clock entrained, or
synchronized, by light to exactly 24 hours (1). By predicting the day-night cycles, the clock
imparts a survival advantage as physiological processes are performed at the appropriate time
to find food, mating partners or to avoid predators (1-3). The clock is self-sustained, but in
the absence of light, the endogenous clock free-runs, generating cycles of either slightly
longer or slightly shorter than 24 hours, hence the term circadian (circadian, circa=about
dies=day). The circadian clock controls numerous cellular, physiological, metabolic,
endocrine and behavioral systems (4,5). Disruption of this control results in fatigue,
disorientation, insomnia, altered hormone profile and high morbidity, seriously influencing

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overall health (4,6-12). Obesity, which is characterized by the excess of fat accumulation in
white adipose tissue, has been related to irregular sleep/wake schedules, high snacking
frequency, or social jet lag known to disrupt the circadian clock (13,14). Bright light at night
has also been associated with obesity, as was shown in 100,000 women of the Breakthrough
Generations Study (15). All these studies as a whole, strongly suggest that impairment of the
circadian system is involved in the etiology of several illnesses (16).
The central circadian clock is located in the anterior hypothalamus and is confined to the
suprachiasmatic nuclei (SCN). Photic information perceived by the retina and transmitted via
the retinohypothalamic tract (RHT) synchronizes SCN neurons to coordinate circadian
outputs (17-20). Clocks, similar to those found in SCN neurons, are found in peripheral
tissues, such as the liver, intestine, heart, adipose tissue, retina and in various brain regions
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(2,21-23). 5-20% of tissue-specific genes are estimated to exhibit an oscillatory expression
profile, emphasizing the circadian control over the function of peripheral tissues (23-32). The
SCN clock regulates peripheral rhythms by neuronal connections, secretion of humoral
factors (33-35), or indirectly by driving rhythmic feeding, locomotor activity and/or body
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temperature, which, in turn, coordinate rhythmic gene expression.
The molecular clock in SCN neurons and peripheral cells is an intracellular mechanism
composed of transcription-translation feedback loops (3). The transcription factor CLOCK
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(Circadian Locomotor Output Cycles Kaput) dimerizes with BMAL1 (brain and muscle
ARNT-like protein 1) and together the CLOCK:BMAL1 heterodimer activates transcription
by binding to E-box (5’- CACGTG -3’) and E-box-like promoter sequences (2). Amongst the
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regulatory targets of CLOCK:BMAL1 are the Period (Per1, Per2) and Cryptochrome (Cry1
and Cry2) genes. Oligomerization and nuclear translocation of PERs:CRYs leads to the
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inhibition of CLOCK:BMAL1-mediated transcription (2,36) (Fig. 1). The nuclear receptor
reverse ERBα (REV-ERBα) negatively regulates Bmal1 expression (37), whereas retinoic
acid receptor-related orphan receptor α (RORα) and RORγ (38) (Fig. 1) positively
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regulates its expression via ROR response elements (RORE) (39). All clock genes exhibit
24-h oscillation in peripheral tissues (40).
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2. White, brown and beige (or brite) adipose tissue

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Classically, adipose tissue has been regarded as a passive reservoir for energy storage, but
this traditional point of view is no longer valid. In 1987, adipose tissue was identified as the
major site for metabolism of sex steroids (41). Several years later, in 1994, the discovery of
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the cytokine-like factor leptin (42) entirely changed our perspectives on adipose tissue. This
was a revolution in the study of obesity, as for the first time it was shown that adipose tissue
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was able to secrete hormones or “adipocytokines” capable of communicating information

from periphery to the central nervous system. Adipose tissue was no longer considered a
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passive reservoir of energy; it was an endocrine organ. Adipocytokines or adipokines, such as

adiponectin, resistin, tumor necrosis factor α (TNFα), visfatin, obestatin have autoendocrine,
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paracrine and endocrine functions. Thus, adipose tissue regulates energy storage and
expenditure and influences systemic metabolic homeostasis through production of adipokines
(43).
In mammals, at least two types of adipose tissue have been described, white adipose
tissue (WAT) and the brown adipose tissue (BAT). WAT and BAT originate from different
stem cells and they have different distribution, gene expression, and function (44) (Fig. 2).
Adipocytes with features similar to BAT found within white adipose depots are termed beige
or brite (brown in white) adipose tissue (44). Nowadays, it is known that the development of
obesity depends not only on the balance between food intake and energy expenditure but also
on the balance between WAT, BAT and brite adipose tissue.

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WAT:
The most abundant type of adipose tissue in mammals and the main energy reservoir, stores
dietary energy as triacylglycerides (TAGs) in unilocular lipid droplets and secretes a huge
number of hormones and cytokines that regulate metabolism and insulin resistance (43,45).
After meals, WAT uptakes dietary fats and carbohydrates from the circulation and, through
lipogenesis, converts them to TAGs (46). During fasting or physical activity, lipolysis ensues
and TAGs are broken down into free fatty acids (FFAs) and glycerol that are released to the
circulation to supply the needs of other tissues (46).
BAT:
This tissue contains multilocular lipid droplets and high mitochondrial density and is able to
convert chemical energy into heat (47). Cold stimulation leads to the release of
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norepinephrine at innervated sites within the BAT, which leads to lipolysis of TAGs and
increase in cellular FFAs. FFAs are subsequently transported to the mitochondria and serve
as substrates for β-oxidation. Heat is generated using uncoupling protein-1 (UCP1), a protein
that is localized on the inner membrane of mitochondria and uncouples electron transport
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from ATP production (47). The resulting energy derived from substrate oxidation is
dissipated as heat. In addition to its key role in maintaining body temperature under cold-
stress, the metabolic function of BAT is important for global energy balance, insulin
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sensitivity and lipid metabolism (48-50). Developmentally, BAT is typified by the activation
of myogenic factor 5 (Myf5) and early paired box 7 (Pax7) expression (51,52), which encode
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two transcription factors that mark myogenic precursor cells. Late Pax7 expression marks
cells that are predominantly restricted to the skeletal muscle lineage (52,53).
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Brite (beige) adipose tissue:
Unlike WAT, which is characterized by the presence of few mitochondria that are devoid of
UCP1, beige adipocytes found within WAT express UCP1 and are mitochondrial-rich. These
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adipocytes are distinct from BAT, but still have a thermogenic capacity by responding to cold
exposure to dissipate heat through UCP1 (54).
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3. The circadian clock and metabolism

The robust and coordinated expression of clock genes in peripheral tissues, such as liver and
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adipose tissue, mediates the activity of nuclear receptors, enzymes, hormones and
transporters involved in carbohydrate, lipid and protein metabolism (21,30,55-61). In turn,
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animal studies have shown that a high-fat diet may contribute to the development of obesity
and insulin resistance via alterations in the circadian period of locomotor activity rhythms
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and changes in the oscillation of clock genes in adipose and liver tissue (62-65). In humans,
results suggest that the modulation of the dietary fat and carbohydrate content alters the
function of the central and peripheral circadian clocks in humans (66). The mutual influence
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of the clock and metabolic regulation is achieved due to the participation of key catabolic,
anabolic and lipid metabolic factors in the core clock mechanism (Fig. 1).
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REV-ERBs and retinoic acid–related orphan receptors (RORs):

Two important families that link the core clock mechanism with lipid metabolism are REV-
ERBs and RORs. These factors are vital for adipocyte differentiation (67), lipogenesis and
lipid storage and exhibit striking circadian rhythms (68,69). In addition to their metabolic
role, REV-ERBs are the negative and RORs the positive regulators of Bmal1 expression
(37,38,70,71) (Fig. 1). In turn, the CLOCK:BMAL1 heterodimer regulates the
expression of Rev-erbs and Rors (37,38,72). Diet-induced obese mice treated with a REV-
ERB agonist showed reduced fat mass and improved dyslipidaemia and hyperglycaemia (73).
In addition, a REV-ERB agonist suppressed mouse orexinergic gene expression, whereas
REV-ERBβ-deficient mice had increased orexinergic transcripts (74). In this line, mice

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deficient for Rev-erbα develop higher adiposity on regular chow and high-fat diet supposedly
due to increased fat uptake by adipose tissue (75). Nevertheless, in BAT, Rev-erbα seems to
have an opposite role on adiposity since deletion of Rev-erbα markedly improved the
thermogenic response to cold and physiological induction of UCP1 by cold temperatures is
preceded by rapid downregulation of Rev-erbα (76). As REV-ERBs negatively regulates
Bmal1 expression, the role of BMAL1 in WAT and BAT function merits further study.
Peroxisome proliferator-activated receptors (PPARs):
PPARs are a nuclear receptor family consisting of 3 isoforms in mammals, α, β/δ, and γ,
which are differentially expressed among tissues (77). PPARs plays a key role in the
transcription of genes involved in lipid and glucose metabolism upon binding of endogenous
free fatty acids (78,79). PPARα is abundant in the liver, BAT, heart and kidney, while
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PPARγ in expressed in the adipose tissue and PPARβ/δ throughout the body (80).
PPARα agonists, such as fibrates, are clinically proven lipid-lowering drugs, whereas
PPARγ ligands such as thiazolidinediones (TZDs), improve glycemic control via insulin
sensitization in patients with type 2 diabetes (81). PPARs are connected to the core clock
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mechanism, as their expression is mediated by the CLOCK:BMAL heterodimer. In turn,
PPARα activates Bmal1 expression (Fig. 1) by binding to the peroxisome-proliferator
response element (PPRE) (82-84). The role of PPARs in the circadian regulation has been
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demonstrated through experiments performed with PPARα agonists, that showed advanced
locomotor activity and feeding daily rhythms in mice (85), whereas experiments in PPARγ
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deletion showed dampened behavioral and cellular circadian rhythms (86).
PPARγ coactivator 1 (PGC1):
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The PPARγ coactivator 1 (PGC1) is a family of transcriptional coactivators that induce
mitochondrial oxidative metabolism. Indeed, PGC-1α is a catabolic factor, whose expression
is induced in the liver during starvation, in BAT during cold exposure, and in skeletal muscle
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during physical exercise (87). PGC-1α null mice show abnormal diurnal rhythms of body
temperature, activity and metabolic rate, due to disrupted clock and metabolic gene
expression (88). Moreover, PGC1 family members exhibit circadian expression and, in turn,
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PGC1α stimulates the expression of Bmal1, Clock, Per2 and Rev-erbα, through co-activation
of the ROR family (88,89).
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AMP-activated protein kinase (AMPK):

AMPK is a sensor of the energy status within cells, whose phosphorylation activates cellular
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catabolism (90,91). When activated, AMPK phosphorylates and hence activates casein kinase
I ε (CKIε), which leads to PERs degradation (92). AMPK also phosphorylates and, as a
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result, destabilizes CRY1 in mouse fibroblasts (93) (Fig. 1). PERs and CRYs degradation
relieves CLOCK:BMAL1 inhibition leading to a phase advance in the circadian expression in
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some tissues (63). Indeed, metformin, an indirect AMPK activator, leads to alterations of the
circadian phase in a tissue-specific manner (94).
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Sirtuin 1 (SIRT1):
SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase, is a
catabolic factor involved in transcriptional silencing (95,96). SIRT1 is connected to the core
clock mechanism by interacting with CLOCK to deacetylate BMAL1 and PER2 (97,98).
Deacetylated PER2 is further phosphorylated and degraded relieving CLOCK:BMAL1
inhibition (Fig. 1). Hepatic circadian transcriptome analyses revealed that differently from
SIRT1, SIRT6 interacts with CLOCK:BMAL1 and controls their recruitment to gene
promoters. In addition, SIRT6 also controls circadian chromatin recruitment of sterol
regulatory element-binding protein 1 (SREBP-1), resulting in the cyclic regulation of genes
involved in fatty acid and cholesterol metabolism (99). It turns out that the CLOCK:BMAL1
heterodimer as well as SIRT1 regulate the circadian expression of NAMPT (nicotinamide

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phosphoribosyltransferase), a rate-limiting enzyme in the NAD+ salvage pathway, therefore,

enabling the circadian synthesis of the coenzyme required for SIRT1 activity (100,101).
SIRT1 activity is also enhanced by AMPK, which increases cellular NAD+ levels (102). High
levels of NAD+ inhibit DNA binding of CLOCK:BMAL1 (103).
Mammalian target of rapamycin (mTOR):
mTOR is an anabolic factor involved in protein synthesis and integrates the input from
multiple upstream pathways, including insulin, growth factors, and mitogens, and functions
as a sensor of cellular nutrient and energy levels (104). The mTOR pathway is regulated by
light in the SCN (105). One of the key factors in the mTOR pathway, protein 70 S6 kinase 1
(P70S6K1), rhythmically phosphorylates BMAL1 allowing it to both associate with the
translational machinery and stimulate circadian oscillations of protein synthesis (106) (Fig.
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1). Thus, the mTOR signaling pathway, in addition to its role in protein translation, it is
linked to the circadian clock mechanism.
4. Chronodisruption and metabolic disorders
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Chronodisruption is defined as the chronic desynchronization of the 24-h rhythms, resulting
in adverse health effects (107). More specifically, chronodisruption occurs when the
synchronization between external environmental cues and internal physiological processes is
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lost. This can either result in a total loss of rhythmicity, a reduction in rhythm amplitudes or
in phase differences between the SCN and peripheral clocks (108). The interrelations
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between the key metabolic factors mentioned above and the core clock mechanism may
explain why metabolic chronodisruption is detrimental.
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Clock gene mutants and knockouts demonstrate the most compelling linkage between
metabolic disorders and the circadian clock (Table 1). Mice with a mutated Clock gene
(Clock∆19 mice) have a dampened diurnal feeding rhythm, are obese, and they develop a
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metabolic syndrome of hyperleptinemia, hyperlipidemia, hepatic steatosis and hyperglycemia

(109). Combination of the Clock∆19 mutation with the leptin knockout (ob/ob) leads to
significantly heavier mice than the ob/ob phenotype (110), reiterating the contribution of
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clock disruption to the obese phenotype (55,57,111). Compared with wild type mice, Per2-/-
mice fed a high-fat diet developed significant obesity (112).
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Bmal1-/- knockout mice exhibit suppressed diurnal changes in triglycerides and glucose
levels as well as no gluconeogenesis (113). Hyperinsulinemic-euglycemic clamps showed
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that Bmal1-/- mice exhibit no circadian rhythm in insulin action (114). Adipocyte-specific
deletion of Bmal1 resulted in obesity in mice. As adipocytes signal the levels of stored energy
to the brain, Bmal1 deletion led to changes in the expression of hypothalamic neuropeptides
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that regulate appetite emphasizing the role the adipocyte clock in the temporal organization
of energy regulation (115).
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In mouse studies, circadian changes in hepatic Cry1/2 expression were sufficient to

modulate gluconeogenesis (116), emphasizing the important role CRY proteins play in
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regulating hepatic glucose output (117). In addition, Cry1/2-/- mice had an increased
vulnerability to high-fat diet-induced obesity that correlated with increased insulin secretion
and lipid storage in adipose tissue (118,119) and constitutive elevation of proinflammatory
cytokines (120). SCN-mutant mice showed a significant number of rhythmic transcripts
(121). Thus, although it is known that the SCN clock regulates lipolytic and lipogenic
processes (122), recent findings in tissue-specific knockouts indicate that peripheral clocks
play a crucial role in regulation of whole body energy homeostasis.
Consistent with these findings, genetic polymorphisms in human clock genes have been
associated with metabolic alteration (Table 1). Although mutations are rare in humans, it is

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rather common to have a single nucleotide polymorphism (SNP) that underlies differences in
our vulnerability to disease.
CLOCK:
It was first demonstrated that several variants at CLOCK were associated with obesity,
especially abdominal obesity (123). Later that year, it was confirmed that CLOCK could play
a relevant role in the development of the metabolic syndrome (MetS), type 2 diabetes and
cardiovascular disease (124). Subsequently, we demonstrated that several genetic variants in
clock genes were related to obesity and related diseases, such as MetS. For example, CLOCK
SNPs (rs3749474, rs4580704 and rs1801260 (3111T>C)) were associated with obesity and
insulin resistance-related variables (125). Systolic and diastolic blood pressure values were
also strongly associated with SNP rs4580704 (125). Furthermore, these genetic variants were
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associated with energy intake (126). Minor alleles ate more, specifically fat, and were more
obese, whereas minor allele carriers (A) of CLOCK rs4580704 showed decreased risk of
developing diabetes (31% lower) and hypertension (46% lower) than non-carriers (125).
Some of these associations were functionally explained by the presence of a polymorphism
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involving a change in the structure of the mRNA leading to a change in gene expression
(127). In addition, people carrying CLOCK 3111C (risk C carriers: including TC and CC) are
more likely to be obese, and exhibit greater difficulties in losing weight. Further studies
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performed to understand the connections between CLOCK rs1801260 (3111T>C) and
resistance to weight loss showed that risk carriers (C) displayed significant abnormalities at
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the daily rhythms of wrist temperature and activity. These abnormalities included a) lower
amplitude; b) greater rhythm fragmentation; c) less stable patterns; and d) significantly
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decreased circadian function. C carriers were also less active, started their activities later in
the morning and were sleepier during the day, showing a delayed acrophase (maximum
expression) that characterizes 'evening-type' subjects (128). Moreover, C carriers have higher
parasympathetic activity during daytime (when it is supposed to be low) than TT carriers and
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reduced daily rhythms of autonomic nervous system (ANS) function (129). These changes
correlated with weight loss resistance, as low sympathetic activity and high parasympathetic
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activity reduce energy expenditure and predispose onset of obesity (MONA LISA theory)
(130).
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PER2:
PER2 SNPs (rs2304672C>G and rs4663302C>T) has been associated with abdominal
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obesity (131). In particular, PER2 rs2304672C>G minor allele carriers (6% of the
population) displayed several obesogenic behaviors, such as an increased attrition of the
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weight loss treatment, increased frequency of snacking, stress while dieting, eating while
bored and skipping breakfast, when compared to non-carriers (131).
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BMAL2:
BMAL2 SNPs have also been associated with high risk of developing type 2 diabetes in obese
patients. The A/G and A/A genotypes of BMAL2 rs7958822 showed significantly higher odds
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ratios for type 2 diabetes than the G/G genotype among obese men and women. There were
no significant associations between BMAL1 rs11022775 or rs2290035 genotypes and type 2
diabetes (132).
REV-ERBα:
REVERBα rs2314339 has been associated with obesity in two independent populations:
Mediterranean and North American. However, as opposed to other clock gene variants,
which are associated with obesity through changes in dietary intake, REVERBα rs2314339 is
associated with obesity through a decrease in physical activity (133). These findings were
consistent with those obtained in experimental animals showing that Rev-erbα−/− mice
showed a significantly lower spontaneous locomotor activity than their wild-type littermates

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(134). However, mice lacking Rev-erbα displayed marked hyperactivity and impaired
response habituation when introduced to a new environment (135).
CRY1:
Genetic variants at CRY1 have been connected to glucose metabolism. Meta-analysis
performed in two independent populations, Mediterranean and European origin North
American, indicated that an increase in carbohydrate intake was associated with a significant
increase in homeostasis model assessment of insulin resistance (HOMA-IR), fasting insulin
and a decrease in quantitative insulin sensitivity check index (QUICKI), only among
individuals homozygous for the minor C allele at CRY1 rs2287161. These findings support
the notion that there is a strong link between the circadian system and glucose metabolism
and suggests the importance this CRY1 locus for insulin resistance and diabetes risk (136).
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5. Meal timing, chronodisruption and metabolic alterations
Several behaviors also help to explain the connections between chronodisruption and
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metabolic alterations. Meal timing, such as restricting food to a particular time of day,
defined as time restricting feeding (TRF) has profound effects on the behavior and
physiology of animals. 2-4 h before the meal, the animals display food anticipatory behavior,
which is demonstrated by an increase in locomotor activity, body temperature, corticosterone
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secretion, gastrointestinal motility, and activity of digestive enzymes (137-140), all are
known output systems of the biological clock. Moreover, changing the time of food intake to
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unusual times as happens when nocturnal mice are fed a high-fat diet only during the 12-h
sleep (light) phase, is associated with an increase in weight gain (141). In humans, unusual
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feeding time produces a disruption of the circadian system, which might produce unhealthy
consequences (142). The timing of food intake may have a significant role in obesity (141),
as well as in weight loss and glucose metabolism (143). For example, mice with adipocyte-
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specific deletion of Bmal1 fed a high-fat diet during the light period gained significantly more
weight compared to mice fed during the dark period. Thus, disruption of the adipocyte clock
leads to obesity without an overall increase in daily caloric intake when mice are fed during
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the inactive phase (115). In humans, late eating has been shown to be predictive of weight
loss difficulties during a 20-week dietary intervention conducted in 420 obese and overweight
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individuals (144). The effect was independent of the total 24-h caloric intake (144). In
addition, insulin sensitivity was lower in late eaters compared to early eaters. Moreover, a
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twelve-week clinical study showed that those subjects assigned to high caloric intake during
dinner lost significantly less weight than those assigned to high caloric intake during
breakfast. Interestingly, there were no significant differences in the total amount of food or in
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energy expenditure as assayed by a physical activity questionnaire (145).

The physiological explanation for the metabolic alterations related to late eating is that
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when feeding patterns are changed, for example during shift work in humans or by means of
restricted access to chow in rodents, this has profound effects on peripheral oscillator
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regulation, while SCN rhythms remain largely unaltered (146,147). Indeed, in animal models
it has been demonstrated that temporal feeding restriction can change the phase
of circadian gene expression in peripheral cell types by up to 12 h while leaving the phase of
cyclic gene expression in the SCN unaffected (146). Similarly, a recent in-lab study has
demonstrated that a 5-h delay in meals leads to a 1.5-h delay in the circadian rhythm of the
clock gene PER2 in adipose tissue, while no significant changes were found in the central
clock (148).
6. Circadian regulation of adipose tissue in health and disease

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As all peripheral tissues, the adipocyte clock mechanism regulates cellular functions (30).
More importantly, these circadian rhythms persist in the absence of the SCN, for example, in
adipose tissue explant cultures (149). To ensure proper nutrient/energy flux and substrate use
by the organism, the circulating levels of secreted adipokines, such as leptin and adiponectin,
key players in the regulation of energy metabolism, display an oscillatory profile with a 24-h
period (122) (Fig. 3). Indeed, it was shown that BAT glucose uptake in mice and humans
reveals a diurnal rhythm that could in part be generated by signals from plasma hormones
(150,151). Alterations of this timing may have important detrimental metabolic
consequences. Interestingly, the circadian rhythms of adipokine expression/secretion differ
depending on the location of the adipose tissue, as significant differences were found both in
their relative acrophase and amplitude between subcutaneous and visceral adipose tissues
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(152). Analyses of various genes implicated in metabolic processes, such as energy intake
and expenditure, insulin resistance, adipocyte differentiation, dyslipidemia, and body fat
distribution indicated that circadian rhythmicity followed a predictable physiological pattern,
particularly for subcutaneous depots (25,152). Visceral depots also show a higher amplitude
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for all glucocorticoid-related genes and (152) it secretes more pro-inflammatory factors than
subcutaneous adipose tissue (153). However, induction of lipolysis is easier in visceral fat
depots, due to the increased sensitivity to catecholamines (154).
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Leptin:
The adipocyte-derived leptin, which acts at specific receptors in the hypothalamus to
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suppress appetite and increase metabolism, is extremely important in obesity. Plasma leptin
levels are circadian with leptin peaking early in the non-active phase, that is during the early
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dark phase in diurnal animals, such as monkeys and humans (155,156), and during the early
to mid-light phase in nocturnal animals, such as rats and mice (157,158) (Fig. 3). SCN
ablation was shown to eliminate leptin circadian rhythmicity in rodents, suggesting that the
central circadian clock regulates leptin expression (157). In obese subjects, leptin retains
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diurnal variation in release, but with lower amplitude (159), suggesting that blunted circadian
variation may play a role in leptin resistance and obesity (160). If exogenous leptin is
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continuously applied, desynchronized feeding during the light-phase failed to cause excessive
weight gain in ob/ob mice, but if it was applied in a timed pulse during the light phase,
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obesity ensued (161). Thus, desynchronized feeding can affect the endogenous rhythm of
leptin to influence metabolic signals that lead to weight gain.
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Adiponectin:
In human adipose tissue, the expression of adiponectin achieved its zenith (maximum) during
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the morning, which could be implicated in the maximal withdrawal of fatty acids and the
improvement in glucose tolerance at that time of the day (162). As adiponectin promotes
insulin sensitivity, its acrophase should precede insulin sensitivity, which reaches its
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maximum level at noon (152,163,164). PPARγ, which participates both in the clock
mechanism (Fig. 1) and adiponectin expression, could also be related to adiponectin circadian
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expression. In fact, the high expression of PPARγ during the morning is consistent with
results obtained in nocturnal mammals (56) and could be influencing the further increase in
adiponectin expression and the increase in insulin sensitivity during this time of the day.
Corticosteroids:
It has been described that in all species the maximum of circulating corticosteroid rhythms
occurs just before or at the onset of activity (165,166). Over the course of the day, they fall,
reaching low or undetectable levels an hour or two before bedtime. In human adipose tissue,
the antiphase relationship between leptin and glucocorticoids receptors (Fig. 3) (152) is
reasonable considering that both hormones are strongly interrelated and they exert opposite
functions in food intake regulation. While leptin displays an anorexigenic role,

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glucocorticoids increase appetite (orexigenic function). Indeed, plasma leptin ultradian pulses
were inversely correlated with those of adrenocorticotropic hormone (ACTH) and cortisol
(164).
Insulin:
Adipose tissue lipogenesis is highly regulated by insulin. Intake of sugars, like glucose, leads
to elevated insulin concentrations in the blood, which, in turn, increase glucose uptake by
upregulating glucose transporter 4 (GLUT4) expression on adipocyte membranes (167). This
process increases the substrate for glycolysis and, consequently, fatty acid biosynthesis in
adipocytes (168). As mentioned above, insulin sensitivity varies according to time of day,
with decreased values in the evening and at night in humans. Indeed, it was demonstrated that
insulin sensitivity in human subcutaneous adipose tissue displays a circadian rhythm and that
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it reaches its maximum (acrophase) around noon with sensitivity 54% higher than during
midnight (Fig. 3). In contrast, no circadian rhythms in insulin sensitivity were detected in
visceral adipose tissue (163). Mechanisms responsible for the diurnal variation in insulin
sensitivity warrant further study.
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7. Role of the circadian clock in adipose tissue differentiation
Currently, it is known that key clock proteins, not only regulate the “time specific” functions
T
in adipose tissue, but they also play a direct role in adipogenesis in both WAT and BAT.
However, some of the reported results are still contradictory.
WAT adipogenesis:
R
PER2, a negative regulator of BMAL1 expression, was found to interact with PPARγ and
A
repress its proadipogenic activity (169) (Fig. 4). Indeed, PER2-deficient mice display altered
lipid metabolism with drastic reduction of total triacylglycerol and non-esterified fatty acids.
PER2 exerts its inhibitory function by blocking PPARγ recruitment to target promoters and
E
thereby inhibiting transcriptional activation (169). As mentioned above, PPARγ expression is

positively regulated by the CLOCK:BMAL1 heterodimer and it positively regulates Bmal1
C
gene expression. Indeed, as mentioned above, an agonist for REV-ERB, which negatively
regulates Bmal1 expression, suppressed mouse orexinergic gene expression, whereas REV-
N
ERBβ-deficient mice had increased orexinergic transcripts (74). In addition, cell-based

studies demonstrate that activation of REV-ERBα with either synthetic or natural (heme)
ligands is required for adipocyte differentiation (158). Thus, PPARγ repression by PER2 and
A
activation of REV-ERB, which lead to BMAL1 downregulation, would be expected to

attenuate adipogenesis. However, surprisingly, knockdown of BMAL1 led to increased
V
adipogenesis, adipocyte hypertrophy and obesity in mice and promoted adipogenic

differentiation in preadipocyte and mesenchymal stem cells (115,170,171) (Fig. 4). This was
D
achieved by down-regulation of genes in the canonical Wnt pathway, an evolutionarily

conserved pathway that regulates crucial aspects of cell fate determination (172). Wnt
pathway is also known to suppress adipogenesis, as BMAL1 binds to the promoter of genes
A
at Wnt pathway to increase their transcription. These contradictory results could stem from a
change in the levels of the involved proteins throughout the adipogenic process. Further
studies are needed in order to delineate the role of BMAL1 in adipogenesis and how other
clock regulators fit into this mechanism.
BAT adipogenesis:
BMAL1 inhibits adipogenesis and thermogenic capacity (173). Global ablation of BMAL1 in
mice and adipocyte-selective inactivation increases brown fat mass and cold tolerance.
BMAL1 inhibits brown adipogenesis through direct transcriptional control of key
components of the cellular differentiation signaling pathways of transforming growth factor β
(TGFβ) and bone morphogenetic protein (BMP); activation of TGFβ or blockade of BMP

on 17 March 2018
pathways suppresses enhanced differentiation in BMAL1-deficient brown adipocytes (173).

REV-ERBα, the negative regulator of BMAL1 expression, promotes brown adipose tissue
development and adipogenesis. As opposed to BMAL1, REV-ERBα represses key
components of the TGFβ signaling pathway (174), an inhibitory pathway of brown fat
development (175,176). Thus, in WAT, BMAL1 primarily regulates the canonical Wnt
cascade, whereas in BAT, regulation of the TGFβ pathway is predominant (Fig. 4). Whether
the effects of BMAL1 and REV-ERBα are pleotropic or demonstrate the physiological
significance of the clock in BAT thermogenesis remain to be determined.
8. Future perspectives
Research during the last years has focused on characterizing the molecular mechanisms by
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which circadian clocks impact adipose tissue physiology. Synchronization of the molecular
clocks in the different types of adipose tissues with the rest of the body still merits further
investigation. The connections of circadian genetic variants and obesity now help to
understand the importance of the circadian system in adipose-related illnesses, such as
IC
obesity. More importantly, they may help to design individual therapies to obesity based on
the circadian system in order to increase the effectiveness of the treatments. Identification of
certain compounds capable of manipulating clock function at the cellular level is critical to
T
reprogram adipose tissue physiology.
Despite the immense knowledge achieved in the relevance of the internal clock in health
R
and disease, there is still a big gap between this molecular knowledge and clinical practice.
As explained above, a big effort has been done in the last years in order to connect the results
observed in experimental models (animals, tissues, cells) to humans. Currently, large-scale
A
epidemiological studies are repeatedly demonstrating that the alteration of the circadian
system has an impact on health and is associated with several adipose tissue-related
metabolic illnesses, such as obesity, metabolic syndrome or diabetes. Nevertheless, the
E
success in translating this knowledge to the clinical practice is still limited.

To achieve this goal, it is necessary a) to increase the understanding of the importance of
C
the circadian regulation of the endocrine system in medical practice; b) to develop new tools
that help to assess the circadian system performance in clinical practice; c) to design new
N
therapies based on the regulation of the circadian system in order to increase the effectiveness
of treatments of adipose tissue-related pathologies. Salivary samples, which may be non-
invasively collected at several time-points throughout the day, are useful to determine the
A
daily pattern of hormones that connect the circadian system and adipose tissue. In addition,
chronodisruption scores to assess metabolic syndrome in the clinical practice (177) or the use
V
of devices that allow to continuously measure activity, body or wrist skin temperature or
heart rates may help to assess daily patterns in the habitual life of the patient (178,179) and,
D
as a result, the circadian system performance.

In conclusion, timing is a crucial aspect in adipose tissue metabolism in order to ensure
A
the correct accumulation of fat during periods of excess energy or TAG mobilization when
energy requirements are not achieved. Therefore, different adipokines have to be secreted at
the correct time to confer the proper endocrine, paracrine or autocrine function. The current
knowledge of the presence of an internal clock in the different adipose tissue types, white,
brown and beige, and its connection with key elements in metabolism may help us to achieve
a better understanding of adipose tissue function, and to design novel strategies to combat
obesity.
Corresponding authors: *Oren Froy: Tel: +972-8-9489746 Fax: +972-8-9363208
E-mail: oren.froy@mail.huji.ac.il, *Marta Garaulet: Tel: +34-868-88-39-30 Fax:
+34-868-88-39-63 E-mail: garaulet@um.es

on 17 March 2018
Disclosure statement:
The authors have nothing to disclose
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Figure 1: The relationship between the core clock mechanism and metabolic factors.
CLOCK and BMAL1 mediate the expression of clock- and clock-controlled genes regulating
metabolism. PER1, PER2 and CRY1 serve as the negative feedback loop that inhibits
CLOCK:BMAL1-mediated expression. The catabolic factors SIRT1, PGC1 and AMPK,
when activated under low cellular energy levels, relieve the inhibition mediated by the
negative feedback loop. BMAL1 expression is positively regulated by RORs and PPARs and

on 17 March 2018
negatively regulated by REV-ERBs. mTOR, an anabolic factor interacts with BMAL1 and
suppresses the activity of the catabolic factors. The green and red arrows denote possible
pathways that activate or inhibit adipocyte metabolism, respectively.
Figure 2: Lineage of white, brown and “brite/beige” adipocytes from mesenchymal stem
cells and their cellular and molecular markers. UCP1 – uncoupling protein 1
Figure 3: Temporal expression of adipocytokines in rodents and humans. The peak

expression of each adipokine is depicted. Representation of the acrophase or timing of the
maximum levels of adipokines expression in human adipose tissue: comparison with human
circulating levels and with rodent adipose tissue mRNA levels. Data is represented as the five
LE
hours of active and resting phase in both humans and rodents because this is the timing when
most adipokines peak.
Figure 4: The relationship between the core clock mechanism and differentiation signaling
IC
pathways in brown and white adipose tissues. BMAL1 activates the transcription of PPARγ
and PER2. PER2 inhibits BMAL1 activity and PPARγ increases BMAL1 expression. Wnt
signaling represses WAT adipogenesis, whereas TGFβ/Smad3 signaling represses BAT
formation. BMP signaling induces BAT formation. Reduced levels of BMAL1 lead to
T
increased WAT and BAT formation.
R
Table 1: Clock gene mutations in mice and genetic variants in humans and their effect on
metabolism.
A
Clock Gene Mutant mice Metabolic alterations Human genetic Metabolic alterations in humans
in mutant mice variant
CLOCK Clock∆19 (109) Hyperphagic and obese, CLOCK SNPs Obesity, metabolic syndrome, higher
develop a metabolic (rs3749474, energy and fat intake, higher risk of
E
syndrome of rs4580704, and developing diabetes and hypertension,

hyperleptinemia, rs1801260 alterations in the autonomic nervous
hyperlipidemia, hepatic (3111T>C)) (123- system
steatosis, and 129)
C
hyperglycemia
BMAL1 Bmal1-/- (113) Suppressed diurnal BMAL2 rs7958822 High risk of developing type 2 diabetes
variations in glucose and (132) in obese patients
N
triglycerides and
abolished
gluconeogenesis
Cry1/2(-/-) (116-120)
A
CRYs Increased insulin CRY1 rs2287161 With high intake of carbohydrates,

secretion and lipid (136) carriers of the risk variant have
storage in adipose tissue, increased insulin resistance.
elevation of
V
proinflammatory
cytokines
PER2 Per2-/- (112) On high-fat diet, eat as PER2 SNPs Abdominal obesity, several obesogenic
D
much during the light (rs2304672C>G and behaviors

period as the dark period rs4663302C>T) (131)
and develop
significant obesity
A
REV-ERBα Rev-erbα-/- (134) Reduced spontaneous REV-ERBα Obesity resulting from decreased
locomotor activity rs2314339 (133-135) physical activity

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Froy and Garaulet, Figure 1
Reviews
Catabolic Factors:
IC
AMPK, PGC1, SIRT1
Endocrinology
T
Clock Negative Feedback Loop
ADVANCE ARTICLE: Endocrine
R
PER1, PER2, CRY1
A
Adipose tissue Anabolic Factor:
CLOCK BMAL1
metabolism mTOR
E
C
Lipid Metabolism Factors:

REV-ERBs
N
Lipid Metabolism Factors:

A
PPARs, RORs
V
D
A

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Froy and Garaule
Reviews
IC
Mesenchymal Precursors
Endocrinology
Myf5- Myf5+
T
WAT Precursor
R BAT Precursor
A
Mitochondrion
Nucleus
Mature Adipocytes
Nucleus
Mitochondrion
E
Lipid droplet
Lipid droplet
White Brite (Beige) Brown
C
UCP1- UCP1+ UCP1+

N
A
V
D
A

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LE
Froy and Garaulet, Figure 3
Reviews
Resting Active
IC
phase
Endocrinology
phase
Rodents
T
Humans
R
A
E
Adiponectin
C
Chemerin
Corticosteroids
N
IL-6
Insulin sensitivity
A
Leptin
PAI-1
V
PPARƳ
Visfatin
D
A

on 17 March 2018
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Froy and Garaule
REV-ERBα
Reviews
IC
BMAL1
Endocrinology
PER2
T
PPARγ
R
A
E
TGFβ/Smad3 signaling BMP signaling Wnt signaling

C
N
A
V
BAT WAT
D
A

on 17 March 2018

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The circadian clock in white and brown adipose tissue: mechanistic,

endocrine and clinical aspects

Oren Froy, Marta Garaulet

Submitted: July 24, 2017

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The circadian clock in adipose tissue

The circadian clock in white and brown adipose tissue: mechanistic,

activity of enzymes, hormones, and transport systems involved in metabolism.

1. Circadian rhythms and the circadian clock

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2. White, brown and beige (or brite) adipose tissue

was able to secrete hormones or “adipocytokines” capable of communicating information

passive reservoir of energy; it was an endocrine organ. Adipocytokines or adipokines, such as

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3. The circadian clock and metabolism

REV-ERBs and retinoic acid–related orphan receptors (RORs):

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AMP-activated protein kinase (AMPK):

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phosphoribosyltransferase), a rate-limiting enzyme in the NAD+ salvage pathway, therefore,

4. Chronodisruption and metabolic disorders

metabolic syndrome of hyperleptinemia, hyperlipidemia, hepatic steatosis and hyperglycemia

In mouse studies, circadian changes in hepatic Cry1/2 expression were sufficient to

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Some of these associations were functionally explained by the presence of a polymorphism

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energy expenditure as assayed by a physical activity questionnaire (145).

6. Circadian regulation of adipose tissue in health and disease

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sensitivity warrant further study.

thereby inhibiting transcriptional activation (169). As mentioned above, PPARγ expression is

ERBβ-deficient mice had increased orexinergic transcripts (74). In addition, cell-based

activation of REV-ERB, which lead to BMAL1 downregulation, would be expected to

adipogenesis, adipocyte hypertrophy and obesity in mice and promoted adipogenic

achieved by down-regulation of genes in the canonical Wnt pathway, an evolutionarily

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pathways suppresses enhanced differentiation in BMAL1-deficient brown adipocytes (173).

understand the importance of the circadian system in adipose-related illnesses, such as

success in translating this knowledge to the clinical practice is still limited.

as a result, the circadian system performance.

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disease in mice with a dysfunctional circadian clock. Circulation 2009; 119:1510-1517

health. J Physiol Paris 2013; 107:323-326

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