A

SEMINAR REPORT ON
ADAPTIVE FUNCTIONAL SIGNIFICANCE OF
CIRCADIAN CLOCK

SUBMITTED IN PARTIAL FULFILLMENT FOR THE AWARD OF DEGREE

MASTER OF SCIENCE IN ZOOLOGY (2021 -23)

GUIDED BY: - PRESENTED BY: -

Dr. MUNTAZ KHAN SUBHASMITA SAHOO

ROLL NO. - 2101PGZOO42

4th SEMESTER
M. Sc. Zoology

DEPARTMENT OF ZOOLOGY

KALAHANDI UNIVERSITY BHAWANIPATNA, KALAHANDI

 ACKNOWLEDGEMENT

I am greatly indebted to Dr. Subrat Ku. Panigrahi Sir, HOD of Dept.

of Zoology and Dr. Jatindra Ku. Pradhan Sir, Seminar in-charge of Dept. of
zoology, Kalahandi University; for giving me this opportunity to present a
seminar on the topic “Adaptive Functional Significance of Circadian
Clock”.

I wish to extend my sincere gratitude to my Seminar Guide Dr. Muntaz

Khan, Assistant Professor of Dept. of Zoology, Kalahandi University; for
his valuable guidance and encouragement which has been absolutely
helpful in successful completion of the seminar.

I am also grateful to my parents and friends for their timely aid.

And last but not least, I thank God Almighty for his blessings without
which the completion of the seminar would not have been possible.

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 CONTENT

I. GLIMPSES OF SLIDES OF SEMINAR PRESENTATION

II. DESCRIBED IN DETAIL:

i. INTRODUCTION
ii. DIFFERENCE BETWEEN CIRCADIAN CLOCK AND CIRCADIAN
CYCLE
iii. IMPORTANCE OF CIRCADIAN CLOCK
iv. MECHANISM OF CIRCADIAN CLOCK IN MAMMALS
v. PHYSIOLOGICAL FUNCTIONS CONTROLLED BY CIRCADIAN
CLOCK
vi. FACTORS AFFECTING THE CIRCADIAN CLOCK
vii. ADAPTIVE FUNCTIONAL SIGNIFICANCE

III. REFERENCE

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 INTRODUCTION
The environmental changes brought about by the rotation of earth around its axis
promote the development of endogenous timekeeper. The ability to adapt and respond
to changes in environmental conditions is essential for the survival and overall health of
all living things. Daily fluctuations in light and temperature provide predictable
changes in the environment. Mammals and other organisms have evolved a mechanism
by which they coordinate physiological processes, such as sleep and meals, with the
light/dark cycle. The evolutionarily-conserved circadian clock integrates external cues,
also known as zeitgebers (time-givers), to coordinate behaviors, metabolism and
physiology with the light/dark cycle.

The term circadian was coined by Franz Halberg in 1959. According to Halberg's
original definition:

• The term circadian comes from the Latin circa, meaning "approximately", and
dies, meaning "day". It may serve to imply that certain physiologic periods are
close to 24 hours, if not exactly that length. Herein, "circadian" might be applied
to all "24-hour" rhythms, whether or not their periods, individually or on the
average, are different from 24 hours, longer or shorter, by a few minutes or
hours.

In 1977, the International Committee on Nomenclature of the International Society

for Chronobiology formally adopted the definition:

• Circadian: relating to biologic variations or rhythms with a frequency of 1 cycle

in 24 ± 4 h; circa (about, approximately) and dies (day or 24 h).

Note: term describes rhythms with an about 24-h cycle length, whether they are
frequency-synchronized with (acceptable) or are desynchronized or free-running from
the local environmental time scale, with periods of slightly yet consistently different
from 24-hr.

To be called circadian, a biological rhythm must meet these three general criteria:

1) The rhythm has an endogenous free-running period that lasts approximately

24 hours.
2) The rhythms are entrainable.
3) The rhythms exhibit temperature compensation.

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 DIFFERENCE BETWEEN CIRCADIAN RHYTHM AND
CIRCADIAN CLOCK

Circadian Rhythms:

• These are internally driven cycles that rise and fall during the 24-hour day
• Help you fall asleep at night and wake you up in the morning
• The master circadian clock in the brain synchronizes and controls these cycles so
they work together.

Circadian Clock:

The circadian clock has an internally driven 24-hour rhythm that tends to run longer
than 24 hours but resets every day by the sun’s light/dark cycle. Taking melatonin
supplements can also shift the timing of the body’s “clock.”

The internal body clock sets the timing for many circadian rhythms, which regulate
processes such as:

• Sleep/wake cycles
• Hormonal activity
• Body temperature rhythm
• Eating and digesting

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 IMPORTANCE OF CIRCADIAN CLOCK

Circadian clock regulates various aspects of metabolism, physiology and behavior.

Circadian rhythmicity is present in the sleeping and feeding patterns of animals,

including human beings. There are also clear patterns of core body temperature, brain
wave activity, hormone production, cell regeneration, and other biological activities.

In addition, photoperiodism, the physiological reaction of organisms to the length

of day or night, is vital to both plants and animals, and the circadian system plays a role
in the measurement and interpretation of day length.

Timely prediction of seasonal periods of weather conditions, food availability, or

predator activity is crucial for survival of many species. Although not the only
parameter, the changing length of the photoperiod (day length) is the most predictive
environmental cue for the seasonal timing of physiology and behavior, most notably for
timing of migration, hibernation, and reproduction.

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 MOLECULAR BASIS OF CIRCADIAN CLOCK IN MAMMALS

Circadian rhythms are driven by an internal timing system regulated at the

transcriptional level that gives rise to gene networks that oscillate with a 24-h cycle.
Within these networks are clock genes that control rhythms in physiology and behavior.

The circadian clock in mammals is cell-autonomous and depends on transcriptional

autoregulatory feedback loops. Circadian rhythms are also tuned at the post-
transcriptional and post-translational levels, although gene transcription remains vital
for making the clock ‘tick’.

Genome-wide approaches have found that rhythmic transcription is accompanied

by rhythmic transcription factor binding and histone modifications in enhancer regions,
as well as by circadian recruitment of RNA polymerase II (Pol II) to DNA. An
additional layer of regulation involves chromosome organization, with interactions of
active and repressive chromosomal domains undergoing circadian oscillations.

The molecular mechanisms regulating circadian rhythms is based upon

Transcription-translation feedback loop (TTFL). Circadian Clocks in different
organisms use different set of genes that are similar in terms of their function in the
molecular feedback loop. Clock controlled genes ( CCGs) regulated by the TTFL are
largely tissue specific and underlie the sleep/wake cycle, feeding and fasting ; and
other complex physiologies.

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Fig. 1.Transcription-Translation Oscillating (TTO) Loop model. In the positive arm of
the TTO loop, Clock, and Bmal1 heterodimerize to activate transcription of circadian
target genes, including Per(homologs: 1–3), Cry (homologs:1–2), ROR, and Nr1d1(REV-
ERB-α). In the negative arm of the TTO loop, Per and Cry are thought to interact and
inhibit the action of Bmal1 and Clock, thereby decreasing their own transcription. ROR
and REV-ERBα mediate opposing actions on Bmal1 gene expression. Per1 is
phosphorylated by the circadian regulatory kinases casein kinase 1δ/ε, which allows it
to enter the nucleus.

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 THE MOLECULAR CLOCK MECHANISM IN MAMMALS
In nearly every cell in the body, there is an evolutionarily-conserved time-keeping
mechanism that is generated by transcriptional activators and repressors. The activators
drive expression of their own repressors, creating a self-regulating negative feedback
loop. The roughly 24-h oscillating transcriptional activity, regulated by activators and
repressors within this transcriptional-translational feedback loop (TTFL), gives rise to
circadian rhythms of gene expression.

In mammals, the TTFL is formed by core clock proteins that work in pairs to
activate and repress transcription.

The positive regulatory arm of the TTFL consists of Brain and Muscle Arnt-Like
protein 1 (BMAL1), also known as ARNTL, and Circadian Locomotor Output Cycles
Kaput (CLOCK).

• These transcriptional activators heterodimerize in the cytosol before

translocating to the nucleus.
• BMAL1/CLOCK complex recognize the consensus E-box elements in
promoter regions to regulate gene expression. The intrinsically disordered C-
termini of BMAL1 and CLOCK allow for promiscuous binding with
transcriptional coactivators and repressors to form large complexes.
• Following DNA binding, the transactivation domain (TAD) in the distal C-
terminus of BMAL1 initiates transcription of targeted genes, including
Ø PER 1/2/3
Ø CRY 1/2
Ø NR1D 1/2 (REV-ERB α/β)

• Coactivators CBP and p300 assist with transcription by stabilizing the BMAL1
TAD.

Posttranslational modifications and timely nuclear localization are key

regulatory steps of the clock to introduce delay in transcriptional repression and control
of periodicity. The negative regulatory arm of the TTFL consists of repressors PERIOD
(PER1/2/3) and CRYPTOCHROME (CRY1/2).

• PER and CRY form a heterodimer as they accumulate in the cytosol before
translocating to the nucleus.

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 • The PER and CRY proteins are post-translationally regulated by parallel E3
ubiquitin ligase pathways (FBXL3 and FBXL21 for CRY and β-TrCP for PER),
with PER levels being also regulated by CK1.
• CRY acts as the primary BMAL1/CLOCK repressor, binding the CLOCK PAS-B
domain and occupying the BMAL1 TAD, and can repress E-box transcription via
a blocking-type mechanism in the absence of PER.
• Then PER/CRY recruit casein kinases (CKs), which phosphorylate
BMAL1/CLOCK, disrupting its association with the E-box.
• PER acts predominantly as a facilitator for the formation of the
BMAL1/CLOCK/CRY complex, while CRY can act independently to inhibit
transcription by BMAL1/CLOCK complex.
• PER and CRY thereby inhibit their own expression. Gradually, PER and CRY
degrade, allowing CLOCK and BMAL1 complex to resume transcription
activities.

An additional oscillating feedback loop exists, in which rhythmic expression of

BMAL1 and CLOCK is regulated by two nuclear receptor subfamilies: activators,
retinoic acid receptor-related orphan receptors (RORα/β/γ) and repressors, REV-
ERBα/β (encoded by NR1D1/2 genes).

• ROR binds the ROR response elements (RRE) in the BMAL1 and CLOCK
promoters and is inhibited by REV-ERB. That is REV-ERB rhythmically repress
the transcription of Bmal1 and Nfil3, two genes that are activated by retinoic
acid-related orphan receptor-α/β (RORα/β).
• REV-ERB is regulated in turn by NFIL3 together with D-box binding protein
(DBP), as well as CLOCK and BMAL1 through the E-box in its promoter,
regulate a rhythm in the REV-ERBα/β nuclear receptors.
• Further, circadian expression of CRY1 is also mediated by the RRE and the D-
box, in addition to the E-box.
• Rhythmic DBP expression is regulated through the DBP promoter E-box by
BMAL1/CLOCK.

These three interlocked transcriptional feedback loops regulate the majority of

cycling genes. Timely trafficking and degradation of core clock proteins BMAL1,
CLOCK, PER, and CRY is what drives generation of 24-h rhythms.

The BMAL1/CLOCK complex transcriptionally regulates thousands of genes,

giving rise to a hierarchical network of oscillators at the cellular, tissue, and organismal
levels. This network allows for clock regulation of cellular processes, including growth

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and metabolism, tissue outputs, such as muscle strength and endocrine signaling, and
complex physiological outputs like the sleep/wake cycle.

The mammalian circadian clock is able to coordinate these and many other
processes with predictable daily changes in the environment. The negative feedback
time-keeping mechanism of the TTFL is highly conserved in many organisms and
works using the same mechanism in virtually every type of tissue in the body,
highlighting its functional significance.

SUPRACHIASMATIC NUCLEUS (SCN)

• The circadian system is hierarchical and begins at the suprachiasmatic nucleus

(SCN), which is a bilateral structure located in the anterior hypothalamus, that
connects nervous system with endocrine systems.

• The SCN is considered the master circadian pacemaker in mammals because it

synchronizes all downstream peripheral clocks to the light/dark cycle.

• Multiple afferent neuronal tracts projects to the SCN. Its major tract is the retino-
hypothalmic tract originating from photosensitive ganglion cells of the retina.
Efferent projections from the suprachiasmatic nucleus innervate structure such
as the pineal gland, producing melatonin during the night for induction of sleep.

• The SCN regulates daily various behavioral and physiological rhythms through
a complex neural circuitry within the hypothalamus.

• Internally, the SCN can be delineated into two subregions composed of various
cell types expressing different neuropeptides:
Ø the ventral core, an
Ø the dorsal shell.

• In the core region, vasoactive intestinal polypeptide (VIP) is the predominant

neuropeptide and in the SCN shell region, arginine vasopressin (AVP) is
primarily expressed.

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 (a)

(b)

Fig- 2 (a) and (b): Diagrammatic representation of hypothalamus gland and location of
Suprachiasmatic Nucleus in human brain.

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Entrainment of the Central Suprachiasmatic Nucleus Clock by
Light:

• The light input pathway of the SCN starts at the retina, which is essential for
synchronizing the circadian clock to the local light/dark cycle.

• The retina contains intrinsically photosensitive retinal ganglion cells (ipRGCs),

which respond to short-wave light through the G-coupled protein receptor
(GPCR), melanopsin.

• ipRGCs can be separated into six classes (M1-6), of which the M1 class contains
the highest expression of melanopsin.

• While melanopsin phototransduction appears to differ between classes, the

photoisomerization reaction that occurs is the same. Melanopsin readily
interconverts between three states: two resting states, R and E, and an excited
state, metamelanopsin (M), that is the intermediate between R and E . Because
short-wave light is more easily absorbed by resting states than the M state, it
drives melanopsin to the M state.

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• When excited, the activated GPCR signals downstream effectors to depolarize
cells by opening intracellular ion channels. However, the melanopsin-activated
second-messengers have not clearly been identified and evidence suggests that
they differ between ipRGC classes.

• Light strikes the retina and excites melanopsin, driving it to the M configuration.
The signal travels along the retinohypothalamic tract (RHT), resulting in
increased intracellular levels of Ca2+ and cAMP in the SCN. Ca2+ activates
calmodulin (CaM) andcalmodulin-dependent kinase II (CaMKII). cAMP
activates PKA. CaMKII and PKA activate cAMP response element-binding
protein (CREB), which drives PER1/2 transcription. Rhythmic cAMP-CREB
signaling and control of PER1/2 expression drives SCN synchronization to the
light/dark cycle.

• Melatonin binds the melatonin receptor (MT), which inhibits CREB activation.
There is bidirectional regulation between melatonin secretion and sleep, sleep
and core body temperature, and core body temperature and SCN signaling.

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 PHYSIOLOGICAL FUNCTIONS CONTROLLED BY CIRCADIAN
CLOCK
1) Circadian control of sleep:

Human circadian sleep disorders and their genetic causes –

• In humans, mutation in circadian clocks have been associated with circadian

rhythm sleep disorder.
a) Familial advanced sleep phase disorder (FASPD):
Ø FASPD is a circadian rhythm sleep disorder with habitual sleep times that
are earlier than the societal norm.
Ø Causes identified for FASPD are
§ A missense mutation (S662G) in the PER2 gene.
§ A missense mutation (T44A) in the human CKId gene.
§ A missense mutation in the human CRY2 gene.

b) Delayed sleep phase disorder (DSPD):

Ø DSPD is characterized as a type of insomnia with inconsistent and
delayed sleep onset and offset times, compared to the societal norm.
Ø Familial cases of DSPD have been described, suggesting that Mendelian
inheritance of DSPD gene may exist with polymorphisms in the CLOCK or
PER3 gene; which lengthen the period of Circadian molecular rhythms.

2) Circadian control of Metabolism:

• The Circadian clock controls metabolism directly by driving transcriptional
programs for certain metabolic pathways. For example, CRY1 suppresses
Hepatic-gluconeogenesis during fasting through the regulation of cAMP/CREB
signaling, the rhythmic repression of the Glucocorticoid receptor gene, and the
suppression of nuclear FOXO1 that in turn downregulates Gluconeogenesis.
• Another clock repressor, PER2, controls lipid metabolism by direct regulation of
peroxisome proliferator-activated receptor gamma (PPARg) and mitochondrial
rate limiting enzymes.
• The nuclear hormone receptors, REVERBs regulate the transcription of several
key rate-limiting enzymes for fatty acid and cholesterol metabolism.
• Disruption of CLOCK and BMAL1 has also been associated with obesity, hyper-
insulinemia, and diabetes.

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3) Circadian control of the Immune system:
• Circadian clock controls many aspects of immune response, from trafficking of
immune cell to the activation of innate and adaptive immunity.
• Neutrophils and Monocytes, exhibit circadian patterns of migration from the
blood to tissues.
• T and B Lymphocytes, also exhibit strong circadian oscillation in the blood, with
their numbers peaking during an organism’s resting phase.
• Glucocorticoids induce Interleukin-7 receptors (IL-7R) with a diurnal rhythm,
thereby increasing Chemokine receptor CXCR4 expression and supporting T cell
survival and recruitment to various tissues. The diurnal variation in T cell
distribution enhances immune responses to soluble antigens and systemic
bacterial infection at night.

4) Circadian clock and the kidney:

• Multiple renal functions exhibit a circadian rhythm including renal blood flow,
glomerular filtration rate, potassium excretion, and sodium excretion.
• Per1 is involved in the basal and aldosterone-induced regulation of the α-subunit
of the epithelial sodium channel (ENaC) in the renal collecting duct . ENaC is the
key mediator of sodium reabsorption in the renal collecting duct and is
responsible for the fine-tuning of sodium reabsorption and blood volume control
by the kidney.

5) Circadian control of Endocrine System:

• The circadian clock plays a key role in the regulation of the endocrine system, but
in turn, the endocrine system plays an important role in the synchronization and
regulation of the peripheral clocks.

• The glucocorticoids, a group of steroid hormones synthesized in the adrenal

cortex, are involved in mediating anti-inflammation, the stress response,
metabolism, cardiovascular, and neurological function. One of the proposed roles
of the glucocorticoids is in synchronization of the circadian clock.

• Melatonin is a hormone secreted from the pineal gland and other tissues, which is
controlled by light. It behaves as a feedback mechanism for the central clock in
the SCN by inhibiting neuron firing.

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6) Circadian control of Reproductive System:

• Release of GnRH and GnRH neuron activity is known to display a circadian

pattern.
• BMAL1 protein is responsible for ovulation, estrous cycle and implantation in
female; and spermatoid maturation in male.

7) Circadian control over Cardio-Vascular System:

• Cardiovascular complications are higher incidence in the morning, with daily
variation in blood pressure.
• Pharmacological targeting of REV-ERB decreases atherosclerotic plaque burden in
mice.
• Deletion of Bmal1 in myeloid cells increased monocyte recruitment and
atherosclerosis lesion size.

8) Circadian control of Nervous System:

• Suprachiasmatic nucleus (SCN) imposes rhythmicity throughout the body.
• Astrocyte cell help in maintaining circadian rhythm.
• Permeability of blood-brain-barrier is controlled by circadian rhythm.
• There are two Retina-brain pathways that integrate light and highlight its
influence on learning and mood.

Summary:

The circadian timing system or circadian clock plays a crucial role in many biological
processes, but the increase in activities that operate 24/7 and the common usage of
television, internet, and mobile phones almost 24 h a day leads to a gradual decrease in
the adequate sleeping time. According to recent research, long-term circadian disruptions
are associated with many pathological conditions such as premature mortality, obesity,
impaired glucose tolerance, diabetes, psychiatric disorders, anxiety, depression, and
cancer progression, whereas short-term disruptions are associated with impaired
wellness, fatigue, and loss of concentration. In this review, the circadian rhythm in
metabolic processes and their effect on energy balance were examined.

Key Messages: Circadian rhythm has a bidirectional interaction with almost all metabolic
processes. Therefore, understanding the main reason affecting the circadian clock and
creating treatment guidelines using circadian rhythm may increase the success of disease

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treatment. Chronopharmacology, chrononutrition, and chronoexercise are the novel
treatment approaches in metabolic balance.

Fig. 2: Integrative role for the circadian clock in the regulation of physiological function.
Circadian proteins listed in parentheses dictate which proteins have been implicated in
regulating the process in question. If a protein is not listed, it does not imply that it is
not involved, but that it has not yet been tested. Green arrows represent induction by
the circadian protein; red arrows represent repression. The time shown on the clock is
for illustrative purposes only.

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 FACTORS AFFECTING CIRCADIAN CLOCK

Circadian rhythmicity is regulated by following environmental factors:

• Light-dark cycle (most important)

• Temperature
• Oxygen availability
• Arousal stimuli

Environmental chemicals affecting circadian rhythm:

• Steroid hormone
• Metals
• Pesticides and biocides
• Neuroscience drugs
• Oxidative stress

Other factors influencing circadian clock:

• Molecular Clock
• Gene expression
• Metabolic regulation
• Nutrients
• Physical movements

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 ADAPTIVE FUNCTIONAL SIGNIFICANCE OF CIRCADIAN CLOCK
Circadian clock serves as an adaptation for a rotating world by creating an
opportunity for an organism to anticipate daily external environmental changes and
prepare accordingly. The process of natural selection leads to adaptation. Darwin
proposed that individuals with variations which are better suited to the environment
they inhabit, manage to survive and reproduce and in turn leave more offspring (and
thus more contribution to the gene pool) to the next generation than those with less
suitable or harmful variations. Darwin refereed to this process of selective enrichment
of advantageous variation as Natural Selection. Therefore, a train which comes into
being through a process of natural selection under a given environment suggests its
suitability to that environment and is referred to as an adaptation.

It is believed that an organism possessing circadian clocks gains fitness advantage

in two ways:

• (i) by synchronizing its behavioral and physiological processes to cyclic

environmental factors (extrinsic adaptive value);
• (ii) by coordinating its internal metabolic processes (intrinsic adaptive value).

a) EXTRINSIC ADAPTIVE VALUE:

• Circadian rhythms confer fitness advantage by scheduling various biological

functions at specific times of the day.
• This is done by avoiding harsh conditions, procuring more mates, finding more
food. E.g. Probability of successful mating can be increased by synchronizing
activity schedules of conspecifics.
• Chances of being preyed upon can be reduced by scheduling activities at a time
when predators are asleep.

b) INTRINSIC ADAPTIVE VALUE:

• Circadian rhythms benefit organism by coordinating their internal metabolic

processes.
• Synchronization of internal rhythms (sleep-wake, hormone concentration,
immune response, metabolic enzymes) is an essential aspect of physiology.
Sleeping at night and feeding during the day.
• Body reaches lowest temperature at night when we sleep, the blood pressure
drops and rate of metabolic reactions is low. It would be physiologically
compatible if we experienced hunger and sleep at same time as we would not be
in the right state to metabolize food efficiently.

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 REFERENCE

• Adaptive significance of circadian clocks, VK Sharma - Chronobiology

international, 2003 Taylor & Francis
• Genomics of circadian rhythms in health and disease, F Rijo-Ferreira, JS
Takahashi - Genome medicine, 2019 - Springer
• Circadian synchrony: sleep, nutrition, and physical activity, KL Healy, AR
Morris, AC Liu - Frontiers in network physiology, 2021 - frontiersin.org
• Circadian Clocks and Metabolism; Biliana Marcheva, Kathryn M. Ramsey,
Clara B. Peek, Alison Affinati, Eleonore Maury, and Joseph Bass, PMCID:
PMC4089089NIHMSID: NIHMS594044PMID: 23604478
• Mechanism of the circadian clock in physiology; Am J Physiol Regul Integr
Comp Physiol. 2013 Jun 15; 304(12): R1053–R1064.
Published online 2013 Apr 10. doi: 10.1152/ajpregu.00066.2013
PMCID: PMC4073891,PMID: 23576606

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