Melatonin as a Hormone: New Physiological and Clinical ... › pluginfile.php › 4605959...Melatonin as a Hormone: New Physiological and Clinical Insights Jos´e Cipolla-Neto 1 and

Melatonin as a Hormone: New Physiologicaland Clinical Insights

José Cipolla-Neto1 and Fernanda Gaspar do Amaral2

1Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, 05508-000 São Paulo, Brazil; and 2Department of Physiology, Federal University of São Paulo, 04023-900 São Paulo,Brazil

ORCiD numbers: 0000-0003-3748-3731 (J. Cipolla-Neto); 0000-0003-4492-4822 (F. G. Amaral).

ABSTRACT Melatonin is a ubiquitous molecule present in almost every live being from bacteria to humans. In vertebrates, besides being

produced in peripheral tissues and acting as an autocrine and paracrine signal, melatonin is centrally synthetized by a neuroendocrine organ,

the pineal gland. Independently of the considered species, pineal hormonemelatonin is always produced during the night and its production

and secretory episode duration are directly dependent on the length of the night. As its production is tightly linked to the light/dark cycle,

melatonin main hormonal systemic integrative action is to coordinate behavioral and physiological adaptations to the environmental

geophysical day and season. The circadian signal is dependent on its daily production regularity, on the contrast between day and night

concentrations, and on specially developed ways of action. During its daily secretory episode, melatonin coordinates the night adaptive

physiology through immediate effects and primes the day adaptive responses through prospective effects that will only appear at daytime,

when melatonin is absent. Similarly, the annual history of the daily melatonin secretory episode duration primes the central nervous/

endocrine system to the seasons to come. Remarkably, maternal melatonin programs the fetuses’ behavior and physiology to cope with the

environmental light/dark cycle and season after birth. These unique ways of action turn melatonin into a biological time-domain–acting

molecule. The present review focuses on the above considerations, proposes a putative classification of clinical melatonin dysfunctions, and

discusses general guidelines to the therapeutic use of melatonin. (Endocrine Reviews 39: 990 – 1028, 2018)

“We wish to report isolation from beefpineal glands of the active factor thatcan lighten skin color and inhibit MSH.It is suggested that this substance becalled ‘melatonin’. ” (1)

I t was and the hard-working team of re-searchers led by Aaron Lerner finally managedto isolate the pineal active factor that had beenstudied for years and was named for its ability toaggregate melanin granules within the melanocytes.Sixty years later, those researchers’ work (), alongwith more than , other published researcharticles, have shown that melatonin has myriadfunctions that encompass every live organism inwhich its presence was attested. From bacteria tohumans, this rather simple molecule has proven itsskill to be involved in the sustainment of life. Thisreview is dedicated to the study of melatonin as a

mammalian pineal hormone and focuses on humanphysiology and pathophysiology.

It is noteworthy that in spite of the impressiveamount of data on melatonin and pineal research, astandard and systematic theoretical framework ofanalysis is lacking, among researchers and clinicians,that would lead to appropriate interpretation of theobtained data and adequate understanding of the roleplayed by the hormone melatonin in human physi-ology and pathophysiology. To contribute to thisdiscussion, it is the authors’ intention to propose aframework of analysis that would help researchers andhealth professionals to analyze, understand, and in-terpret the effects of melatonin and its putative role inseveral pathologies. The first point is the proposal of aclassification of melatonin’s well-known ways of actionand ensuing effects based on the present experimentaland clinical knowledge. It intends to clarify that thehormonal effects of melatonin cannot be deduced and

ISSN Print: 0163-769X

ISSN Online: 1945-7189

Printed: in USA

Copyright © 2018

Endocrine Society

Received: 5 March 2018

Accepted: 21 June 2018

First Published Online:

12 September 2018

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interpreted exclusively, as is usually done for otherclassic hormones, as a result of its ongoing immediateinteraction with its molecular effectors. As presentedhere, several of its effects are primed by this interactionbut will appear only several hours afterward, providedmelatonin is not present anymore. Additionally,several of its hormonal effects directly depend on thecircadian and seasonal characteristics of pineal mel-atonin synthesis and secretion, which are dependent onits daily repetition, daily duration of nocturnal signal,and seasonal direction of changing (increasing or de-creasing period of synthesis), resulting in the timing ofthe physiological phenomena in the circadian andannual timescale. The second point, based on theunderstanding of melatonin’s ways of action, is topropose a classification of melatonin-related putative

clinical syndromes, considering the classic hypo-production and hyperproduction or function, andintroducing the dysfunctions that directly depend onmelatonin temporal signal organization and melatonintiming action. Finally, as a third point, the authorsintroduce a discussion about the therapeutic use ofmelatonin, taking into consideration its characteristicways of action.

The definition of a common intellectual frame-work is essential to guide the planning of experi-mental and clinical research and data interpretation,allowing the construction of a solid foundation toproperly understand the melatonin functional roleas a hormone in human physiology and conse-quently to interpret and deal with its dysfunction inhuman pathology.

Melatonin—the Basics

Melatonin, chemically characterized in (), is anamphiphilic tryptophan-derived indoleamine (.molecular weight) proficient in free radical scavengingwith noteworthy antioxidant properties due to itsadditional ability to stimulate antioxidant enzymesin different tissues. This ancient role has been pro-posed as melatonin’s primary function that is con-served throughout evolution, as melatonin has beenfound in numerous live organisms from differenttaxa, including cyanobacteria, dinoflagellates, fungi,flatworms, molluscs, starfish, insects, yeast, plants, fish,amphibians and reptiles, and birds and mammals(–).

Melatonin synthesis has been described in all of theabove-mentioned organisms, presenting autocrine andparacrine actions. It is also valid for several tissuesand organs of multicellular organisms that presentlocal melatonin production with specific intracrine,autocrine, and paracrine effects, such as retina,gastrointestinal tract, bone marrow, lymphocytes,and skin (, ). Vertebrates, in addition to that,present a specialized gland, the pineal gland, that

synthesizes melatonin to serve as a hormone withendocrine actions.

Pineal gland melatonin synthesis in mammals istimed by the hypothalamic suprachiasmatic nucleus(SCN) master clock and synchronized to the light/darkcycle by the retinal intrinsic photosensitive ganglioncells whose projections to the SCN convey the envi-ronment photoperiodic information so that melatoninproduction is confined to the dark phase of the night(). Importantly, note that melatonin synthesis isblocked by light at night, an effect mediated by theretinal melanopsinergic system and a complex neuralsystem (–) that culminates in inhibition of thesympathetic projection to the pineal gland. In humansthis photoinhibition phenomenon is determined bylight preferably in the blue range ( to nm) andin intensities starting at , lux ( to lux)(–). These two facts (synchronization by the light/dark cycle and nocturnal photoinhibition) are im-perative for the role of melatonin as a time domainmolecule that synchronizes the organism’s internaltemporal order to the external daily and seasonal light/dark environment, as presented here (see “Chrono-biotic effects” and “Seasonal effects”).

ESSENTIAL POINTS

· Melatonin is considered a pineal hormone· Melatonin is a biological time–domain molecule acting on the circadian, seasonal, and transgenerational timescales· Melatonin developed special ways of action· The ways of action determine immediate effects expressed during the night and prospective effects expressed during thefollowing day

· The prospective effects are classified as proximal effects, expressed immediately after melatonin ceases in the earlymorning, and distal lengthy effects, expressed throughout the following day

· The following melatonin-related syndromes are described: hypomelatoninemia, hypermelatoninemia, circadiandisplacement, and inappropriate melatonin receptor–mediated responses

· General guidelines about therapeutic uses of melatonin are discussed

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Pineal melatonin synthesis timing by the SCN isachieved by its projections to the paraventricularhypothalamic nucleus, which communicates with thehigher thoracic segments of the intermediolateralspinal column, conveying information to the superiorcervical ganglion from where sympathetic postsynapticfibers reach the pineal gland, releasing norepinephrineexclusively during the dark phase of the night, trig-gering the enzymatic conversion of tryptophan tomelatonin (, ). Tryptophan hydroxylase convertstryptophan to -hydroxytryptophan, which is convertedto serotonin, which, in turn, is acetylated to N-acetylserotonin by arylalkylamine N-acetyltransferase(AANAT), and N-acetylserotonin is converted to mel-atonin by acetylserotonin O-methyltransferase. Nor-epinephrine activates b and ab adrenergic receptorsthat increase cAMP and protein kinase A (PKA) ac-tivity, ultimately increasing cAMP response elementbinding protein (CREB) phosphorylation and AANATactivity, among other mechanisms, leading tomelatoninsynthesis activation (, ). The above-mentionedneural control of melatonin production is also seenin humans, as neurologic patients showing tetraplegiaor lesions on the cervical spinal cord or superior cervicalganglion and its sympathetic trunks and patientssubmitted to surgical sympathectomy or who are undertreatment with beta-blockers show very low levels ofmelatonin production, losing the expected nocturnalincrease and, consequently, its circadian rhythm(–). Additionally, note that this particular area ofsympathetic innervation is not activated by the well-known characteristic mass mobilization of the sym-pathetic system, both in humans and in experimentalanimals ().

Pineal melatonin is not stored, being readily re-leased to the bloodstream (where it is bound to al-bumin) and to the cerebrospinal fluid (CSF), reachingseveral areas of the central nervous system (CNS) and allperipheral organs, where it will trigger different effectsby various mechanisms of action that are pointed outbelow. Melatonin half-life in the blood is ~minutesin humans, as it is converted to -hydroxymelatoninby cytochrome P isoforms (mainly CYPA) andconjugated to -sulfatoxymelatonin in the liver andkidneys for subsequent urinary excretion (). In theCNS, melatonin is metabolized toN-acetyl-N-formyl--methoxykynuramine that is deformylated to N-acetyl--methoxykynuramine (, ).

Maternal melatonin is the only source of thishormone both to the mammalian fetus (via placentalcirculation) and to the mammalian newborn (viabreastfeeding), as their pineal glands do not producemelatonin until later after birth (–). The ontogenyof human melatonin production was well studied(–) and pineal melatonin was found in - to-month-old full-term infants, reaching its peak inprepubertal children, reducing after puberty andreaching the young adult level (, ). After years

of age according to some researchers () and after years of age according to others (), pineal melatoninproduction declines to % of the young adult level.From there on, there is a continuous decline to valuesas low as % of the young adult level in people $years of age (). Importantly, note that pineal mel-atonin production is always higher in women at allages after puberty ().

Melatonin, Mechanisms of Action, Ways ofAction, and Effects

Melatonin mechanisms of actionAs for its amphiphilicity, melatonin is able to cross thecell, organelles, and nuclear membranes and directlyinteract with intracellular molecules in the so-callednon–receptor-mediated actions. In addition to that,melatonin also presents receptor-mediated actionsthat result from the interaction of this hormone withboth membrane and nuclear receptors.

Non–receptor-mediated actionsMelatonin is a well-known effective antioxidant, as it isboth a proficient direct free radical scavenger [and so areits metabolites ()] and an activator of a series ofscavenging mechanisms such as stimulation of thetranscription and activity of antioxidative enzymes (, )and binding to transition metals that inhibits the for-mation of the hydroxyl radical (). Besides that, mela-tonin protects lipids, protein, and DNA from oxidativedamage (, ), being highly concentrated in the mi-tochondria (). The mechanisms of melatonin antioxi-dant actions are extensively reviewed elsewhere (, ).

The antioxidant properties of melatonin are ofcrucial importance for the mitochondrial functions, asite were free radicals are naturally formed as a result ofcellular respiration [reviewed in ()]. Indeed, mela-tonin plays critical roles in mitochondrial functionbesides the antioxidant protection such as regulationof respiratory chain complexes I and IV activities ()and protection of mitochondrial DNA frommutationsand deletions (). It was recently demonstrated thatmelatonin is synthetized in mice brain mitochondriaand acts through the mitochondrial external mem-brane melatonin receptor (MT), preventing cyto-chrome c leakage and subsequent apoptosis (an actiondefined as automitocrine) ().

Some of the above-mentioned effects are usually aconsequence of melatonin–protein direct interaction.It is also notable that melatonin plays a role in theregulation of the ubiquitin–proteasome system thatultimately controls protein degradation. Melatoninwas reported to inhibit Ca+/calmodulin-dependentprotein kinase II activity and autophosphorylation by adirect interaction with Ca+-activated calmodulin,acting as an antagonist (). It has also been suggestedthat melatonin influences clock genes expression (see

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“Prospective effects”) by acting as a direct proteasomeinhibitor ().

Protection against DNA damage is fundamentaland melatonin has shown to be efficient in doing sodue to its antioxidant properties, once elevated reactiveoxygen species (ROS) levels are a major cause of DNAdamage. Additional mechanisms include the decreaseof ATM (a phosphoinositide -kinase–related kinase)expression and of the histone HAX phosphorylation, astep involved in the DNA damage response, amongothers [reviewed in ()].

Receptor-mediated actionsThe discovery, cloning, and characterization of mel-atonin membrane receptors were performed in the lates and early s (–). MT and melatoninreceptor (MT), formerly named Mela and Melb,are high-affinity specific G protein–coupled receptorsencoded by theMTNRA (human chromosome q.)and MTNRB (human chromosome q–q) genes.Human MT is a –amino acid protein, and humanMT is a –amino acid protein with predicted mo-lecular masses of , and , Da, respectively, thatwere found in several areas of the CNS, including theSCN, mediobasal hypothalamus, thalamus, temporal,parietal, and frontal cortex, hippocampus, the preopticarea, basal ganglia, area postrema, retina, cerebellum, andpars tuberalis (PT) [reviewed in (, )]. Peripheralorgans such as adipose tissue (), kidney (), pancreaticislets (), parotid glands (), adrenal glands (), liver(), bone (), skin (), reproductive tract (–),immune cells (), and cardiovascular system (CVS)[reviewed in ()], among others, also present MT andMT melatonin receptors.

MT and MT melatonin receptors are hetero-trimetric Gi/Go and Gq/ protein–coupled receptors thatinteract with downstream messengers such as adenylylcyclase, phospholipase A, phospholipase C, and calciumand potassium channels, generally decreasing cAMP andcGMP production and/or activating phospholipase C.MT andMT usually dimerize, forming homodimers orheterodimers that keep both melatonin binding sitesfunctional and with the respective selectivity (). GPRis another G protein–coupled receptor that may dimerizeto MT, reducing its affinity to melatonin and to mel-atonin agonists, being a potential regulatory step of thissignaling mechanism (, ).

MT signaling pathways involve, for example, ()activation of Kir./. potassium ion channels thatmediate the inhibition of neuronal firing in the SCN(, ); () modulation of protein kinase C (PKC) andphospholipase A (); () modulation of specific ionchannels by MT coupling to Gq/ proteins (); ()mitogen-activated protein kinase kinase /–ERK/pathway stimulation in nonneuronal cells (, ); and() vasoconstriction (, ).

Complementary MT signaling pathways involve,for example, () inhibition of cGMP formation and

stimulation of PKC activity in SCN (); () regulationof uterus contractility (); and () vasodilatation (). Itwas recently suggested that MT melatonin receptors inthe SCN might correspond to a G protein–coupledinwardly rectifying potassium channel ().

MT (previously named ML) is a third charac-terized mammalian melatonin binding site (not con-sidered a receptor) that is a form of quinone reductase ,a detoxifying enzyme (, ), and was reported to beinvolved, for example, in the melatonin-derived in-crease of chemotherapeutic-induced cytotoxicity andapoptosis in tumor cell lines ().

Melatonin may also interact with nuclear receptorsof the retinoic acid–related orphan receptor (ROR)/retinoid Z receptor group, although that is still con-troversial in the literature (–). Melatonin-induceddecrease of -lipoxygenase gene transcription wasshown to involve ROR/retinoid Z receptor melatoninsignaling ().

Melatonin ways of action and effectsIn view of the aforementioned general characteristicsof pineal melatonin production and its several specificmechanisms of action, it is noteworthy that to ac-complish its physiological role, melatonin presentsseveral ways of action that will determine differenttime-allocated effects. It is important to note thatmelatonin’s ways of action and ensuing effects mightvary according to the considered physiological system;however, they should be broadly taken into account tofully understand and interpret melatonin physiologyand pathophysiology.

Immediate effectsImmediate effects are the consequence of what canbe called the classic hormonal way of action and arerelated to melatonin being present in biologicalfluids and its instant interaction with correspondingmolecular effectors. Therefore, these effects areexpressed during the night when melatonin is re-leased by the pineal gland and is present in bloodand CSF.

Examples of these immediate effects are describedin the previous section and are mediated or not bymelatonin receptors. The receptor-mediated immedi-ate effects are expected to be quantitatively and/orqualitatively different depending on the target organs,local concentration of the hormone, the type of cellularreceptors and signaling system, the duration of thesignal, and the affinity and desensitization of the dif-ferent receptor types. Therefore, the immediate effectsof melatonin depend on, among other factors, thephase of the melatonin circadian production cycle(the rising, evening phase; the phase of nocturnal dailypeak; and the dawn falling phase at the end of thenight) that will determine the concentration of ex-tracellular melatonin and the duration of its interactionwith its targets and their sensitivity (–).


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Prospective effectsProspective effects are dependent on a special mela-tonin’s way of action, as they are primed during thenight (), through the immediate effects, but areexpressed only during the following day when mel-atonin is no longer present. In other words, thenocturnal action of melatonin triggers cellular andmolecular mechanisms that will determine effects thatare expressed only after cessation of the melatoninsignal and, as a rule, the absence of melatonin duringthe following day is a necessary condition for theiroccurrence.

Two types of prospective effects should be con-sidered (Fig. ), as presently discussed.

Proximal or consecutive effects. Proximal orconsecutive effects are expressed immediately aftercessation of melatonin signal. Assuming an adequatesynchronization to the light/dark cycle, these effectsare seen in the beginning of the following day andmight last for hours, given that melatonin is notpresent.

An example of these proximal effects is the hyper-sensitivity or supersensitivity of the intracellular adenylylcyclase/cAMP/PKA/CREB transduction pathway that isseen after a long-lasting inhibition induced by the in-teraction of melatonin and its Gi protein–linked re-ceptor, as was demonstrated in several central andperipheral melatonin-responsive systems of mammalianspecies. Depending on the duration of this interaction,and consequently the duration of a sustained inhibitionof the adenylyl cyclase/cAMP/PKA/CREB transductionsystem, a rebound effect is seen immediately aftercessation of the melatonin signal. As a consequence, thisproximal effect is maximal during the first hours of theday, when there is a greater effectiveness of the in-teraction of any agonist of Gsa-linked receptors, in-creasing the adenylyl cyclase intracellular transductionpathway activation owing to its supersensitization anddetermining a larger effect than would be observed ifmelatonin was not present during the preceding night.

This melatonin proximal effect was originallydemonstrated in cells of the ovine PT where the effectsof different durations of melatonin signal modulatedcAMP intracellular signaling sensitization (, ).This sensitization effect determined by melatonin,reflecting basal and forskolin-stimulated responses,was demonstrated to gradually intensify depending onthe increasing duration of melatonin pre-exposure,being up to % higher at the maximal sensitizationtime as compared with a preparation that was notpreincubated with melatonin. The time-dependentsensitization process in this particular system is trig-gered after hours of melatonin pre-exposure, beingmaximal after hours; importantly, note that it wasnot dependent on melatonin concentration or on denovo protein synthesis.

In the PT of mice and hamsters, another cellularphenomenon regulated by melatonin proximal effects isthe diurnal rhythm of Per transcription and translation(). This was shown to be a consequence of thenocturnal melatonin Gi/-mediated immediate effectthat primes sensitization of the adenylyl cyclase/cAMPsignaling system that appears when the melatonin signalends at the beginning of the day. At this precise momentand for a few subsequent hours, pituitary PT cells aresensitized to the induction of Per gene expression bythe adenosine Ab receptor–mediated cAMP trans-duction signal. As a consequence, melatonin, through itsproximal effects, is fine-tuning the proper expression ofthe clock genes transcription/translation inhibitory loopthat is critical for the circadian function of this system.Importantly, note that the same kind of proximal effectcontrolling clock genes expression during the dayfollowing a long-lasting melatonin signal is also seen inpancreatic b cells Rev-erb (reverse erythroblastosisvirus)a and Bmal (brain and muscle ARNT-like )gene expression ().

The same kind of melatonin proximal effectresulting in sensitization of the adenylyl cyclase/cAMP

Melatonin ways of action

Immediate effects(and priming of

prospective effects)

Prospective effects

Distant or lengthy(Clock and clock-controlled genes)

Proximal orconsecutive

effects(sensitization)

Night Day

Biological night Biological day

Melatonin

© 2018 Illustration Presentation ENDOCRINE SOCIETY

Figure 1. Melatonin ways of action. The daily temporal allocation of immediate, prospective,distant/lengthy, and proximal/consecutive melatonin effects is shown. Using these two different ofways of action (immediate and prospective), melatonin can control cell function distributionduring night and day. Immediate effects are all measurable effects occurring during the ongoingmelatonin interaction with its effectors. These effects are seen during the night. At the same timeand still dependent on these immediate effects, melatonin primes other effects that will only beseen during the following day, if and when melatonin is not present anymore. These prospectiveeffects are divided into two categories depending on the time of occurrence and the primedmechanisms. Proximal effects are dependent on the duration of the nocturnal melatonin signal anddepend on the longstanding cAMP synthesis inhibition mediated by Gi/0 protein coupled to themelatonin receptor. The sensitization of adenylyl cyclase/cAMP signaling system will appearimmediately after the ending of the melatonin signal, at the beginning of the day. The distal or lengthyprospective effects depend on the control of the clock genes expression. These genes are the molecularsubstrate of the circadian rhythmicity in every cell, and their effects are spread throughout the 24-h daymediated by CCGs. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]


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signal was determined in several other central andperipheral systems. In rat Leydig cells, LH-inducedtestosterone production was % to % higher after hours of preincubation with melatonin comparedwith non–pre-exposed Leydig cells (, ). InChinese hamster ovary cells expressing human MTreceptor, the pre-exposure to a physiological con-centration of melatonin for a length of time thatmimics the period of darkness induced supersensitizationof the cAMP-dependent signaling cascade during thewithdrawal period, as determined by a potentiation offorskolin-mediated stimulation of cAMP formation,activation of PKA, and CREB phosphorylation ().This sensitization effect was time-dependent, beingmaximal at hours after preincubation with melatoninfor some parameters (e.g., cAMP) and lasting for at least hours after melatonin withdrawal for other param-eters (e.g., phosphorylated CREB).

Among the peripheral systems where the mela-tonin proximal effect of cAMP signaling sensitizationis best studied are the pancreatic b cells and pancreaticisolated islets, with evidence also found in humans. Inthis system the melatonin proximal effect is shown tobe involved in b cell survival, function, and circadianrhythmicity (, –).

Melatonin may also play a direct facilitatory role ofthe b cell function by sensitizing the cells to respond toglucagon-like peptide (GLP-) that results in in-creased insulin secretion, as shown in the INS- ratinsulinoma cell line and isolated pancreatic islets ().Moreover, there is some evidence that a long-durationovernight exposure to melatonin might preventcAMP-dependent GLP- receptor rapid homologousdesensitization (), allowing the receptors to be fullyavailable (if not upregulated) for mobilization the nextmorning. More recently, it was shown that melatoninpreincubation of rat insulinoma cell line INS /(in a time frame that mimics the typical night ex-posure), in addition to significantly enhancing theactivation of the cAMP-dependent signal transductionpathway as previously demonstrated, attenuatedproteotoxicity-induced b cell apoptosis, decreasedactivation of stress-activated protein kinase/c-JunN-terminal kinase, and diminished the oxidativestress response (). Moreover, activation of this kindof melatonin proximal effects ( to hours ofpreincubation) in hyperglycemia chronically exposedisolated human islets was shown to significantly reducethe oxidative stress as well as to partially restore theglucose-stimulated and incretin-stimulated insulinresponse. Similar proximal effects of melatonin weredemonstrated in postmortem islets obtained frompatients with type diabetes mellitus, except for theincretin-mediated insulin release ().

Distal or lengthy effects. Distal or lengthyeffects are primed during the night and expressed atany time or during the following day, in the absence ofcirculating melatonin. These effects will be reset by the

new episode of melatonin secretion during the fol-lowing night and are, in general, mediated by theregulation of gene expression and protein translationand degradation, mainly involving clock genes. Theclock genes products regulate expression of the tissue-specific output of clock-controlled genes (CCGs) tocontrol the cellular/tissue/organ circadian function.Such rhythmic CCG expression is directly responsiblefor the daily oscillation of cell metabolism and organ/system function. Therefore, ultimately, melatonin isable to regulate several functions expressed for hours of the day by regulating the dynamic of the clockgenes–CCGs interaction (Fig. ). In other words, throughthese distal lengthy effects melatonin modulates theperiod, amplitude, and/or phase of daily expression ofthese rhythmic genes, participating as an active de-terminant of the daily functions of central and peripheralsystems. In some of them, the absence or amplitudereduction of melatonin (e.g., pinealectomy, light at night,aging) or the absence of receptor-mediated melatoninfunction (as in single or double MT/MT knockoutmice or, eventually, single nucleotide polymorphisms)might result in clock genes arrhythmicity. In several cases,adequate melatonin replacement therapy reverts theobserved effects on clock and CCGs expression. Inaddition to these in vivo observations, in vitro melatoninincubation was shown to be able to trigger, block, and/ormodulate clock genes transcription, depending on the celltype and experimental design considered.

A detailed description of these melatonin distaleffects is given in the next section.

Chronobiotic effectsA chronobiotic agent is defined as an agent that is ableto synchronize and reset biological oscillations ().The first convincing evidence that melatonin was ableto synchronize the behavioral activity/rest circadianrhythm (see Box ) in mammals came from studies infree-running nocturnal rats that were entrained by mg/kg melatonin when it was injected at or im-mediately before the beginning of the active circadianphase (). In addition to showing the chronobioticproperty of melatonin, exogenous melatonin entrain-ing action was shown to be critically dependent on thephase of the circadian cycle. A complementary study() demonstrated that the entrainment of free-running rats under constant light was achieved ateven lower doses of melatonin (~mg/kg), pointing to aputative participation of the daily physiological pinealmelatonin production in activity/rest circadian rhythmsynchronization and stability. It was even demon-strated that melatonin is able to entrain the restoredcircadian activity rhythms of hamsters bearing fetalSCN grafts (). In humans, the chronobiotic way ofaction of melatonin is classically seen in the entrain-ment of the non–-hour sleep–wake rhythm disordereither in blind or sighted patients, as discussed below(, ).


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Owing to these functional characteristics, mela-tonin is properly defined as a chronobiotic or internalzeitgeber (, –). As would be expected from azeitgeber (), melatonin must act on oscillatorsaccording to a well-defined phase-response curve(PRC) (). A PRC shows the magnitude of response,in terms of phase advance or phase delay, derived fromthe action of the zeitgeber on the oscillator, beingdirectly dependent on the moment (defined as phase)of its incidence along the intrinsic period of oscillationof the internal clock. Figure shows a classic PRC ofcircadian activity/rest cycle responses to light pulses.Melatonin PRCs were demonstrated for lizards (),birds, and mammals (), besides being clearly andcompletely demonstrated for humans (), as shownin Fig. . In this case, a melatonin PRC is characterized,as expected, by two opposing regions (phase-advanceand phase-delay zones) and a nonresponsive deadzone. The dead zone occurs during the night whenendogenous levels of melatonin are usually high. Thephase-advance zone is usually located early in theevening, to hours (maximum effect at hours)before the beginning of the nocturnal episode ofmelatonin production that is characterized by the dim-light melatonin onset (DLMO) or, as in the blindpatient, the melatonin onset (, ). The phase-delay zone is located in the late night/early morninghours, around (6 hour) the usual endogenousmelatonin offset. This melatonin PRC guides theclinical administration of melatonin as a chronobioticagent or in replacement therapy. Depending on thedesired effect (phase advance, phase delay, or no phaseshift), such should be the moment of melatonin ad-ministration to the patients.

The chronobiotic effect of melatonin (see Box )depends on its putative action in several levels of thecircadian timing system. The circadian timing system iscomposed of a number of structures of the CNS, mainlythe hypothalamic SCNs, defined as the central oscillator,that times the peripheral oscillators through neuraland/or humoral/hormonal mediators, determining

their phase, amplitude, and period. At the cellularlevel, one basic mechanism of the circadian timingprocess, present in almost every cell, either central orperipheral, is the cellular molecular oscillatory systemrepresented by the so-called clock genes.

The clock genes oscillatory system is characterizedby interconnected negative, positive, and regulatoryfeedback loops (). The core clock genes are part ofthe primary positive/negative transcription/translationmolecular loop. In the positive feedback limb, theprotein products of the genes Clock (circadian loco-motor output cycles kaput) and Bmal heterodimerize,and the dimer acts as a transcription factor thatpositively regulates the transcription of two othergroups of core clock genes, Per (from Period; Per,Per, and Per) and Cry (from Cryptochrome; Cry andCry). In the negative feedback limb of the primarymolecular loop, the protein products of these genes,PERs and CRYs, heterodimerize and translocate intothe nucleus to inhibit the positive transcriptionalactivities of CLOCK/BMAL, resulting in inhibition oftheir own transcription, causing the cycle to rerunfrom a low level of transcriptional activity (). Itshould be taken into account that the inactivation ordegradation of the negative limb proteins PER andCRY is required to terminate the repression phase andrestart a new cycle of transcription, setting the periodof the clock ().

The second molecular loop, which is regulatoryand stabilizes the circadian period of the first loop(), is composed of two families of nuclear receptors,REV-ERB a or b and ROR a, b, or g. The CLOCK/BMAL dimer also initiates the transcription of bothRev-erba or b and Ror a or b. The REV-ERB andROR proteins then compete for ROR responseelement binding sites within the promoter of Bmalwhere ROR proteins initiate Bmal transcription andREV-ERB proteins inhibit it. One should considerat least a third, more complex transcriptional loopinvolving several regulatory elements such as DBP(D-box binding protein), PARzip protein (proline

BOX 1. Circadian RhythmsCircadian rhythms are endogenously generated ~24-hour biological rhythms. The circadian timing system is organized inseveral levels, from the molecular one, represented by the clock genes, to the systemic regulatory timing neuroendocrinesystem. This circadian oscillation is directly synchronized by external environmental cycles, mainly the typical light/dark cycleof the geophysical day and night. These external rhythmic events that are able to synchronize biological rhythms are calledzeitgebers or synchronizers. Synchronization between oscillators is obtained when the phase relationship between them iskept constant. “Entrainment” is defined as the particular case of synchronization between oscillators (with different butsimilar periods) that occurs when one of the oscillators imposes its period on the other. That is the case of the circadianrhythms. The endogenous period (shown as free-running rhythms in constant conditions) is always slightly different from 24hours. The day/night light/dark cycle presents a 24-hour period. During the synchronization, the endogenous masteroscillator assumes the 24-hour period of the external zeitgeber, thus being entrained by it. This entrainment process dependson phase advances and phase delays of the endogenous oscillator that are strictly dependent on the moment of incidence ofthe external stimulus, defining what is called the phase-response curve (see Figs. 3 and 4).

Excellent books about circadian rhythmicity are theHandbook of Behavioral Neurobiology, Vol. 4, Biological Rhythms, editedby Jurgen Aschoff (New York, NY: Plenum Press, 1981), in particular chapters 5 through 7, and the classic The Clocks ThatTime Us, by Martin C. Moore-Ede, Frank M. Sulzman, and Charles Fuller (Cambridge, MA: Harvard University Press, 1982).


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and acidic amino acid–rich basic leucine zipper), andNAD+-dependent SIRT that add further regulationto the main two clock genes transcription/translationloops ().

As can be deduced from the above, virtually everystage of the aforementioned gene expression should beconsidered nodal points for the circadian control ofclock genes and CCGs expression. In this context,melatonin, owing to its functional pleiotropic ways ofaction that regulate basic cellular metabolism, chromatinintegrity and gene transcription and translation, nuclearexport, microRNA regulation, and mRNA and proteindegradation, might modulate almost every stage in clockgenes and CCGs expression and function (, –).

In the SCN, the central circadian pacemaker, in vivoexperiments and quantitative in situ hybridization analysisshowed that melatonin systemic injection in rats imme-diately prior to the light–dark transition is able to modifythe mRNA expression of Rorb (preventing the otherwisedecreasing expression) and Rev-erba (provoking its phaseadvance) and does not modify Rora expression ().Note that although the above-mentioned effects werenoticed in the night immediately after melatonin injection(melatonin immediate effects), phase shifting of BmalmRNA expression is only observed in the following night(melatonin distal and lengthy effect).

In addition to that, melatonin administration tomice at the light–dark transition, in a short-photoperiodregimen, is able to modulate the amplitude (and not thephase) of Per, Per, Bmal, and Clock expression in theSCN (). The classic in vitro rat’s brain slice prepa-ration containing SCNs was used to study the effect ofmelatonin on neuronal electrical activity and clockgenes expression, and the results demonstrated thatmelatonin being present at the subjective day-to-nighttransition alters the SCN clock phasing via the regu-lation of Per and Per clock genes in an immediateeffect mediated by PKC ().

Additionally, considered that melatonin is able tocontrol the circadian timing of the SCN throughprospective proximal effects. The circadian rhythm ofSCN neuronal excitability presents two temporaldomains that are characterized by distinct sensitivity tospecific signal transduction pathways [reviewed in()]. The nighttime domain is characterized by thepredominance of elevated cGMP level and subsequentactivation of protein kinase G, the cGMP-dependentprotein kinase. Alternatively, the daytime domain canbe characterized by its sensitivity to direct activation ofthe pituitary adenylate cyclase-activating polypeptide/cAMP/PKA pathway. The presence of MT and/orMT melatonin receptors in the SCN is undisputed(, –), and considering the ability of bothreceptors to inhibit adenylyl cyclase, it is conceivable thatthe long-lasting nocturnal melatonin signal may beconsidered one important factor determining, throughits proximal effects, the daytime domain predominanceof supersensitivity to the adenylyl cyclase/cAMP/PKA

pathway in the SCN (). In this context, the role playedby the regulator of G protein–signaling protein (RGS), that functions as a GTPase-accelerating pro-tein for Gi (), should be additionally considered, as itpromotes GTP hydrolysis and arrests GTP-bound Gisignaling. It is shown that melatonin might upregulatethe RGS gene expression () and that RGSprotein accumulation peaks during daytime (), actingto overcome the melatonin Gi-mediated effect andcontributing to the putative daytime domain of cAMPsignaling sensitization.

In addition to the control of clock genes tran-scription and translation in the central circadianpacemaker, it is noteworthy that melatonin is able tomodulate clock genes expression in several other CNSstructures as other hypothalamic nuclei, hippocampus,and striatum (–).

As far as peripheral oscillator clock genes expressionand circadian function control are concerned, pancreaticb cells and islets and the metabolically most relevanttissues (liver, muscle, and adipose tissue) are all targetsfor melatonin. Mouse pancreatic islets present self-sustained clock gene and protein oscillations that aredirectly involved with growth, survival, glucose meta-bolism, and insulin synthesis (). Ramelteon®, amelatonin receptor synthetic agonist that mimics themelatonin receptor–mediated cAMP signaling sensiti-zation proximal effect, controls Rev-erba and Bmalexpressions in rat pancreatic INS- b cells (), il-lustrating the role played by melatonin in the dailyregulation of glucose-stimulated insulin release (). Inrodents, visceral white adipose tissue core clock geneexpression (Clock, Bmal, Per, Per, Cry, and Rev-erba)

Distal and lengthy prospective effects dependent onclock genes and clock-controlled genes

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Figure 2. Distal and lengthy prospective effects are dependent on clock genes and CCGs.Melatonin during the night, by immediate effects, might stimulate and/or inhibit the expression ofthe clock genes. During the day, in its absence, the inverse occurs. Either way, the clock machinerywill cycle in accordance to melatonin regulation of the expression of genes either from the positiveor the negative loop. Once the 24-hour cycle of the clock genes is defined, the clock genes will, bythemselves, control the expression of the CCGs at different phases of the daily cycle. These genesare the effector genes of the cell and will trigger different functions according to the time of the24-hour cycle. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]


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is under the control of melatonin as well as the dailydistribution of its functions such as lipolysis, lipogenesis,leptin production, and adipocyte proliferation (–).The same daily organization of functions and clockgenes expression is seen in humans (–) andseems to be, as well, undermelatonin regulation (). Inskeletal muscle, melatonin acting through MT seems tocontrol the amplitude of the clock genes and CCGsReverba and Dbp and to control the phase of Bmalexpression (). In sheep, liver clock genes ex-pression is under photoperiodic control. Under shortnights, Per expression peaks at the end of the night,whereas under long nights there is a phase advance ofPer and the peak occurs at the beginning of thenight (). In mice liver, a significant reduction ofPer daily amplitude in MT knockout animals was

demonstrated (). Moreover, melatonin importantlyparticipates in the daily distribution of liver gluconeo-genesis (, ).

Reproductive organ circadian rhythms and phys-iological functions are also under the control ofmelatonin. In rat Leydig cells () the absence ofpineal melatonin abolishes Per daily rhythm andincreases the daily amplitude of Per gene expression.In this case, melatonin replacement was able to revertthe deleterious effects. It was also demonstrated ()that pinealectomy altered the daily mRNA expressionprofile of several clock genes in the rat cumulus–oocyte complex. The absence of melatonin abolishedthe daily rhythm of Clock, Per, Cry, and Rora incumulus cells, altered the amplitude of Clock, Bmal,and Cry in oocytes, and phase shifted Per and Cry

Figure 3. Light PRC. A PRC shows the magnitude of response, in terms of phase advance or phase delay, derived from the action of thezeitgeber on the oscillator, being directly dependent on the moment (defined as phase) of its incidence along the intrinsic period ofoscillation of the internal clock. The figure shows the PRC (black curve) derived from light stimulation (importantly, note that the PRCdepends on the wavelength, intensity, and duration of the light pulse) of young adults under dim light (with gray bar and yellow starsrepresenting light pulses). In this free-running condition, the DLMO or the moment of occurrence of minimal core body temperature istaken as the internal circadian reference time to plan the moment of incidence of the light pulses (measured in units of circadiantime—that is, the free-running period divided by 24). DLMO is usually attributed to circadian time 14. The difference between theinstant of occurrence of the internal marker (beginning of melatonin secretion or moment of minimal temperature) on the control dayand on the day or days after the light pulse defines the phase shift induced by light exposure at that particular circadian time. Despitea light PRC being derived in an experimental free-running condition, the same effects of light on phase-shifting the internal circadianclock can be seen in a day-by-day entrained situation represented by the yellow/dark blue bar. Also shown is the light PRC referenced tothe endogenous melatonin rhythm (red dashed curve). As shown, the beginning of the night until about 1 to 2 hours after the DLMO(stated at 2100 h) defines the time zone (green) when the light pulses provoke the maximum phase delays of the circadian clock.Alternatively, light pulses given at the end of the night, near the DLMOff (stated at 0600 h), evokes the maximum phase advanceresponses (blue). Notably, there is a time zone, usually corresponding to the middle of the day, when light pulses do not phase-shift thecircadian clock, which is defined as the dead zone (purple). [© 2018 Illustration Presentation ENDOCRINE SOCIETY]

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in oocytes and Bmal in cumulus cells. Melatoninreplacement therapy was able to counteract several ofthe above-mentioned effects.

The adrenal gland seems to be another pe-ripheral target for the chronobiotic role of mela-tonin, as it was shown to synchronize and triggercircadian clock genes oscillation. In the capuchinmonkey, for example, it regulates the daily adrenalfunction and Bmal and Per circadian expressionpeak ().

Also, note that the literature demonstrated thatmelatonin controls peripheral clock genes and CCGsoscillation in several other systems such as retina (,), skin fibroblasts (), and the CVS (), particularlyin cardiomyocytes (), as well as PT (, ) andhuman myometrial smooth muscle cells (). As clockgenes are well demonstrated in several human cells (,, –), it is conceivable that melatonin might actas one of their synchronizers (, ).

Even the mammalian fetus is entrainable by thematernal melatonin signal. It was shown () thatmaternal melatonin regulates SCN Bmal and Per clockgenes expression in the primate capuchin monkey fetus.Corroborating the importance of maternal melatonincircadian rhythm as a key signal for the generation and/orsynchronization of the circadian rhythms in the mam-malian fetus, the absence ofmaternal melatonin from day to day of gestation in rats markedly affected themRNA expression level of clock genes and CCGs in thefetus adrenal gland so that Bmal and Per circadianoscillations were abolished and, additionally, the fetaladrenal circadian rhythm of corticosterone synthesis wasalso abolished (, ). All of these effects wereovercome in the adrenal glands from fetuses whosemothers received melatonin replacement therapy.

Finally, even the organisms that cohabitate thehuman body may be synchronized by the host’smelatonin profile, as was shown for the malarial

Figure 4. Melatonin PRC. The melatonin PRC is derived from the phase shifts obtained by the difference between the DLMOmomentof occurrence in a control situation and after oral melatonin exposure, usually taken by individuals in the day-by-day entrainedcondition. Melatonin PRC (black curve) is characterized by two opposing regions (phase-advance and phase-delay zones) anda nonresponsive dead zone. Melatonin pulses in the dead zone (purple area) occur during the night when endogenous levels ofmelatonin are usually high (red dashed curve). The phase-advance zone (blue area) is usually located late in the afternoon and early inthe evening, 2 to 7 hours (best seen around 3 to 4 hours) before the beginning of the nocturnal episode of melatonin production. Thephase-delay zone (green) is located in the late night/early morning hours,61 hour around the usual endogenous melatonin offset. Thefigure also shows the best (safe zone) and the worst (forbidden zone) times to use melatonin in chronic conditions, depending onthe desired effects (phase advance, phase delays, or no phase shift at all). It is interesting to compare the melatonin PRC to the light PRCin Fig. 3. At the beginning of the night, light pulses evoke the biggest phase delays; alternatively, melatonin administration would causethe biggest phase advances. The opposite is seen at the end of the night, when light evokes phase advances and melatonin evokes phasedelays. [© 2018 Illustration Presentation ENDOCRINE SOCIETY]

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Plasmodium parasite () and for a commensalbacterium from the human gastrointestinal system, thenoncyanobacterial prokaryote Enterobacter aerogenes,of which the circadian clock and daily activity patternare synchronized by the host’s pineal and gastroin-testinal melatonin profile (, ).

In addition to controlling clock genes/CCGs expres-sion, there are at least two other ways that melatonin canregulate/induce circadian rhythmicity. The first one isregulating the cellular redox state either in central or pe-ripheral oscillators because it is well known that that there isan important functional interplay between the cellularmolecular circadian clock machinery and the cellular redoxstate (, ). The other way is through the previouslydefined supersensitization of adenylyl cyclase and cAMPsignaling that appears when melatonin levels decline atdawn. As demonstrated in the PT (), this prospectiveeffect of melatonin might amplify clock gene expressionrhythms, providing an additional mechanism for rein-forcing rhythmicity in central and peripheral tissues.

The appropriate interpretation of the chronobioticeffect of melatonin should additionally consider that theeffectiveness of a chronobiotic agent or zeitgeber (andmelatonin is not an exception) is directly dependent ontwo other factors: the regularity of the daily repetition ofits signal (, , , ) and its strength (),which is represented here by the contrast betweennocturnal and diurnal melatonin concentration values.

In summary, given its periodic circadian releasedriven by the SCN, the great contrast between nightand day circulating concentrations, in addition to thepleiotropic mechanisms of action controlling centraland peripheral oscillators, melatonin acts as a powerfulchronobiotic hormone and ultimately participates asone of the most important unifying agents that isresponsible for the synchrony between the multitudeof circadian rhythms at several levels (cell, tissue,organ, and system). Therefore, pineal melatoninhormone is an important player in the determinationand stabilization of the internal circadian temporalorder, being crucial to the physiological and thera-peutic prevention and treatment of chronodisruption(, –). Moreover, as stated previously, pinealmelatonin, due to its SCN-controlled regularly timedsynthesis, is mainly linked to the external light/darkcycle rather than to the activity/rest cycle (as arecortisol or corticosterone) of the mammalian organ-ism. Therefore, acting as a photo-neuroendocrinemediator of the light/dark cycle and as an internalzeitgeber, its chronobiotic effect guarantees the ade-quate daily rhythmic physiological and behavioralfluctuations that are fundamental to the proper cir-cadian temporal relationship between the organismand the environmental day/night cyclic changes.

Seasonal effectsAs the chronobiotic circadian effect of melatonin isimportant for the adequate daily relationship of the

organisms and their ecological niche, the seasonaleffect mediated by melatonin is fundamentally im-portant to synchronize physiological and behavioralseasonal adaptations to the expected changes in ex-ternal environmental conditions that are typical of theseasons of the year. These adaptations include annualcycles of reproduction and metabolism, as well as, forexample, the consequent growth and body weightcontrol, thermogenesis and brown adipose tissue func-tion, hibernation, migration, and immune responses.

The cyclic photoperiodic annual changes in theduration of day and night are the most importantenvironmental factor for synchronization of circan-nual rhythms. As the nocturnal melatonin profilevaries according to the duration of the night, it in-ternally encodes the photoperiodic annual change inthe day and night length. In this manner, melatoninis the internal hormonal mediator that was “sculpted”by the environmental photoperiodic changes so thatany seasonal physiological and behavioral adaptationsare dependent on this predictable annual regular var-iation on the daily duration of melatonin signal(–), which is also present in humans (see below).

As is well known, there is a critical day/night lengthratio (critical photoperiod) that most members of acertain animal population detect as a signal ofchanging season and switch from one physiologicalstate to another, which will be fundamental for futureadaptation to the upcoming season (, ). As theduration of the melatonin nocturnal profile encodesthis environmental photoperiod, it is well known thatthere is a critical duration of the nocturnal episode ofmelatonin secretion capable of triggering the seasonalphysiological changes (e.g., brown adipose tissue ac-tivation and recruitment, increasing food intake andbody weight, modification of the reproductive axisaccording to the species reproductive profile) that arenecessary for the proper adaptation to the seasonalfluctuations of the external environment (e.g., tem-perature changes, food availability, day and nightduration) ().

Moreover, another fundamental element in sig-naling seasonal changes is the direction of the dailychange in photoperiod (increasing or decreasing daylength) and, consequently, the direction of the dailychange in the duration of the nocturnal episode ofmelatonin production. Therefore, the history of thephotoperiodic changes and the consequent melatoninnocturnal secretory profile are the major determinantsof the seasonal physiological changes that are fun-damental for the adaptation of the organism to theexpected future seasonal environment. The impor-tance of the history of the annual changes to melatoninsecretory nocturnal profile is shown in Fig. . Asdepicted, any given duration of the nocturnal mela-tonin signal occurs at two different phases of theannual cycle, one in the direction of the winter sol-stice (increasing night length/increasing duration of


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nocturnal melatonin signal) and another in the di-rection of the summer solstice (decreasing nightlength/decreasing duration of nocturnal melatoninsignal). However, from the biological point of view, thefirst one triggers physiological and behavioral prepa-ration for the following winter and the second one forthe following summer, depending on how the signal isread and translated by the PT, transmitted to thepituitary pars distalis, and, via the third-ventricletanycytes, transmitted to the hypothalamus (see be-low) (–). Accordingly, the different adaptiveeffects of melatonin depend on the different phases ofthe year and are ultimately determined by the hy-pothalamic detection of the history of the melatoninsecretory episode duration, day after day, during theannual photoperiodic cycle. This was exemplified byanalysis of two groups of Suffolk ewes that werepreviously adapted to or hours of light per day(). These animals are reproductively active duringthe winter (long night length). The -hour light/-hourdark cycle–adapted group presented reproductive sys-tem inhibition whereas the -hour light/-hour darkcycle–adapted group presented reproductive systemstimulation. Both groups were transferred to an

intermediate -hour light/-hour dark cycle. Thefirst one (previously adapted to hours of light and hours of dark per day) became reproductivelyactive and the other one (previously adapted to hours of light and hours of dark per day) becamereproductively inhibited. In other words, in the firstgroup the CNS read the -hour melatonin signal as“preparation for the winter” (moving from hoursof dark to hours of dark; longer night length),whereas in the second group, the same -hourmelatonin signal was read in opposite direction andwas translated as “preparation for the summer”(moving from hours of dark to hours of dark;shorter night length).

According to the previous discussion, in mammals,the annual sequential variation of melatonin profileduration is the internal representative of the environ-mental photoperiod (day/night length ratio) that is themain synchronizer of the circannual rhythmicity butnot the only one (). Similar to the circadian chro-nobiotic effect, the seasonal effect of melatonin dependson its putative action as an “internal circannual zeit-geber or synchronizer” acting on several levels of thecircannual timing system that is composed of a number

Figure 5. Melatonin seasonal profile. This figure shows the annual seasons and the correspondent photoperiodic change in theduration of the day and the night. At the same time, it is possible to see the annual historic evolution of the duration of the dailyepisode of melatonin production. The first observation is that the classic calendar of four seasons is reduced to the biological point ofview of two seasons, one determined by increasing duration of the melatonin nocturnal episode and the other defined by the increasingreduction of the nocturnal episode. The second point to be observed is the importance of the historical perception of the annualevolution of the environmental photoperiod. Any given duration of the nocturnal melatonin signal occurs at two different phases of theannual cycle, one in the direction of the winter solstice and another in the direction of the summer solstice. However, from thebiological point of view, the first one triggers physiological and behavioral preparation for the following winter and the second one forthe following summer, depending on how the signal is read and translated by the PT/third-ventricle tanycytes. Finally, note that there isa critical day/night length relationship—the critical photoperiod that is detected as the signal of changing season and switching fromone physiological state to another—that is internally represented by the critical duration of the melatonin nocturnal profile. [© 2018Illustration Presentation ENDOCRINE SOCIETY]

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of structures, mainly the PT, third ventricle tanycytes,and several hypothalamic nuclei (, , –).

The mechanism involved with the synchronizingeffect of melatonin that will trigger the physiologicaladaptation according to the respective seasonal en-vironment depends on melatonin interaction withMT receptors (through immediate and prospectiveeffects as inhibition of cAMP synthesis and its sub-sequent supersensitization and clock genes dailytranscription/translation cycle) at the PT- specificthyrotroph cells (, –). These specific PT cellsexpress both the a and b subunits of TSH, but they arenot under the control of the hypothalamic TSH-releasing hormone, as are pars distalis thyrotrophcells classically. Instead, PT TSH synthesis and releasedo not respond to TSH-releasing hormone (nor tothyroid hormones) and is under strict control ofmelatonin, mainly mediated by its MT membranereceptor and clock genes expression regulation (,–). At night onset, melatonin, through im-mediate effects, induces Cry gene and CRY proteinexpression (, –). Alternatively, PER proteinis induced immediately after melatonin signal offsetdepending on its prospective effects (primed duringthe previous night) mediated by cAMP super-sensitization (, –). Therefore, the PER–CRY phase relationship critically determines theamount of PER–CRY dimer—an essential element ofthe inhibitory limb of the clock genes circadiancycle—being a direct reflex of the seasonal changes inthe duration of the nocturnal melatonin signal (,). In this way, the clock genes machinery seems tobe the link between melatonin and TSH expression byPT-specific thyrotrophs (, ).

In addition to TSH, melatonin regulates neuromedinU (NMU) expression and release in PT-specific thyro-trophs (). NMU seems to be an important mediatorof the seasonal effects of melatonin on energy meta-bolism as it is detailed below and elsewhere (). It isinteresting to observe that obesity in humans is related togenetic variants of the NMU encoding gene but not ofthe NMU receptor encoding gene (, ).

Physiological and behavioral adaptations to theseasonal changing environment are ultimately de-termined by the hypothalamus. The retrogradefunctional connection between PT (“season sensor ordecoder”) and the hypothalamus includes anotherfundamental element, the third-ventricle tanycytes.Tanycytes are radial glial cells whose cell somas areinterposed between the vascular bed of the medianeminence and the third-ventricle ependymal cells,whose cellular processes project to the PT and toseveral hypothalamic nuclei (paraventricular, dorso-medial, ventromedial, arcuate) (, ). Tanycytesseem to be key elements, as they are settled at theinterface between blood, CSF, and brain tissue. In fact,tanycytes express cell membrane receptors for TSH,NMU, GPR, FGFR, IL-, and LPS, and they are

able to transport peptides and hormones by trans-cytosis, playing the role of sensors of nutrients, hor-mones, and immune and inflammatory mediators(–). Therefore, these cells seem to be the func-tional link between PT (and, for extension, melatonin-mediated environmental photoperiodic changes), CSF,blood, and hypothalamus, being an important player inthe seasonal regulation of reproduction, energy meta-bolism (ultimately, body weight), and immune function(, , , ).

PT-specific thyrotroph TSH released in the ex-tracellular space acts as a paracrine factor on tanycytesTSH receptors, inducing an increase in the expressionof type iodothyronine deiodinase (DIO) and re-ducing the expression of type iodothyronine deio-dinase (DIO). DIO converts T in T and DIOconverts T in reverse T, degrading T to diiodo-thyronine. The coordination of both enzyme activitieswill regulate the availability of the active thyroidhormone in the hypothalamus, modulating several ofits nuclei and neuroendocrine systems functions and,consequently, the interplay between the physiologicaland behavioral functions that are necessary for theorganism to cope with the environmental changes(, , ). Long days (short duration of dailynocturnal melatonin episode) enhance TSH pro-duction in PT-specific thyrotrophs, which increasesDIO expression and suppresses DIO expression,increasing thyroid hormone signaling in the medi-obasal hypothalamus. On the contrary, short days(long duration of daily nocturnal melatonin episode)trigger an opposite effect. As far as seasonal re-production is concerned, this T hypothalamicgenerating mechanism appears to be the same inboth long-day and short-day breeders, and thus itmust be complemented by some downstream in-trinsic hypothalamic species-specific mechanisms tothe proper regulation of the final seasonal breedingstatus (and, perhaps, all other seasonal adaptations)(–).

Accordingly, a restricted seasonal differentialhypothalamic gene expression following the above-mentioned hypothalamic thyroid hormone availabilitywas recently shown to be the final step in the propa-gation of the photoperiodic message from PT to themediobasal hypothalamus ().

Note that for certain seasonal physiological ad-aptations such as reproductive activation and in-activation, in addition to the above-described PTretrograde action in the hypothalamus, there is ananterograde action that regulates prolactin productionin the pituitary pars distalis. In this case, themelatonin-driven PT mediators are different fromTSH and NMU. There is no consensus about theircharacterization, but messengers from PT to parsdistalis include tachykinins, vascular endothelialgrowth factor, and endocannabinoids (). An in-trinsic endocannabinoid system was shown to be


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important for communication between PT and parsdistalis, including in the human brain ().

In summary, the melatonin seasonal effect is de-pendent on the regular and predictable change of theduration of its synthesis and blood presence acrosssuccessive nights and how this message is decoded byPT through retrograde and anterograde paracrinemediators. These mediators convey the photoperiodicinformation to the mediobasal hypothalamus andpituitary, triggering the adaptive physiological andbehavioral responses that anticipate the predictablechanges of environmental seasons, guaranteeing healthmaintenance.

The question of whether the melatonin circannual/seasonal synchronizing effect is important in humanphysiology and pathophysiology should be considered.The evolutionary history of photoperiodism ()points to a positive response to this question. Itsdemonstration, however, is not an easy task. All of theexperimental studies described previously depend onan elaborate protocol, using artificial photoperiodcondition and its controlled changes or, alternatively,in a more naturalistic study, the observation andmeasurements of physiological and behavioral pa-rameters for at least full year or more. For obviousreasons this is not directly applicable to humans.Additionally, the human cultural attribute allows theartificial control of the environment conditions by theintroduction of nocturnal lighting, temperature con-trol, food availability throughout the year, and otherfactors. As a consequence, these social conditions areexpected to reduce the impact of a seasonal changingnatural environment to putative natural human cir-cannual physiological and behavioral responses (,). However, using adequately designed epidemio-logical studies, an adequate choice of communitiesliving in different cultural settings, and proper experi-mental conditions, it is possible to show that severalparameters of human physiology (e.g., birth, puberty,metabolism, body weight, eating behavior, glucosehomeostasis, hormonal production, thermoregulation,immune responses, sleep duration) show circannualrhythms so that humans might be characterized asseasonal as any other photoperiodic mammal (,–). Moreover, humans show seasonal changes inmelatonin production (, –), and melatoninseems to act as a circannual synchronizer in humans,despite no direct demonstration (–).

Transgenerational and programming effectsThe daily maternal plasma melatonin rhythm typicallyshows an increase in amplitude from the first to thesecond and to the last third of pregnancy, reaching amaximum at term and returning to basal levels im-mediately after delivery, including in humans (–).This physiological regular increase of melatonin con-centration during pregnancy seems to be dependent,in rats, on the number of fetuses and is under control

of some placental factors such as the vasoactive in-testinal polypeptide, progesterone, estradiol, and others(, ).

Alternatively, it is well known that maternalmelatonin is freely transferred to the fetus via theplacenta (–), and this maternal–fetal transfer ofmelatonin is the only fetal source of this hormone.Moreover, melatonin concentration in fetal umbilicalcirculation reflects the day–night difference andnocturnal duration as seen in the maternal circulation(, –).

This transplacental melatonin has several effects onfetus physiology, including the coordination of pe-ripheral organs and tissues development, neural de-velopment and neural plasticity, and metabolic,cardiovascular, and immunological programming, ananticipatory biological response that prepares theoffspring to cope with forthcoming environmentaldemands. Among these programming effects of ma-ternal melatonin on the developing fetus, one re-markable effect is the timing of the fetus futurephysiology and behavior, another time domain actionof hormonal melatonin. Maternal melatonin sends atemporal circadian (time of the day) and seasonal(photoperiod and its history) signal to the fetus so thatits CNS is able to properly deal with the environmentalday/night fluctuation after birth. It was particularlyshown for the onset of puberty that takes the pho-toperiodic history during gestation time into accountto be adequately triggered (, , –).

This phenomenon might be called maternal cir-cadian predictive adaptive programming or maternalphotoperiodic predictive adaptive programming. Thistransgenerational timing programming effect ofmelatonin is an example of the so-called predictiveadaptive responses (–). The only difference tothe general concept of predictive adaptive responses isthat, thanks to the earth rhythmic geophysical tem-poral organization, the photoperiod and day/nightcycle of the future environment is almost confi-dently predictable and, with a great degree of certainty,there will be no chance of mismatch between thepredicted environment and the real one. Therefore, thedevelopmental plasticity determined by maternalmelatonin is able to generate an almost perfect tem-poral adaptation of the offspring to the future day/night cycle and the evolving seasons of the year.

In , an elegant paper showed for the first timethe effect of maternal melatonin in timing the circa-dian behavior of hamster pups (). They used SCN-lesioned arrhythmic dams (absence of circadian pinealmelatonin rhythm) and injected them with melatoninin antiphase ( hours and hours, differentgroups), using different concentrations, for to daysduring gestation. The pups wheel-running activityperiod and acrophase were measured at weaning, andthe average acrophases differed between groups by ~hours and were correlated to the time of prenatal

“In general, the immediate andprospective effects ofmelatonin determine the sameevents both in nocturnal andin diurnal animals, but inopposite phases of the dailycycle.”


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melatonin injection to the pregnant dams. Moreover,the different peaks of activity of the offspring wereexclusively determined by the time of melatonin in-jection and were not dependent on the dose ( to mg) or the number of repeated days of injection ( or). A similar set of experiments was done in rats tostudy the effects of maternal pinealectomy or superiorcervical ganglionectomy, with or without melatoninreplacement for days in late gestation, on the off-spring circadian drinking behavior evaluated for weeks immediately after weaning and during free-running condition (constant darkness) (). Theresults show that the maternal melatonin circadianrhythm is also the determinant of the drinking be-havior circadian rhythm of the offspring, even thoughit was evaluated nearly month after weaning. It isnoteworthy that scattered drinking behavior acrop-hases are present both in pups born to pinealectomizeddams and in pups born to ganglionectomized ones. It iswell known that pinealectomized mammals present nocirculating melatonin and that ganglionectomized onespresent a residual circulating melatonin throughout the-hour cycle of ~% of the amount of intact animals(). This might indicate that the critical factor de-termining the transplacental circadian timing of mel-atonin is the maternal pineal melatonin circadianfluctuation. Therefore, as observed for the chronobioticeffect of melatonin, the transgenerational timing effectdepends on the daily repetition and on the contrastbetween day and night concentrations of maternalmelatonin. This transgenerational timing effect ofmelatonin was explored at molecular levels (), indifferent organs and physiological systems and in bothaltricial and precocious species, and it is addressed inpublished reviews (, ).

The first convincing demonstrations of in uterotransplacental transfer of daylength informationcame from research articles that studied the growthand reproductive development in voles (Microtusmontanus) (, ). Studies of gonadal develop-ment in Djungarian hamsters and the effects ofprepubertal photoperiodic responses showed for thefirst time that maternal transfer of photoperiodicinformation influences prepubertal responses topostnatal different daylengths (). Additionally,this maternal–fetal transfer of photoperiodic informationwas so skillful that vole offspring (Microtus penn-sylvanicus) expressed different postnatal responsesdepending on the night duration experienced bytheir mothers: pups born in the autumn (long nightsof shorter duration) have much thicker coats thando those born in late winter (long nights of longerduration) ().

The study of maternal–fetal transfer of photo-periodic information in Djungarian hamstersshowed for the first time that the maternal pinealmelatonin daily profile was fundamental for theproper maternal–fetal intrauterine transference of

photoperiodic information by using pinealectomy,programmed nocturnal infusion of melatonin, andexposure to different environmental postnatalphotoperiods (). In a complementary study,using the same experimental model and animal, themelatonin signal was efficient in transferring pho-toperiodic information dependent on the daily repe-tition of the maternal melatonin signal (at least consecutive days) and it also showed an intrauterine-sensitive window to the melatonin timing effect be-tween and days before birth ().

The mechanism of this prenatal melatonin-induced photoperiodic programming was recentlyelucidated () as it was shown that in Siberianhamsters, the maternal organism primes the fetal PT/hypothalamic tanycytes system so that TSH geneexpression in the neonatal PT-specific thyrotrophregulates tanycytes deiodinase gene expression inaccordance with the photoperiodic history experi-enced during pregnancy. As shown, this intrauterinemelatonin programming effect uses the same mech-anism that is used to regulate the seasonal effect inadult animals.

Melatonin and the Conundrum of Diurnal vsNocturnal Species: Similarities and Differences

The preceding discussion of the ways of action andeffects of melatonin should be taken into account tounderstand the similarities and differences in thephysiological effects and regulation of melatonin ondiurnal or nocturnal species. When considering this, itis reasonable to recognize that some of the immediateand prospective effects of melatonin might be differentaccording to the daily distribution of the activity/restcycle of the studied species. In fact, in nocturnal speciessuch as rats, the immediate effects of melatonin, de-duced from melatonin receptor knockdown, pineal-ectomy, and/or melatonin replacement experiments,result in increased insulin sensitivity, higher glucosetolerance, and increased activity, temperature, andenergy expenditure, in addition to resetting the bar-oreflex so that the rising of blood pressure is limited.Alternatively, the prospective effects of melatonin innocturnal species include, for example, daytime he-patic insulin resistance and gluconeogenesis.

Conversely, melatonin immediate effects in diurnalspecies involve, for example, the induction of sleep,blood pressure and temperature dipping, cortisol se-cretion blockade, and induction of insulin resistanceand glucose intolerance. The prospective effects are theinduction of daytime insulin sensitivity, pancreatichigher sensitivity to glucose and incretins-inducedinsulin secretion, regulation of daytime blood pres-sure, and energy balance.

As can be seen, despite several immediate effectsbeing different and opposite in direction, the biological


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role played by melatonin is to trigger at the nocturnalphase, by its immediate effects, the proper adaptivemechanisms to the considered species for that phase ofthe day and, additionally, to prepare, by its prospectiveeffects, the physiology and behavior to be adaptivewhen the complementary daytime phase arises.

In other words, in general, the immediate andprospective effects of melatonin determine the sameevents both in nocturnal and in diurnal mammals, butin opposite phases of the daily cycle.

Unfortunately, the immediate effects of melatoninare the only ones usually considered and discussed inthe literature, as is the case with the discussion aboutthe role played by melatonin in the regulation ofenergy metabolism, particularly in insulin production,secretion, and action. It is well demonstrated innocturnal mammals (e.g., rats, mice, bats) that theimmediate effects, caused by the actual presence ofmelatonin, are the sensitization of the organism to theaction of insulin, either by potentiating the insulinreceptor transduction pathways or its peripheral ac-tion, mainly increasing GLUT-dependent glucoseuptake in muscle and adipose tissues. It is also shownthat the absence or reduced production of melatoninduring the night induces insulin resistance and glucoseintolerance. The same effects were demonstrated inMT and/or MT knockout mice.

Alternatively, in humans, the immediate effect ofmelatonin is the opposite: insulin resistance andglucose intolerance. Moreover, nocturnal reduction ofmelatonin production or reduction of its effects due toputative single nucleotide polymorphisms of its re-ceptors induces daytime defective insulin release inresponse to an overload of glucose, owing to insulinresistance and glucose intolerance.

In fact, the nocturnal production of melatonin isone of the major determinants of the physiologicalinsulin sensitivity during the day in humans, andduring the night in nocturnal rodents. The overalleffect of melatonin is the same in both species. If onlythe immediate effects of melatonin were taken intoconsideration, melatonin and insulin would be clas-sified as having opposite effects, which is not the caseas far as regulation of carbohydrate metabolism isconcerned.

The adequate therapeutic use of melatonin ishighly dependent on the proper understanding of itsimmediate and prospective effects. For rodents, noc-turnal administration of melatonin increases noctur-nal insulin sensitivity and daytime hepatic insulinresistance. In humans, nocturnal administration ofmelatonin induces nocturnal insulin resistance anddiurnal insulin sensitivity. So, in both species theaccurate replacement of melatonin during the nightinduces insulin sensitivity in the respective activity/feeding circadian phase. Therefore, the adequatenocturnal presence of melatonin is essential for thedetermination of the daily cycle of insulin action.

Additionally, in both species, melatonin acts as apowerful chronobiotic. It means that the consecutiveand repetitive daily nocturnal presence of melatoninand the contrast between high nocturnal concentra-tion and diurnal absence or very low concentrationhelp to properly set the circadian clock so that thetypical circadian physiology and behavior of theconsidered species is synchronized to the environ-mental light/dark cycle of the day and night. So, inaddition to considering the immediate and prospectiveeffects of melatonin, its chronobiotic effects should beaccounted for to better understand its physiologicalaction and resulting effects to establish a proper pu-tative therapeutic application.

Melatonin, Physiology, Pathophysiology, andClinical Application

The accurate understanding of melatonin physiologicaland clinical effects is challenging, as several aspects shouldbe taken into consideration and properly perceived sothat its functional characteristics will be adequatelyinterpreted in any considered system or function. Thepresent literature often brings limited highlights of fewfunctional aspects of melatonin, for example, mainlyemphasizing the immediate effects, or the super-sentization effect, or only the chronobiotic or seasonaleffects. However, one should keep in mind that mela-tonin physiology is integrative per se and is dependent onthe ontogenetic, daily, and seasonal history of its secretionprofile, and on its vastness of actions and resulting effects.

Owing to its special characteristics, pineal mela-tonin is a privileged molecule acting through severalmechanisms and in almost all levels of the physiologyof the organism. As previously stated, it acts by reg-ulating basic cell biology phenomena (, , , ,–) in almost every cell type. As far as itsfunctional integrative action is concerned, melatoninacts both centrally and peripherally in almost everylevel of several physiological systems: the CNS, everycomponent of the CVS, the energy metabolism system,the reproductive system, the immune system, thehydroeletrolytic regulation system, the respiratorysystem, the endocrine system, bone, and others. Theways of action and integrative role of melatonin allowthe amplification and diversification of its functionalaction, mainly in the time domain [time domainmolecule, “time messenger” ()] so that it enablesthe organism’s physiology for dealing with the presentchallenges (immediate effects) and, at the same time, itprepares the organism to deal with future predictiveevents (prospective effects), in addition to synchronizethe organism’s physiology and behavior to the dailyand annual photoperiodical cycles.

Consequently, it should always be taken intoconsideration, as far as experimental (even in vitroexperiments) or clinical studies and treatment are

“Experimental studies showthat melatonin might affectthe sleep mechanism itself, inaddition to circadian control.”


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concerned, that melatonin effects will depend on

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