Maternal melatonin effects on clock gene expression in a nonhuman primate fetus

In the adult mammal the circadian system, which allows predictive adaptation to daily environmental changes, comprises peripheral oscillators in most tissues, commanded by the suprachiasmatic nucleus (SCN) of the hypothalamus. The external environment of the fetus is provided by its mother. In primates, maternal melatonin is a candidate to entrain fetal circadian rhythms, including the SCN rhythms of metabolic activity. We found in the 90% of gestation capuchin monkey fetus expression of the clock genes Bmal-1, Per-2, Cry-2, and Clock in the SCN, adrenal, pituitary, brown fat, and pineal. Bmal-1, Per-2, and the melatonin 1 receptor (MT1) showed a robust oscillatory expression in SCN and adrenal gland, whereas a circadian rhythm of dehydroepiandrosterone sulphate was found in plasma. Maternal melatonin suppression changed the expression of Bmal-1, Per-2, and MT1 in the fetal SCN. These effects were reversed by maternal melatonin replacement. In contrast, neither maternal melatonin suppression nor its replacement had effects on the expression of Per-2 and Bmal-1 or MT1 in the fetal adrenal gland or the circadian rhythm of fetal plasma dehydroepiandrosterone sulphate. Our data suggest that maternal melatonin is a Zeitgeber for the fetal SCN but probably not for the adrenal gland.     

Melatonin and the circadian entrainment of metabolic and hormonal activities in primary isolated adipocytes

The aim of this work was to investigate the effect of the in vitro circadian-like exposure to melatonin [in the presence or absence of insulin (Ins)] on the metabolism and clock gene expression in adipocytes. To simulate the cyclic characteristics of the daily melatonin profile, isolated rat adipocytes were exposed in a circadian-like pattern to melatonin added to the incubating medium for 12 hr (mimicking the night), followed by an equal period without melatonin (mimicking the day) combined or not with Ins. This intermittent incubation was interrupted when four and a half 24-hr cycles were fulfilled. At the end, either during the induced night (melatonin present) or the induced day (melatonin absent), the rates of lipolysis and D-[U-(14)C]-glucose incorporation into lipids were estimated, in addition to the determination of lipogenic [glucose-6-phosphate dehydrogenase and fatty acid synthase (FAS)] and lipolytic (hormone sensitive lipase) enzymes and clock gene (Bmal-1b, Clock, Per-1 and Cry-1) mRNA expression. The leptin release was also measured. During the induced night, the following effects were observed: an increase in the mRNA expression of Clock, Per-1 and FAS; a rise in lipogenic response and leptin secretion; and a decrease in the lipolytic activity. The intermittent exposure of adipocytes to melatonin temporally and rhythmically synchronized their metabolic and hormonal function in a circadian fashion, mimicking what is observed in vivo in animals during the daily light-dark cycle. Therefore, this work helps to clarify the physiological relevance of the circadian pattern of melatonin secretion and its interactions with Ins, contributing to a better understanding of the adipocyte biology.     

Rhythmic expression of functional MT1 melatonin receptors in the rat adrenal gland

We previously demonstrated that melatonin is involved in the regulation of adrenal glucocorticoid production in diurnal primates through activation of MT1 membrane-bound melatonin receptors. However, whether melatonin has a similar role in nocturnal rodents remains unclear. Using an integrative approach, here we show that the adult rat adrenal gland expresses a functional MT1 melatonin receptor in a rhythmic fashion. We found that: 1) expression of the cognate mRNA encoding for the MT1 membrane-bound melatonin receptor, displaying higher levels in the day/night transition (1800-2200 h); 2) expression of the predicted 37-kDa MT1 polypeptide in immunoblots from adrenals collected at 2200 h but not 1000 h; 3) no expression of the MT2 melatonin receptor mRNA and protein; 4) specific high-affinity 2-[(125)I]iodomelatonin binding in membrane fractions and frozen sections from adrenals collected at 2200 h but not 0800 h (dissociation constant = 14.22 +/- 1.23 pm; maximal binding capacity = 0.88 +/- 0.02 fmol/mg protein); and 5) in vitro clock time-dependent inhibition of ACTH-stimulated corticosterone production by 1-100 nm melatonin, which was reversed by 1 microm luzindole (a melatonin membrane receptor antagonist). Our findings indicate not only expression but also high amplitude diurnal variation of functional MT1 melatonin receptors in the rat adrenal gland. It is conceivable that plasma melatonin may play a role to fine-tune corticosterone production in nocturnal rodents, probably contributing to the down slope of the corticosterone rhythm.     

Circadian variation in placental and hepatic clock genes in rat pregnancy

Clock genes drive circadian rhythms in a range of physiological processes both centrally and in peripheral tissues such as the liver. The aims of this study were to determine whether the two functionally-distinct zones of the rat placenta (junctional and labyrinth) differentially express clock genes and, if so, whether these exhibit circadian patterns. Rats were sampled from d 21 of pregnancy (term = d 23) and from diestrus I of the estrous cycle. Adult liver (all animals), fetal liver, and placental zones (pregnant animals) were collected at 0800, 1400, 2000, and 0200 h. Both zones of the rat placenta expressed all seven canonical clock genes (Clock, Bmal1, Per1, Per2, Per3, Cry1, and Cry2), but there were marked zonal differences and, unlike in maternal liver, circadian variation in placenta was limited. Similarly, placental expression of Vegf varied with zone but not time of day. Pregnancy also led to marked changes in hepatic clock gene expression, with peak levels of Per1, Cry1, and Cry2 all reduced, Per3 increased, and circadian variation in Clock expression lost. All clock genes were expressed in fetal liver, but there was less circadian variation than in maternal liver. Similarly, fetal corticosterone levels remained stable across the day, whereas maternal corticosterone showed clear circadian variation. In conclusion, our data show that the rat placenta expresses all canonical clock genes in a highly zone-specific manner but with relatively little circadian variation. Moreover, pregnancy alters the expression and circadian variation of clock genes in maternal liver, possibly contributing to maternal physiological adaptations.     

Immunocytochemical demonstration of day/night changes of clock gene protein levels in the murine adrenal gland: differences between melatonin-proficient (C3H) and melatonin-deficient (C57BL) mice

The circadian system comprises several peripheral oscillators and a central rhythm generator that, in mammals, is located in the suprachiasmatic nucleus of the hypothalamus. Expression of clock genes is a characteristic feature of the central rhythm generator and the peripheral oscillators. With regard to the rhythmic production of glucocorticoids, the adrenal gland can be considered as peripheral oscillator, but little is known about clock gene expression in this tissue. Therefore, the present study investigates the levels of three clock gene proteins PER1, BMAL1 and CRY2 in the murine adrenal cortex and medulla at seven different time points of a 12-hr light/12-hr dark cycle. To determine a potential role of melatonin we compared the patterns of clock gene proteins in the adrenal gland of melatonin-proficient mice (C3H) with those of melatonin-deficient mice (C57BL). In C3H mice, both, the adrenal cortex and medulla displayed day/night variation in PER1-, CRY2- and BMAL1-protein levels. PER1 and CRY2 peaked in the middle of the light phase, whereas BMAL1 peaked in the dark phase. This pattern was also observed in the adrenal medulla of C57BL, but in the adrenal cortex of C57BL clock gene protein levels did not change with time and were consistently lower than in C3H mice. These results support the hypothesis that the adrenal gland is a peripheral oscillator and raise the possibility that melatonin may be involved in the control of clock gene protein levels in the adrenal cortex of mice.     

Selectively filtering short wavelengths attenuates the disruptive effects of nocturnal light on endocrine and molecular circadian phase markers in rats

Various physiological processes exhibit a circadian rhythm synchronized to the geophysical light/dark cycle. Our study using a rat model demonstrated that exposure to light at night suppressed the expected nocturnal rise in melatonin, increased plasma corticosterone, and disrupted core clock gene expression in the hypothalamus and the adrenal gland. These effects were prevented by filtration of a 10-nm bandwidth of light between 470 and 480 nm, whereas filtration of light between 452 and 462 nm prevented the rise of corticosterone without restoring normal melatonin secretion or hypothalamic clock gene expression. This is the first demonstration of a wavelength dependency of glucocorticoid secretion and clock gene expression. Our results in an animal model suggest that filtering a narrow bandwidth of light from nocturnal lighting may efficiently attenuate overall disruption of circadian endocrine rhythms and clock gene expression in the hypothalamus and adrenal gland. Because a narrow bandwidth of light is filtered, the color distribution of the illumination source is not altered, and this may be of practical importance for potential future studies in shift workers.     

Inhibition of steroidogenic response to adrenocorticotropin by leptin: implications for the adrenal response to maternal separation in neonatal rats

Previous studies have shown that leptin can regulate the adrenocortical axis. Neonatal rodents exhibit a period of adrenal hyporesponsiveness to stress in the first 2 wk of life, and we determined the role of leptin as a mediator of this process. We examined the direct effects of leptin on neonatal adrenal steroidogenic responses to ACTH under basal conditions and after 24-h maternal separation. In isolated adrenocortical cells from as early as postnatal d 5 (PND5) and throughout the neonatal period, acute (2.5 h) incubation with leptin significantly inhibited ACTH-stimulated corticosterone and aldosterone secretion without affecting cAMP production. In PND10 pups, 24-h maternal separation and the resulting rapid decline in plasma leptin levels increased basal corticosterone and aldosterone secretion in vivo and in isolated cells, but did not modify the ability of leptin to inhibit stimulated steroid production in vitro. Maternal separation in PND10 pups increased adrenal expression of steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) proteins as well as all steroidogenic enzymes measured (3beta-hydroxysteroid dehydrogenase, P450C11B1, and P450C11B2). Leptin (1 mg/kg body weight, i.p.) replacement during maternal separation did not affect basal corticosterone output, but reduced corticosterone secretion and StAR and PBR protein expression induced by exogenous ACTH challenge (20 or 80 microg/kg body weight, i.p.). These results indicate that leptin inhibits ACTH-stimulated secretion of corticosterone and aldosterone, at least through a rapid reduction in the expression of StAR and PBR protein in the neonatal adrenal gland. As leptin concentrations in pups are controlled to a large extent by the maternal diet, these results emphasize the key role of leptin to mediate the maternal influence on the adrenocortical axis of the infant.     

Temporal expression of seven clock genes in the suprachiasmatic nucleus and the pars tuberalis of the sheep: evidence for an internal coincidence timer

The 24-h expression of seven clock genes (Bmal1, Clock, Per1, Per2, Cry1, Cry2, and CK1 epsilon ) was assayed by in situ hybridization in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary gland, collected every 4 h throughout 24 h, from female Soay sheep kept under long (16-h light/8-h dark) or short (8-h light/16-h dark) photoperiods. Locomotor activity was diurnal, inversely related to melatonin secretion, and prolactin levels were increased under long days. All clock genes were expressed in the ovine SCN and PT. In the SCN, there was a 24-h rhythm in Clock expression, in parallel with Bmal1, in antiphase with cycles in Per1 and Per2; there was low-amplitude oscillation of Cry1 and Cry2. The waveform of only Per1 and Per2 expression was affected by photoperiod, with extended elevated expression under long days. In the PT, the high-amplitude 24-h cycles in the expression of Bmal1, Clock, Per1, Per2, Cry1, and Cry2, but not CK1 epsilon, were influenced by photoperiod. Per1 and Per2 peaked during the day, whereas Cry1 and Cry2 peaked early in the night. Hence, photoperiod via melatonin had a marked effect on the phase relationship between Per/Cry genes in the PT. This supports the conclusion that an "external coincidence model" best explains the way photoperiod affects the waveform of clock gene expression in the SCN, the central pacemaker, whereas an "internal coincidence model" best explains the way melatonin affects the phasing of clock gene expression in the PT to mediate the photoperiodic control of a summer or winter physiology.     

Adrenal peripheral clock controls the autonomous circadian rhythm of glucocorticoid by causing rhythmic steroid production

Glucocorticoid (GC) is an adrenal steroid with diverse physiological effects. It undergoes a robust daily oscillation, which has been thought to be driven by the master circadian clock in the suprachiasmatic nucleus of the hypothalamus via the hypothalamus–pituitary–adrenal axis. However, we show that the adrenal gland has its own clock and that the peripheral clockwork is tightly linked to steroidogenesis by the steroidogenic acute regulatory protein. Examination of mice with adrenal-specific knockdown of the canonical clock protein BMAL1 reveals that the adrenal clock machinery is required for circadian GC production. Furthermore, behavioral rhythmicity is drastically affected in these animals, together with altered expression of Period1, but not Period2, in several peripheral organs. We conclude that the adrenal peripheral clock plays an essential role in harmonizing the mammalian circadian timing system by generating a robust circadian GC rhythm.     

Mice, melatonin and the circadian system

Melatonin effects are discussed by reviewing results from mice with intact or disrupted melatonin signaling. Melatonin, the neuroendocrine hand of the clock produced in the pineal gland during night, acts upon two receptor subtypes. Melatonin receptors are found in the suprachiasmatic nuclei (SCN), hypophysial pars tuberalis (PT) and adrenal gland. In SCN, melatonin interacts with PACAP, a neuropeptide of the retinohypothalamic tract. Moreover, melatonin acts on the SCN to modulate the activity of the sympathetic nervous system. Melatonin is not required to maintain rhythmic clock gene expression in SCN. By contrast, the rhythmic clock gene expression in PT depends on a melatonin signal interacting with adenosine. Melatonin may also affect clock gene protein levels in the adrenal cortex and influence adrenal functions. In conclusion, melatonin may serve the synchronization of peripheral oscillators by interacting with other neuroactive substances. A stress-reducing potency of melatonin needs to be explored in further studies.     

Zebrafish serotonin N-acetyltransferase-2: marker for development of pineal photoreceptors and circadian clock function.

Serotonin N-acetyltransferase (AANAT), the penultimate enzyme in melatonin synthesis, is typically found only at significant levels in the pineal gland and retina. Large changes in the activity of this enzyme drive the circadian rhythm in circulating melatonin seen in all vertebrates. In this study, we examined the utility of using AANAT messenger RNA (mRNA) as a marker to monitor the very early development of pineal photoreceptors and circadian clock function in zebrafish. Zebrafish AANAT-2 (zfAANAT-2) cDNA was isolated and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expressed exclusively in pineal cells and retinal photoreceptors. Developmental analysis, using whole mount in situ hybridization, indicated that pineal zfAANAT-2 mRNA expression is first detected at 22 h post fertilization. Retinal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appears to be associated with development of the retinal photoreceptors. Time-of-day analysis of 2- to 5-day-old zebrafish larvae indicated that zfAANAT-2 mRNA abundance exhibits a dramatic 24-h rhythm in a 14-h light, 10-h dark cycle, with high levels at night. This rhythm persists in constant darkness, indicating that the zfAANAT-2 mRNA rhythm is driven by a circadian clock at this stage. The techniques described in this report were also used to determine that zfAANAT-2 expression is altered in two well characterized genetic mutants, mindbomb and floating head. The observations described here suggest that zfAANAT-2 mRNA may be a useful marker to study development of the pineal gland and of circadian clock mechanisms in zebrafish.     

mt1 Melatonin receptor in the primate adrenal gland: inhibition of adrenocorticotropin-stimulated cortisol production by melatonin

The pineal hormone melatonin participates in circadian, seasonal, and reproductive physiology. The presence of melatonin binding sites in human brain and peripheral tissues is well documented. However, in the mammalian adrenal gland, low-affinity melatonin binding sites have been detected only in the rat by some but not all authors. Conflicting evidence for a regulatory role of melatonin on adrenal cortisol production, prompted us to investigate this possibility in a New World primate, the capuchin monkey. Expression of melatonin receptors in the adrenal cortex was demonstrated through pharmacological characterization and autoradiographic localization of 2-[125I]iodomelatonin binding sites (dissociation constant = 96.9 +/- 15 pM; maximal binding capacity = 3.8 +/- 0.4 fmol/mg protein). The mt1 identity of these receptors was established by cDNA sequencing. Melatonin treatment of dispersed cells and explants from adrenal gland did not affect basal cortisol production. However, cortisol production stimulated by 100 nM ACTH was significantly inhibited by low melatonin concentrations (0.1-100 nM); this inhibitory effect was reversed by the mt1/MT2 melatonin antagonist luzindole. Melatonin also inhibited dibutyril-cAMP-stimulated cortisol production, suggesting that melatonin acts through a cAMP-independent signaling pathway. The present data demonstrate that the primate adrenal gland cortex expresses functional mt1 melatonin receptors and shows that melatonin inhibits ACTH-stimulated cortisol production.     

Melatonin synthesis and melatonin-membrane receptor (MT1) expression during rat thymus development: role of the pineal gland

To gain insight into the relationship between thymus and pineal gland during rat development, the melatonin content as well as the activity and expression of the two key enzymes for melatonin biosynthesis, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were studied in the thymus at fetal and postnatal stages. Moreover, melatonin-membrane receptor (MT1) expression was also analyzed. We found both the expression and activity of thymic NAT and HIOMT at 18 days of fetal life. Additionally, there is production of melatonin in the thymus as well as MT1 expression at this fetal age. These results show values higher in day-time than at night-time. The pineal gland begins to produce significant levels of melatonin around postnatal day 16, and this synthesis shows a circadian rhythm with high values during the dark period; therefore the nocturnal serum melatonin may inhibit thymic melatonin production. To document this, we report an increased melatonin content of the thymus in pinealectomized rats compared with sham-pinealectomized. In conclusion, these results show, for the first time, the presence of the biosynthetic machinery of melatonin and melatonin production in developing rat thymus and that the pineal gland may regulate this process.     

Multiple effects of melatonin on rhythmic clock gene expression in the mammalian pars tuberalis

In mammals, changing day length modulates endocrine rhythms via nocturnal melatonin secretion. Studies of the pituitary pars tuberalis (PT) suggest that melatonin-regulated clock gene expression is critical to this process. Here, we considered whether clock gene rhythms continue in the PT in the absence of melatonin and whether the effects of melatonin on the expression of these genes are temporally gated. Soay sheep acclimated to long photoperiod (LP) were transferred to constant light for 24 h, suppressing endogenous melatonin secretion. Animals were infused with melatonin at 4-h intervals across the final 24 h, and killed 3 h after infusion. The expression of five clock genes (Per1, Per2, Cry1, Rev-erbalpha, and Bmal1) was measured by in situ hybridization. In sham-treated animals, PT expression of Per1, Per2, and Rev-erbalpha showed pronounced temporal variation despite the absence of melatonin, with peak times occurring earlier than predicted under LP. The time of peak Bmal1 expression remained LP-like, whereas Cry1 expression was continually low. Melatonin infusion induced Cry1 expression at all times and suppressed other genes, but only when they showed high expression in sham-treated animals. Hence, 3 h after melatonin treatment, clock gene profiles were driven to a similar state, irrespective of infusion time. In contrast to the PT, melatonin infusions had no clear effect on clock gene expression in the suprachiasmatic nuclei. Our results provide the first example of acute sensitivity of multiple clock genes to one endocrine stimulus and suggest that rising melatonin levels may reset circadian rhythms in the PT, independently of previous phase.     

The circadian clock stops ticking during deep hibernation in the European hamster

Hibernation is a fascinating, yet enigmatic, physiological phenomenon during which body temperature and metabolism are reduced to save energy. During the harsh season, this strategy allows substantial energy saving by reducing body temperature and metabolism. Accordingly, biological processes are considerably slowed down and reduced to a minimum. However, the persistence of a temperature-compensated, functional biological clock in hibernating mammals has long been debated. Here, we show that the master circadian clock no longer displays 24-h molecular oscillations in hibernating European hamsters. The clock genes Per1, Per2, and Bmal1 and the clock-controlled gene arginine vasopressin were constantly expressed in the suprachiasmatic nucleus during deep torpor, as assessed by radioactive in situ hybridization. Finally, the melatonin rhythm-generating enzyme, arylalkylamine N-acetyltransferase, whose rhythmic expression in the pineal gland is controlled by the master circadian clock, no longer exhibits day/night changes of expression but constantly elevated mRNA levels over 24 h. Overall, these data provide strong evidence that in the European hamster the molecular circadian clock is arrested during hibernation and stops delivering rhythmic output signals.