Retinal rhythms in chicks: circadian variation in melantonin and serotonin N-acetyltransferase activity.

There is a large-amplitude circadian rhythm of indoleamine metabolism in the retina-pigment epithelium of the chicken. N-Acetyltransferase activity (arylamine acetyltransferase; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) and melatonin content are 15-fold higher at night than during the day in a cycle of a 4-fold increase during the subjective night. Light at midnight inactivates N-acetyltransferase and lowers melatonin. N-Acetyltransferase activity is found predominantly in the retina. The circadian rhythm of this enzyme activity persists in pinealectomized chicks. Thus the pineal is not responsible for retinal indoleamine rhythms. Retinal and pineal levels of N-acetyltransferase activity behave similarly under several conditions. In the chicken, the eye is a major site of rhythmic indoleamine metabolic activity.     

Circadian rhythms of pineal function in rats

In pineal glands melatonin is synthesized daily. Melatonin synthesis in rats kept in most light-dark cycles occurs during the subjective night. This rhythm, which persists in constant dark, is a circadian rhythm which may be a consequence of another circadian rhythm in the pineal gland, of N-acetyltransferase activity (NAT). The NAT rhythm has been studied extensively in rats as a possible component of the system timing circadian rhythms. The NAT rhythm is driven by neural signals transmitted to the pineal gland by the sympathetic nervous system. Environmental lighting exerts precise control over the timing of the NAT rhythm. In rats, there is enough data to describe a daily time course of events in the pineal gland and to describe a pineal "life history." Hypothetical schemes for generation of the NAT rhythm and for its control by light are presented.     

Phase-shifting effects of light on the circadian rhythms of 5-methoxytryptophol and melatonin in the chick pineal gland

In the chick pineal gland, 5-methoxytryptophol and melatonin concentrations fluctuate in a rhythmic manner. These rhythms are circadian in nature persisting in constant darkness and have opposite phases. Acute exposure of chicks to white light (30 lux for 5, 10, 20, and 30 min) at night increased the amount of pineal 5-methoxytryptophol and decreased pineal melatonin content. A 6 hr pulse of light (100 lux) applied early in the subjective night (CT12-CT18) caused a delay in the phase of the circadian rhythms of 5-methoxytryptophol and melatonin by 3.7 and 4.5 h, respectively, compared to untreated controls. When the 6 hr light pulse was given during the late subjective night (C18 CT24) it advanced the phase of the 5-methoxytryptophol and melatonin rhythms by 8.1 and 11.9 h, respectively. In the chick pineal the phase-advancing effects of light on the circadian rhythms of 5-methoxytryptophol and melatonin were more pronounced than the phase-delaying effects. Our results provide the first evidence that light is capable of phase shifting the 5-methoxytryptophol rhythm in a manner similar to its action on the melatonin rhythm.     

Twenty-four hour rhythm of melatonin in patients with a history of pineal and/or hypothalamo-neurohypophyseal germinoma

Abstract: Melatonin deficiency after a pinealectomy has been investigated in animals; however, in humans, this status can be assessed solely by investigating patients with a tumor originating in the pineal gland. This study analyzes secretion of melatonin and pituitary hormones in 14 patients with germinoma originating in the pineal or the hypothalamic-neurohypophyseal region. Thirteen patients had been successfully treated prior to this study. One patient was included in this study before the initiation of treatments. Plasma sampling was performed every 2 hr for 24 hr and melatonin concentrations were measured by radioimmunoassay. Melatonin secretion was nearly absent in the patients with pineal germinoma regardless of treatment option, even in the patient who had been untreated. In contrast, melatonin secretion and its circadian rhythms were not affected in patients with a hypothalamo-neurohypophyseal germinoma. The circadian rhythms of growth hormone and adrenocorticotropic hormone were not dysregulated in patients with the melatonin deficiency. We conclude that germinoma cells originating the pineal gland impair the production of melatonin by pineocytes and consequently induce a permanent melatonin deficiency in those patients. Since melatonin exerts multiple physiological functions, once a clinical concept of "melatonin deficiency syndrome" is established, melatonin replacement therapy could be investigated in patients who have a pineal germinoma or who have undergone a neurosurgical pinealectomy.     

Circadian rhythms of melatonin release from individual superfused chicken pineal glands in vitro.

The pineal gland of birds contains one or more circadian oscillators that play a major role in overall temporal organization. We have developed a flow-through culture system for the isolated pineal by which we can measure the release of melatonin continuously from superfused glands over long periods of time. Chicken pineals release melatonin rhythmically, and these rhythms persist in vitro with a circadian oscillation. In light cycles the release of melatonin is strongly rhythmic; however, in constant conditions the amplitude of the rhythm is lower and appears to be damping. Light has at least two effects upon the isolated pineal: cyclic light input synchronizes the rhythm, and acute light exposure at night rapidly inhibits melatonin release. The cultured avian pineal clearly offers great potential as a model system for the study of vertebrate circadian oscillators and may open the way for an analysis of mechanism.     

Melatonin phase-shifts human circadian rhythms with no evidence of changes in the duration of endogenous melatonin secretion or the 24-hour production of reproductive hormones

The pineal hormone melatonin is a popular treatment for sleep and circadian rhythm disruption. Melatonin administered at optimal times of the day for treatment often results in a prolonged melatonin profile. In photoperiodic (day length-dependent) species, changes in melatonin profile duration influence the timing of seasonal rhythms. We investigated the effects of an artificially prolonged melatonin profile on endogenous melatonin and cortisol rhythms, wrist actigraphy, and reproductive hormones in humans. Eight healthy men took part in this double-blind, crossover study. Surge/sustained release melatonin (1.5 mg) or placebo was administered for 8 d at the beginning of a 16-h sleep opportunity (1600 h to 0800 h) in dim light. Compared with placebo, melatonin administration advanced the timing of endogenous melatonin and cortisol rhythms. Activity was reduced in the first half and increased in the second half of the sleep opportunity with melatonin; however, total activity during the sleep opportunities and wake episodes was not affected. Melatonin treatment did not affect the endogenous melatonin profile duration, pituitary/gonadal hormone levels (24-h), or sleepiness and mood levels on the subsequent day. In the short term, suitably timed sustained-release melatonin phase-shifts circadian rhythms and redistributes activity during a 16-h sleep opportunity, with no evidence of changes in the duration of endogenous melatonin secretion or pituitary/gonadal hormones.     

Melatonin site and mechanism of action: Single or multiple?

ABSTRACT: By affecting the entrainment pathways of the biologic clock, melatonin has a major influence on the circadian and seasonal organization of vertebrates. In addition, a number of versatile functions that far transcend melatonin actions on photoperiodic time measurement and circadian entrainment have emerged. Melatonin is a free radical scavenger and antioxidant and it has a significant immunomodulatory activity, being presumably a major factor in an organism's defense toxic agents and invading organisms. Besides affecting specific receptors in cell membranes to exert its effects, the interaction of melatonin with nuclear receptor sites and with intracellular proteins, like calmodulin or tubulin-associated proteins, as well as the direct antioxidant effects of melatonin, may explain many general functions of the pineal hormone.     

Melatonin, endocrine pancreas and diabetes

Melatonin influences insulin secretion both in vivo and in vitro. (i) The effects are MT(1)-and MT(2)-receptor-mediated. (ii) They are specific, high-affinity, pertussis-toxin-sensitive, G(i)-protein-coupled, leading to inhibition of the cAMP-pathway and decrease of insulin release. [Correction added after online publication 4 December 2007: in the preceding sentence, 'increase of insulin release' was changed to 'decrease of insulin release'.] Furthermore, melatonin inhibits the cGMP-pathway, possibly mediated by MT(2) receptors. In this way, melatonin likely inhibits insulin release. A third system, the IP(3)-pathway, is mediated by G(q)-proteins, phospholipase C and IP(3), which mobilize Ca(2+) from intracellular stores, with a resultant increase in insulin. (iii) Insulin secretion in vivo, as well as from isolated islets, exhibits a circadian rhythm. This rhythm, which is apparently generated within the islets, is influenced by melatonin, which induces a phase shift in insulin secretion. (iv) Observation of the circadian expression of clock genes in the pancreas could possibly be an indication of the generation of circadian rhythms in the pancreatic islets themselves. (v) Melatonin influences diabetes and associated metabolic disturbances. The diabetogens, alloxan and streptozotocin, lead to selective destruction of beta-cells through their accumulation in these cells, where they induce the generation of ROS. Beta-cells are very susceptible to oxidative stress because they possess only low-antioxidative capacity. Results suggest that melatonin in pharmacological doses provides protection against ROS. (vi) Finally, melatonin levels in plasma, as well as the arylalkylamine-N-acetyltransferase (AANAT) activity, are lower in diabetic than in nondiabetic rats and humans. In contrast, in the pineal gland, the AANAT mRNA is increased and the insulin receptor mRNA is decreased, which indicates a close interrelationship between insulin and melatonin.     

Daily rhythm of glucose-induced insulin secretion by isolated islets from intact and pinealectomized rat

It is well known that pinealectomy induces in rats a diminished glucose tolerance, insulin resistance, a reduction in GLUT4 content in adipose and muscular tissues, a decrease in hepatic and muscular glycogenesis, impairment of glucagon action and an increase in blood pyruvate concentration. In addition, it has been shown that melatonin suppresses insulin secretion in several experimental conditions. The objective of the present study was to investigate the daily rhythm of glucose-induced insulin secretion and glucose oxidation by isolated pancreatic islets and to investigate the effect of chronic absence of melatonin (30 days of pinealectomy) on this rhythmic process. The data obtained confirmed the presence of a strong 24-hr rhythm of insulin secretion by isolated pancreatic islets. In addition, it was demonstrated that the glucose-metabolizing ability of the B-cell follows a daily rhythm phase locked to insulin secretion rhythm. Most interesting, however, was the demonstration that the daily rhythmic processes of insulin secretion and B-cell -[U-14C]-glucose oxidation by isolated pancreatic islets is completely modified by the chronic absence of the pineal gland. Thus, pinealectomy induced in all groups an increase in 24-hr mean glucose-stimulated insulin secretion and [U-14C]-glucose oxidation, in addition to some alterations in the rhythmic amplitude and a remarkable phase-advancing of the daily curves for 8.3 mm glucose (a condition similar to that observed in fed animals and where the B-cells are supposedly more active). These observations strongly suggest that the presence of the pineal gland may be necessary for the proper synchronization of these metabolic rhythms with other circadian rhythms like activity-rest and feeding.     

Melatonin in relation to physiology in adult humans

Abstract: The role exerted by melatonin in human physiology has not been completely ascertained. Melatonin levels have been measured in different physiopathological conditions, but the effects induced by melatonin administration or withdrawal have been tested only recently. Some effects have been clearly documented. Melatonin has hypothermic properties, and its nocturnal secretion generates about 40% of the amplitude of the circadian body temperature rhythm. Melatonin has sleep inducing properties, and exerts important activities in the regulation of circadian rhythms. Melatonin is capable of phase shifting human circadian rhythms, of entraining free-running circadian rhythms, and of antagonizing phase shifts induced by nighttime exposure to light. Its effect on human reproduction is not completely clear, but stimulatory effects on gonadotropin secretion have been reported in the follicular phase of the menstrual cycle. Direct actions on ovarian cells and spermatozoa have been also documented. Beside these, new important actions for melatonin may be proved. Melatonin may exert protective effects on the cardiovascular system, by reducing the risk of atherosclerosis and hypertension, and may influence immune responses. Finally, by acting as an antioxidant, melatonin could be important in slowing the processes of ageing.     

Gastrointestinal melatonin: localization,function, and clinical relevance

The gastrointestinal tract of vertebrate species is a rich source of extrapineal melatonin. The concentration of melatonin in the gastrointestinal tissues surpasses blood levels by 10–100 times and there is at least 400× more melatonin in the gastrointestinal tract than in the pineal gland. The gastrointestinal tract contributes significantly to circulating concentrations of melatonin, especially during the daytime and melatonin may serve as an endocrine, paracrine, or autocrine hormone influencing the regeneration and function of epithelium, enhancing the immune system of the gut, and reducing the tone of gastrointestinal muscles. As binding sites for melatonin exhibit circadian variation in various species, it has been hypothesized that some melatonin found in the gastrointestinal tract might be of pineal origin. Unlike the photoperiodically regulated production of melatonin in the pineal, the release of gastrointestinal melatonin seems to be related to the periodicity of food intake. Phylogenetically, melatonin and its binding sites were detected in the gastrointestinal tract of lower vertebrates, birds, and mammals. Melatonin was found also in large quantities in the embryonic tissue of the mammalian and avian gastrointestinal tract. Food intake and, paradoxically, also long-term food deprivation resulted in an increase of tissue and plasma concentrations of melatonin. Melatonin release may have a direct effect on many gastrointestinal tissues but may also well influence the digestive tract indirectly, via the central nervous system and the sympathetic and parasympathetic nerves. Melatonin prevents ulcerations of gastrointestinal mucosa by an antioxidant action, reduction of secretion of hydrochloric acid, stimulation of the immune system, fostering epithelial regeneration, and increasing microcirculation. Because of its unique properties, melatonin could be considered for prevention or treatment of colorectal cancer, ulcerative colitis, gastric ulcers, irritable bowel syndrome, and childhood colic.     

Circadian rhythms of melatonin secretion from superfused goldfish (Carassius auratus) pineal glands in vitro

A flow-through, whole-organ culture (superfusion) system was developed, and goldfish pineal glands were maintained at 25 degrees under light-dark (LD) 12:12 cycles, reversed LD 12:12 cycles, continuous dark (DD), or continuous light (LL) conditions for 48 hr. Under LD 12:12 and reversed LD 12:12 cycles, superfused pineal glands showed a rhythmic melatonin secretion: Scotophase was associated with high titers and photophase with low titers. The melatonin secretion rhythms persisted for two cycles under DD conditions, whereas nocturnal rises were suppressed under LL conditions. After the transition from LL to DD conditions on the third day, melatonin showed a nocturnal increase. These results indicate that melatonin secretion from the superfused goldfish pineal gland is directly photosensitive and that the goldfish pineal gland harbors a circadian oscillator which generates melatonin secretion rhythms.     

Melatonin rhythms in the eyes, pineal bodies, and blood of Japanese quail (Coturnix coturnix japonica)

Melatonin levels in the eyes, pineal bodies, and blood of Japanese quail exposed to 12L:12D show robust daily rhythms with high levels occurring in the night and low levels occurring during the day. Since melatonin is synthesized in both the eyes and pineal bodies of birds, the relative contribution of these structures to the blood melatonin levels was determined. A rhythm of blood melatonin persisted in 12L:12D in birds blinded by complete orbital enucleation and in pinealectomized birds but the nighttime levels were reduced by 33 and 54%, respectively, as compared to melatonin levels in control quail. Only a small melatonin rhythm (13% of control levels) was detected in the blood of pinealectomized, blinded quail. This "residual" rhythm could indicate either the contribution of extrapineal, extraocular sources of melatonin or melatonin secretion from remnants (if any) of pineal body tissue remaining after pinealectomy. Blinding did not obviously affect pineal melatonin levels nor did pinealectomy affect ocular melatonin levels. It was concluded that (1) daily rhythms in melatonin content occur in the pineal bodies, the eyes, and the blood of quail; (2) the blood rhythm is the result of melatonin secretion from both the pineal body and the eyes; (3) extraretinal photoreceptors can mediate entrainment of the pineal melatonin rhythm; and (4) obvious compensatory changes in melatonin levels do not occur in the eye following pinealectomy or in the pineal body following blinding.     

Melatonin Rhythms in Quail: Regulation by Photoperiod and Circadian Pacemakers

The profile of melatonin in the eyes, pineal, and blood of Japanese quail was assessed in birds held under LD 16:8 and LD 6: 18 photoperiods. Melatonin levels in all three tissues showed a robust daily rhythm with higher levels occurring at night. The amplitude of the rhythm was depressed and its duration lengthened on LD 6: 18 relative to LD 16:8. The blood melatonin rhythm precisely reflected the rhythms shown by the pineal and eyes, supporting the idea that the blood rhythm is a result of melatonin secretion by both the eyes and pineal.
The ocular melatonin rhythm continued after sectioning of the optic nerve, was reentrainable to a shift in the phase of the LD cycle, and persisted for at least 2 days in constant darkness. It was concluded that either (1) an intraocular circadian clock drives the ocular melatonin rhythm, or (2) an extraocular clock drives the ocular melatonin rhythm via a route other than the efferent innervation (which enters the eye via the optic tract).     

Tired of Diabetes Genetics? Circadian Rhythms and Diabetes: The MTNR1B Story?

Circadian rhythms are ubiquitous in biological systems and regulate metabolic processes throughout the body. Misalliance of these circadian rhythms and the systems they regulate has a profound impact on hormone levels and increases risk of developing metabolic diseases. Melatonin, a hormone secreted by the pineal gland, is one of the major signaling molecules used by the master circadian oscillator to entrain downstream circadian rhythms. Several recent genetic studies have pointed out that a common variant in the gene that encodes the melatonin receptor 2 (MTNR1B) is associated with impaired glucose homeostasis, reduced insulin secretion, and an increased risk of developing type 2 diabetes. Here, we try to review the role of this receptor and its signaling pathways in respect to glucose homeostasis and development of the disease.     

Analysis of the daily changes of melatonin receptors in the rat liver

Melatonin membrane (MT1 and MT2) and nuclear (RORα) receptors have been identified in several mammalian tissues, including the liver. The mechanisms regulating hepatic melatonin receptors are yet unknown. This study investigated whether these receptors exhibit daily changes and the effects of melatonin on their levels. Our results show that mRNAs for MT1/MT2 receptors exhibit circadian rhythms that were followed by rhythms in their respective protein levels; the acrophases for the two rhythms were reached at 04:00 and 05:00 hr, respectively. Pinealectomy blunted the rhythms in both mRNAs and protein levels. In contrast, mRNA and protein levels of nuclear receptor RORα increased significantly after pinealectomy. The cycles of the latter receptor also exhibited circadian rhythms which peaked at 03:00 and 03:45 hr, respectively. Melatonin administration (10–200 mg/kg) increased in a dose‐dependent manner the protein content of MT1/MT2 receptors, with no effects on RORα. Lunzindole treatment, however, did not affect melatonin receptor expression or content of either the membrane or nuclear receptors. Together with previously published findings which demonstrated the intracellular distribution of melatonin in rat liver, the current results support the conclusion that the circadian rhythms of MT1/MT2 and RORα receptors are under the control of the serum and intracellular melatonin levels. Moreover, the induction of MT1/MT2 receptors after the administration of high doses of melatonin further suggests that the therapeutic value of melatonin may not be restricted to only low doses of the indoleamine.     

The human pineal gland and melatonin in aging and Alzheimer's disease

The pineal gland is a central structure in the circadian system which produces melatonin under the control of the central clock, the suprachiasmatic nucleus (SCN). The SCN and the output of the pineal gland, i.e. melatonin, are synchronized to the 24-hr day by environmental light, received by the retina and transmitted to the SCN via the retinohypothalamic tract. Melatonin not only plays an important role in the regulation of circadian rhythms, but also acts as antioxidant and neuroprotector that may be of importance in aging and Alzheimer's disease (AD). Circadian disorders, such as sleep-wake cycle disturbances, are associated with aging, and even more pronounced in AD. Many studies have reported disrupted melatonin production and rhythms in aging and in AD that, as we showed, are taking place as early as in the very first preclinical AD stages (neuropathological Braak stage I-II). Degeneration of the retina-SCN-pineal axis may underlie these changes. Our recent studies indicate that a dysfunction of the sympathetic regulation of pineal melatonin synthesis by the SCN is responsible for melatonin changes during the early AD stages. Reactivation of the circadian system (retina-SCN-pineal pathway) by means of light therapy and melatonin supplementation, to restore the circadian rhythm and to relieve the clinical circadian disturbances, has shown promising positive results.     

Temperature-compensated circadian clock in the pineal of Anolis.

The pineal organ of the lizard Anolis carolinensis can be maintained for up to 10 days in superfused organ culture. During this time it synthesizes and releases melatonin into the medium flowing slowly over it. Collection of timed aliquots of medium and subsequent analysis for melatonin by radioimmunoassay reveal circadian rhythms of melatonin output by the isolated pineal. These rhythms persist for many cycles in constant darkness and at several constant ambient temperatures ranging from 22 to 37 degrees C. The period of the rhythm is temperature compensated (Q10 approximately equal to 1.14) and the rhythm is synchronized by light-dark cycles imposed on the cultured gland. This isolated vertebrate organ displays the three major properties of circadian systems and must therefore contain one or more circadian oscillators.     

Bovine large luteal cells show increasing de novo DNA methylation of the main ovarian CYP19A1 promoter P2

Wrasse species exhibit a definite daily rhythm in locomotor activity and bury themselves in the sand at the bottom of the ocean at night. It remains unclear how their behavior in locomotor activity is endogenously regulated. The aim of the present study was to clarify the involvement of melatonin and clock genes (Per1, Per2, Bmal1, and Cry1) in daily and circadian rhythms of the threespot wrasse, Halichoeres trimaculatus, which is a common species in coral reefs. Daily and circadian rhythms in locomotor activity were monitored under conditions of light-dark cycle (LD=12:12), constant light (LL), and darkness (DD). Daily rhythms in locomotor activity were observed under LD and persisted under LL and DD. Melatonin from a cultured pineal gland showed daily variations with an increase during the nighttime and a decrease during daytime, which persisted under DD. Melatonin treatment induced decreases in locomotor activity and respiratory rate, suggesting that melatonin has a sleep-inducing effect. Per1 and Per2 mRNA abundance in the brain under LD showed daily rhythms with an increase around lights on. Robust oscillation of Per1 and Per2 mRNA expression persisted under DD and LL, respectively. Expression of Bmal1 and Cry1 mRNA also showed daily and circadian patterns. These results suggest that clock genes are related to circadian rhythms in locomotor activity and that melatonin plays a role in inducing a sleep-like state after fish bury themselves in the sand. We conclude that the sleep-wake rhythm of the wrasse is regulated by a coordination of melatonin and clock genes.