Circadian rhythm in diurnal and nocturnal animals, especially mammals

Elevated blood melatonin functions differently in diurnal and nocturnal animals, although in both of them, melatonin secretion in the pineal glands occurs only at night (Schomerus and Korf, 2005; Bilu and Kronfeld-Schor, 2013). Nocturnal melatonin secretion induces the resting phase in diurnal animals, including human (Lewy et al., 1992; McArthur et al., 1996). Melatonin exerts its action on a timing mechanism controlling the switch from behavioral activity to quiescence in diurnal animals. On the other hand, in nocturnal animals, melatonin injections can influence the daily activity cycle of rats, and daily injections are followed by the onset of activity in constant darkness (Redman et al., 1983). When melatonin injection was stopped in rats, activity rhythms started free-running again (Redman et al., 1983). The circadian timing system controlling the rhythm of wheel-running activity in the C3H/HeN mouse was responsive to both light and melatonin (Benloucif and Dubocovich, 1996). Wild mice (nocturnal) produce melatonin, but they lost the ability to produce melatonin when they were laboratory animalized (Kasahara et al., 2010). Only recently have they lost the ability to produce melatonin, but they have maintained their genetically nocturnal nature. Thus, in nocturnal animals, melatonin acts as an entraining agent, as does light. The mechanisms by which melatonin regulates behavior in diurnal and nocturnal animals remain unclear.

Circadian rhythm in nonmammalian vertebrates

Melatonin secreted into the blood performs significant roles in the circadian organizations of many nonmammalian vertebrates, compared to mammals (Cassone, 1998). The removal of the pineal gland (pinealectomy) in the house sparrow, Passer domesticus, altered the free-running locomotor period and abolished their circadian rhythms (Chabot and Menaker, 1992). Similarly, in male European seabass, Dicentrarchus labrax, locomotor activity revealed a change from light-synchronization to free-running rhythmic patterns following pinealectomy (Cowan et al., 2017). Therefore, the melatonin secreted from the pineal gland likely influences behavior and physiology.

The significance of retinal melatonin synthesis is well known (Wiechmann, 1986; Zawilska and Nowak, 1992; Wiechmann and Sherry, 2013). In the retinas of nonmammalian species, such as the goldfish, Carassius auratus, frog Xenopus laevis, lizard Anolis carolinensis, and chicken, Gallus gallus domesticus, melatonin and the capacity for its synthesis and metabolism have been detected (Cahill and Besharse, 1989; Nowak et al., 1990; Grace et al., 1991). Diurnal variations in melatonin levels and AANAT activity in the retina have also been observed in Xenopus laevis and Gallus gallus domesticus (Hamm and Menaker, 1980; Besharse and Iuvone, 1983). Melatonin secreted from the retina may regulate the function of the retina, although there is no evidence that retinal melatonin regulates their behavior.

Seasonal rhythm regulation of melatonin in mammals

Pinealectomy in the male hamster causes an inability to sense changes in day length and the loss of photoperiodic responses, resulting in atrophy of the gonads (Hoffman and Reiter, 1965). Therefore, melatonin has an important function in regulating seasonal reproductive physiology in mammals. Long-day breeding animals (e.g., hamsters) are sexually depressed from autumn to winter and active from spring to summer, while short-day breeders, such as sheep and deer are actually sexually most active and capable during the shortest days of the year (autumn to winter). The elevated melatonin levels regulate sexual maturation in these animals (Fig. 1). Long-day shortens the duration of melatonin secretion in both long-day and short-day breeders. In addition, the changing duration of nocturnal melatonin secretion determines reproductive capability in seasonal breeders through the hypothalamic-pituitary-gonadal axis (Reiter et al., 2009) (Fig. 1). In mammals, kisspeptin (Kiss) and RFamide-related peptide (RFRP, the mammalian homo-logs of gonadotropin-inhibitory hormone: GnIH), are hypothalamic neuropeptides that stimulate and inhibit the synthesis and release of GnRH, respectively (viviD and Bentley, 2018; Ubuka, 2021). Administration of peripheral KISS to short-day-housed Syrian hamsters with regressed testes significantly increased testicular volume and testosterone secretion, while this response was blocked by the GnRH receptor antagonist acyline (Ansel et al., 2011). Melatonin injections reduce Kiss1 expression, and pinealectomy prevents short-day inhibition of Kiss1 in the arc of the Syrian hamster hypothalamus (Revel et al., 2007; Simonneaux et al., 2009). Thus, melatonin affects Kiss1 expression and mediates a reproductive signal via a pathway responsive to GnRH (viviD and Bentley, 2018).

On the other hand, the melatonin binding site in the PT is altered by the photoperiod. Namely, in the PT, the Per gene expression peaks during the daytime, while the Cry gene expression peaks early in the nighttime. Thus, the phase relationship between Per and Cry changes during the different photoperiods, and the PER/CRY protein/protein interactions due to the photoperiodic differences are potential mechanisms for producing photoperiodic responses (Ikegami and Yoshimura, 2012) (Fig. 1). As the thyroid hormone–stimulating hormone is secreted in the PT, thyroid hormones are also involved in seasonal reproduction (Watanabe et al., 2004; Ono et al., 2008; viviD and Bentley, 2018) (Fig. 1). Local thyroid hormone catabolism in the mediobasal hypothalamus by the deiodinase DIO2 and DIO3 enzymes that activate or inactivate thyroid hormones regulates the seasonal reproduction in birds and mammals (Ono et al., 2008). DIO3 inactivates the thyroid hormones triiodothyronine (T3, active form) and thyroxin (T4, prohormone of T3), while DIO2 converts T4 into T3. Long days induce thyroid-stimulating hormone production in the PT of the pituitary gland, which triggers DIO2 expression in the ependymal cells of the mediobasal hypothalamus (Fig. 1). Melatonin is indirectly involved in the regulation of Dio2 expression via thyroid-stimulating hormones. Furthermore, the Dio2 expression levels in the Djungarian hamster (Phodopus sungorus) are high under long-day conditions and low under short-day conditions and can be reduced further by melatonin injections (Watanabe et al., 2004). Therefore, melatonin regulates Dio2 and Dio3 expression and reproductive physiology through the thyroid hormone (Fig. 1).

Considering the above information, studies using long-day and short-day rearing animals have significantly contributed to our understanding of the mechanisms through which day length and melatonin can regulate seasonal reproduction (Fig. 1). As a result of these findings, melatonin has been successfully used as a pharmacological agent to accelerate the reproductive season in sheep, induce estrous cycles at a time when they would normally be in seasonal anestrus, and increase lambing (Haresign et al., 1990; Haresign, 1992; Abecia et al., 2007). Furthermore, daily afternoon injections of melatonin in Syrian hamsters under long-day conditions caused genital atrophy and hormonal changes similar to those induced by short-day exposures (Reiter et al., 1980). Thus, melatonin is an effective agent for regulating maturation in seasonally breeding animals.

Seasonal rhythm regulation of melatonin in non-mammals

Among birds, quail (Coturnix japonica) are the most sensitive to GnRH and LH alterations—changes occur from the first day—when transferred from short-day to long-day conditions. Further, GnRH and LH levels increase from the first day when birds are moved from short-day to long-day conditions, causing the testis weight to change dramatically in a short period of time (Perera and Follett, 1992; Yoshimura, 2006). This is why it has been continuously used in photoperiodic experiments. PT-derived TSH may be the key factor in regulating seasonal reproduction in birds as well as mammals (Nakane and Yoshimura, 2019), as shown in Fig. 1. TSH secreted from the PT influences ependymal cells and increases the expression of dio2. T3, augmented by Dio2, regulates the seasonal morphological change in neuroglial interactions. This morphological change photoperiodically regulates GnRH secretion. Melatonin regulates GnIH secretion and is involved in seasonal reproductive physiology (Chowdhury et al., 2013; Tsutsui and Ubuka, 2021). Melatonin appears to act on GnIH neurons by stimulating GnIH synthesis and release, and then inhibiting plasma LH concentration in birds (Chowdhury et al., 2013).

In the ruin lizard, Podarcis sicula, melatonin may play an important role in the seasonal reorganization of the circadian system (Bertolucci et al., 2002; Foà et al., 2002; Foà and Bertolucci, 2003). Pinealectomy in ruin lizards did not abolish circadian locomotor rhythmicity; instead, it induced marked changes in both the free-running period and circadian activity time (Foà, 1991). Either pinealectomy or melatonin implants caused an immediate transition from the typical summer circadian locomotor pattern to the typical spring or autumn circadian locomotor pattern, respectively (Foà et al., 1997). In addition, the degrees of melatonin and pineal involvement in the circadian organization are highest in summer compared to the other three seasons (Bertolucci et al., 2002; Foà et al., 2002; Foà and Bertolucci, 2003).

In the female skipper frog, Rana cyanophlyctis, continuous light stimulates, and melatonin inhibits, reproductive function (Udaykumar and Joshi, 1997). Continuous light exposure for 30 days stimulated the gonad somatic index (GSI), while daily injections of melatonin prevented the increase in GSI caused by continuous light. Oviductal weights decreased only in the melatonin-injected group. In the male frog Rana hexadactyla melatonin also inhibits testicular function and spermatogenesis when administered at an optimum dose for a stipulated period (Kasinathan and Gregalatchoumi, 1988). The inhibition of spermatogenesis by melatonin has also been reported in toads (Bufo melanostictus), where it suppresses light-induced spermatogenesis (Biswas et al., 1978; Chanda and Biswas, 1982). In both female and male frogs, therefore, melatonin inhibits gonad functions and regulates reproductive physiology.

Fig. 3.

Inhibitory action of calcitonin (A) and melatonin (B) on osteoclasts and detection of melatonin produced in goldfish scales (C). (A, B) Both calcitonin and melatonin significantly reduce osteoclastic activity (*P < 0.05), starting in the nanomolar range (Ikegame et al., 2019). One-way repeated measures analysis of variance followed by Dunnett's test was used to examine differences in the values of the experimental groups. (C) Melatonin produced in the goldfish scales was analyzed by liquid chromatography–tandem mass spectrometry (LCMS-8050; Shimadzu Co., Kyoto, Japan). Analysis of the extract from goldfish scales revealed the presence of melatonin product ions (red and blue) at the same elution time as the melatonin standard (Ikegame et al., 2019).

Melatonin is involved in the seasonal reproductive physiology in fish. For example, in the blue damselfish (Sapphire devil), the expression of aanat2 was higher during short-day conditions than long-day, suggesting that there is a seasonal change in melatonin levels at night (Imamura et al., 2022). Therefore, kiss1 and kiss2, gonadotropin-releasing hormone 1 (gnrh1), and the β-subunit of gonadotropins (fshβ and lhβ) levels were examined in the brain. Feeding mature females melatonin-containing pellets lowered kiss1, gnrh1, and lhβ mRNA levels within 3 h. Continuous melatonin treatment for 1-week resulted in oocyte regression and decreased kiss2, gnrh1, fshβ, and lhβ mRNA expressions. In catfish (Heteropneustes fossilis) and blue damselfish, melatonin inhibited kiss2 and gnrh2 expression in the brain (Chaube et al., 2020).

Bone metabolism regulated by melatonin

Spinal malformations have been reported in chickens (Machida et al., 1993), rats (Machida et al., 1999), and fish (Fjelldal et al., 2004), after surgical ablation of the pineal gland. Experimental scoliosis observed in pinealectomized chickens is similar to that in human idiopathic scoliosis. These results suggest that melatonin is involved in bone metabolism. Subsequently, in vitro studies have been performed to elucidate the direct influence of melatonin on bone metabolism. Melatonin has a strong inhibitory action on osteoclasts (Fig. 3A, B). Calcitonin is well known as a hypocalcemic hormone that inhibits osteoclasts and is used as a therapy for osteoporosis (Body, 2002; Suzuki, 2021). The ability of melatonin to exert an inhibitory action on osteoclasts is very similar to that exerted by calcitonin, as detected using a fish-scale (bone model) bioassay (Suzuki and Hattori, 2002; Suzuki et al., 2000). Melatonin (10^–9–10^–7 M) suppressed osteoclastic activity (Fig. 3B). As Mel1b is detected in osteoclasts immunohistochemically (Igarashi-Migitaka et al., 2020), melatonin directly acts on osteoclasts. In addition, the indirect influence of melatonin on osteoclasts has been reported. For example, the receptor activators of the nuclear factor-κB (RANK)/receptor activators of the nuclear factor-κB ligand (RANKL) system regarding the interaction between osteoclasts and osteoblasts are involved in the inhibitory action of melatonin on osteoclasts (Koyama et al., 2002; Kim et al., 2017; Li et al., 2019). Since RANKL induces osteoclast activation, it appears to be an important factor in osteoclastic bone resorption (Maria and Witt-Enderby, 2014; Nagy and Penninger, 2015). By suppressing RANKL expression, melatonin inhibited osteoclast activation (Koyama et al., 2002; Ikegame et al., 2019). A novel pathway was recently identified for the inhibitory effect of melatonin on osteoclasts (Ikegame et al., 2019; Nakano et al., 2019). As shown in Fig. 4, melatonin prevents bone resorption by increasing the expression of calcitonin by osteoblasts in fish (Ikegame et al., 2019) and chickens (Nakano et al., 2019).

Physical stimuli play an essential role in the regulation of bone remodeling (bone resorption and formation), either directly or indirectly (Carmeliet et al., 2001; Civitelli, 2008). Astronauts are exposed to the microgravity of space, which is known to reduce bone density (Collet et al., 1997; Caillot-Augusseau et al., 2000). Melatonin synthesized in osteoblasts in goldfish scales (Fig. 3C) was found to be involved in the progressive bone density loss effect in space (Fig. 4). Using fish scale culture systems (Fig. 4), containing co-existing osteoblasts and osteoclasts, in vitro space experiments have been performed on the International Space Station (ISS) since 2010 to examine the effect of microgravity on bone metabolism (Ikegame et al., 2019; Yamamoto et al., 2020; Yamamoto et al., 2022). Moreover, aanat expression decreases in microgravity (Ikegame et al., 2019). Furthermore, bone resorption under microgravity was inhibited by melatonin treatments via the promotive action of calcitonin production in osteoblasts (Fig. 4), suggesting that melatonin can be used as a potential drug for bone disease in astronauts (Hirayama et al., 2023).

Fig. 4.

Summary of the space experiment using goldfish scales. Lower frame: We observed the expression of the melatonin receptor, melatonin synthetic enzymes, and calcitonin (an osteoclast-inhibiting hormone) in osteoblasts of the regenerating scales. Melatonin stimulates calcitonin production in the scales, which has also been detected in mammalian bones. The melatonin-induced calcitonin suppresses osteoclast activity. Upper frame: Microgravity during space flight accelerated the multinucleation and resorption activity in scale osteoclasts, which was associated with a decrease in melatonin synthesis, the stimulated gene expression of rankl (a major factor for osteoclastogenesis), and the suppressed gene expression of calcitonin. Melatonin administration maintained the normal gene expression levels of the factors during space flight and suppressed microgravity-stimulated osteoclast activity. The results suggest that melatonin could be used as a prophylactic drug to prevent bone loss in astronauts during space flight. Abbreviations: OB: osteoblasts; OC: osteoclasts; POC: preosteoclasts; Rankl: receptor activator of nuclear factor κB ligand; Mel: melatonin; Mel-R: melatonin receptor; Calcitonin-R: calcitonin receptor.

Glucose metabolism regulated by melatonin

Since melatonin receptors are found in human islet β-cells, cohort analysis of single-nucleotide polymorphisms (SNPs) in the melatonin receptor (Mel1b) and type 2 diabetes mellitus (T2DM) has shown a link between melatonin and glucose metabolism (Karamitri and Jockers, 2019).

It has been reported that the rs1387153 SNP, near MTNR1B (which encodes Mel1b), is associated with increased fasting plasma glucose levels and an elevated risk of T2DM (Bouatia-Naji et al., 2009). Furthermore, MTNR1B is expressed in human islet β-cells, while insulin release in INS-1 832/13 clonal β-cells, in response to stimulation with glucose, was inhibited in the presence of melatonin (Lyssenko et al., 2009). Since MTNR1B expression increases in individuals at risk of T2DM (Lyssenko et al., 2009), its pathological effects are likely exerted through the direct inhibition of insulin release from β-cells by melatonin.

Melatonin regulates basal glucose metabolism in fish. In fasted goldfish individuals, we found that plasma melatonin levels were significantly higher and insulin levels were significantly lower at night. Furthermore, glucose uptake in the brain, liver, and muscle tissues also significantly increases at night. After intraperitoneal administration of melatonin, glucose uptake by the brain and liver showed significantly greater increases than in the control group, although it failed to alter plasma insulin levels (Watanabe et al., 2023). Using an insulin-free medium, we further showed that melatonin treatment increased glucose uptake in primary cell cultures of goldfish brain and liver cells, in a dose-dependent manner (Watanabe et al., 2023). The administration of melatonin also significantly decreased plasma glucose levels in hyperglycemic goldfish but failed to alter insulin mRNA expression or plasma insulin levels, suggesting that melatonin has the same potency as a therapeutic drug as insulin does for diabetes.

Therapeutic benefits of melatonin through antioxidative action

Therapeutic effects of melatonin have been reported for several disorders, such as different types of tumors (intestinal tumors, melanoma, breast cancer, etc.), caused by its antioxidative action because oxidative stress is involved in the initiation, promotion, and progression of carcinogenesis (Karbownik et al., 2001; Tordjman et al., 2017). The inhibitory action of melatonin by its oral administration, after being dissolved in tap water at 20 mg/L, has been reported in intestinal carcinogenesis in rats, induced by the carcinogen 1,2-dimethylhydrazine (Anisimov et al., 1997).

Melatonin is effective in cardiovascular diseases (Tordjman et al., 2017). Urinary noradrenaline increases at night in patients with coronary heart disease (Brugger et al., 1995). The increase in noradrenaline in these patients may be related to the suppression of sympathetic activity by melatonin. Therefore, melatonin levels in the blood of these patients (6.5–11.8 pg/mL) were significantly lower than those in 10 healthy controls (32.2–42.5 pg/mL) (Brugger et al., 1995). Additionally, the administration of melatonin (1 mg) significantly reduced blood pressure in comparison to the placebo (Arangino et al., 1999).

Fig. 5.

Schematic diagram of therapeutic benefits of melatonin by receptor-independent action. Melatonin and its metabolites (AFMK: N¹-acetyl-N²-formyl-5-methoxykynuramine; AMK: N¹-acetyl-5-methoxykynuramine) have some therapeutic effects, such as anti-tumor, anti-cardiovascular disease, anti-bone resorption, and anti-memory disorder by radical scavenging function.

On the other hand, as described above, melatonin inhibits osteoclastic activity via the membrane receptor Mel1b. Since reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, promote bone resorption (Cardinali et al., 2003), melatonin suppresses osteoclastic activity through its free radical scavenging function. Therefore, we hypothesize that melatonin can provide a therapeutic effect on osteoporosis by inhibiting bone resorption through both receptor-mediated and receptor-independent actions.

Protection against space radiation by melatonin

During space flight, astronauts are constantly exposed to radiation, including high linear energy transfer radiation, which differs from the radiation on Earth (Takahashi et al., 2018; Hirayama et al., 2023). The radioprotective effect of melatonin is well known, whereby many in vitro and in vivo studies have shown that melatonin protects mammalian cells from the toxic effects of ionizing radiation (Vijayalaxmi, 2004; Farhood et al., 2019; Nuszkiewicz et al., 2020). Here, we consider that melatonin provides a potent protective effect against space radiation. However, there have been no previous studies on this topic. Therefore, we performed a space experiment using goldfish scales (Fig. 4) with the ISS to determine the radioprotective potential of melatonin during space flight (Furusawa et al., 2020). After returning to Earth, RNA-sequencing analysis with subsequent de novo transcriptome assembly and computational gene expression analysis were performed. Genes coding for heat shock proteins, DNA repair markers, and those involved in the oxidative stress response comprised the majority of the identified upregulated genes in response to space radiation (Furusawa et al., 2020). Fish scales treated with melatonin counteracted the upregulation of those genes in response to space radiation. The effects of melatonin on gene expression are expected to enhance cell survival. In our space experiment, melatonin improved the expression of genes that support cell survival in response to space radiation, such as the antiapoptotic bcl-2 gene (Furusawa et al., 2020).

Fig. 6.

Evolutionary implications of melatonin. The antioxidant properties of melatonin indicate that this action is extremely important for aerobic organisms to remove harmful reactive oxygen species. In Annelida, melatonin signaling regulates circadian swimming by rhythmically activating cholinergic neurons (Schippers and Nichols, 2014). As a result, circadian regulation occurs in invertebrates. In vertebrates, melatonin is mainly secreted from the pineal gland and regulates the circadian and seasonal rhythms systematically via melatonin receptors. Due to rhythmical regulation, we believe that several processes of metabolism must be controlled by melatonin in vertebrates.

Novel functions of melatonin derivatives

AFMK is endogenously generated from melatonin by indoleamine 2,3-dioxygenase or metabolized from melatonin by free-radical scavenging action (Tan et al., 2002). This metabolized AFMK possesses antioxidative ability in calf thymus, rat liver, and HT22 cells, a subclone of the HT4 hippocampal cell line (Tan et al., 2001; Manda et al., 2008). Moreover, Tan et al. (2001) showed that AFMK inhibited the formation of 8-hydroxydeoxyguanosine induced by the incubation of DNA with oxidants in calf thymus. Lipid peroxidation resulting from free radical damage in rat liver homogenates was also inhibited by the addition of AFMK (Tan et al., 2001). In radiation-induced oxidation, AFMK also suppressed the loss of proliferating and immature neurons in the dentate gyrus of the brain by inhibiting oxidative stress in mice irradiated with high-LET ⁵⁶Fe particles (Manda et al., 2008). We recently found a novel action of AFMK and AMK regarding the enhancement of learning and memory in mice (Iwashita et al., 2021). Both AFMK and AMK enhanced long-term memory in the object recognition test. Moreover, melatonin metabolites serve the same physiological functions as melatonin, which may contribute to the endurance of its effects.