MicroRNA‐7 inhibits melatonin synthesis by acting as a linking molecule between leptin and norepinephrine signaling pathways in pig pineal gland

MicroRNAs, including microRNA‐7 (miR‐7), are important modulators of numerous gene expressions and the related biological processes. Melatonin is a key hormone regulating daily and seasonal rhythms, in which a variety of positive and negative regulatory factors, such as norepinephrine (NE) and leptin, are involved. However, the interactions among these factors and the mechanisms remain to be elucidated. The aims of the present study were to identify the functions and the related mechanisms of miR‐7 in regulating melatonin synthesis and secretion through in vitro and in vivo experiments in pineal gland of pigs, which is an important animal model for agricultural and biomedical studies. Our results firstly show that miR‐7 is specifically expressed in porcine pinealocytes and negatively regulates melatonin synthesis. The further functional studies show that the dynamic expression levels of miR‐7 are contrary to the melatonin levels throughout the day, and the forced inhibition of endogenous miR‐7 in porcine pinealocytes sharply increases arylalkylamine N‐acetyltransferase (AANAT) expression by 80.0% (P = 0.0031) and melatonin levels by 81.0% (P = 0.0421), whereas miR‐7 over‐expression down‐regulates AANAT expression by 38.6% (P = 0.0004) and melatonin levels by 37.6% (P = 0.0212). In addition, the miR‐7 expression is up‐regulated by leptin through the JAK/STAT3 signaling pathway, and the in vivo intracerebroventricular injection of leptin increases miR‐7 expression by 80.0% (P = 0.0044) in porcine pineal glands and reduces melatonin levels by 57.1% (P = 0.0060) compared with the controls. This functional inhibition of melatonin synthesis by miR‐7 is accomplished by its binding to the 3′‐UTR of Raf1. Further, our results demonstrate that the RAF1/MEK/ERK signaling pathway mediates NE‐induced AANAT expression, whereas leptin attenuates NE's function through miR‐7. Taken together, the results demonstrated that leptin activates the JAK/STAT3 signaling pathway to increase the expression of miR‐7, which acts as a negative regulatory molecule inhibiting NE‐activated RAF1/MEK/ERK signaling pathway by targeting Raf1, resulting in decreased AANAT expression and melatonin synthesis. These findings suggest that miR‐7 is a novel negative regulator of melatonin synthesis and links leptin‐ and NE‐mediated signaling pathways in porcine pineal glands, which will contribute to our understanding in the establishment of the biological rhythms resulting from melatonin.     

Heterogeneous ribonucleoprotein R regulates arylalkylamine N‐acetyltransferase synthesis via internal ribosomal entry site‐mediated translation in a circadian manner

Rhythmic arylalkylamine N‐acetyltransferase (AANAT) synthesis is a prominent circadian‐controlled response that occurs in most mammals. AANAT is the core enzyme in melatonin production; because melatonin participates in many physiological processes, the regulation of AANAT is an important research topic. In this study, we focused on the role of heterogeneous ribonucleoprotein R (hnRNP R) in the translation of AANAT. A novel RNA‐binding protein hnRNP R widely interacted with the 5′ untranslated region (UTR) of AANAT mRNA and contributed to translation through an internal ribosomal entry site (IRES). Fine‐tuning of AANAT protein synthesis occurred in response to knockdown and overexpression of hnRNP R. Nocturnal elevation of AANAT protein was dependent on the rhythmic changes of hnRNP R, whose levels are elevated in the pineal gland during nighttime. Increases in hnRNP R additionally improved AANAT production in rat pinealocytes under norepinephrine (NE) treatment. These results suggest that cap‐independent translation of AANAT mRNA plays a role in the rhythmic synthesis of melatonin through the recruitment of translational machinery to hnRNP R‐bound AANAT mRNA.     

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): I. Rhythmic pattern and effect of light

Serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] is the rate-limiting enzyme in melatonin synthesis, and its activity exhibits a diurnal rhythm similar to that of the melatonin content in the pineal gland and retina of Japanese quail. Studies were conducted to characterize the Japanese quail AANAT cDNA, and to evaluate the expression of AANAT mRNA in the pineal gland, the retina, and other peripheral tissues. The nucleic acid sequence of a 400 bp cDNA clone obtained by RT-PCR manifested 78 and 95% homology compared to the rat and chicken AANAT cDNA, respectively, while the deduced amino acid sequence homology was 82 and 99%, respectively. AANAT mRNA content in a single pineal gland or an aliquot of eye lysate was measured by a micro-lysate protection assay. The expression of AANAT mRNA in the pineal gland and the retina exhibited circadian rhythm with peak levels at night. AANAT mRNA was also detected in the testis, but did not display a rhythmic change over a 24 hr period. AANAT mRNA was not detected in other tissues studied. Darkness during the day did not increase the pineal AANAT mRNA levels. However, unexpected light-exposure for 2 hr just after lights-off blocked the increase in AANAT mRNA, and at midnight remarkably decreased AANAT mRNA by 50%.     

Regulation of the expression of serotonin N-acetyltransferase gene in Japanese quail (Coturnix japonica): II. Effect of vitamin A deficiency

Levels of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AANAT); EC2.3.1.87] mRNA, AANAT activity, and melatonin display a rhythmic pattern in both the pineal gland and the retina. It has been shown that vitamin A is required to maintain the rhythm of melatonin synthesis in the pineal gland of Japanese quail. To understand the mechanism underlying the direct relationship among these factors, we developed an assay system sensitive enough to determine AANAT mRNA, AANAT activity, and melatonin content from a single pineal gland of Japanese quail. Positive direct relationships were found among these three parameters. We next deprived Japanese quail of vitamin A by feeding them a vitamin A-free diet supplemented with retinoic acid, and examined the effects of vitamin A deficiency on the expression of AANAT mRNA in the pineal gland and the retina. Vitamin A deficiency reduced both the expression of AANAT mRNA and melatonin content in the pineal gland. Retinal AANAT mRNA rhythm disappeared in vitamin A-deficient quails. Moreover, the responsiveness of the pineal gland and the retina to light was reduced by vitamin A deficiency when compared with the control group.     

New light is shining on the melatonin rhythm enzyme The first postcloning view.

One of the most interesting molecules in circadian biology is serotonin N-acetyltransferase (arylalkyfamine N-acetyltransferase, AANAT), the enzyme that controls the daily rhythm in pineal melatonin production and blood melatonin. The recent cloning of AANAT cDNA has led to the characterization of the human gene; the realization that AANAT represents a unique gene family; the discovery of circadian rhythms in AANAT mRNA; the determination of the basis of transsynaptic and cellular regulation of expression of the AANAT gene; a new understanding of the relationship of AANAT mRNA and activity; and the surprising finding of strong expression of the AANAT gene in the retina and significant levels in select brain regions, the pituitary gland, and testes. The cloning of AANAT cDNA has not only made it possible to answer longstanding questions in circadian biology, but has also raised stimulating new issues.     

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.     

Diurnal and circadian rhythms in melatonin synthesis in the turkey pineal gland and retina

The pineal gland and retina of the turkey rhythmically produce melatonin. In birds kept under a daily light-dark (LD) illumination cycle melatonin concentrations in the pineal gland and retina were low during the light phase and high during the dark phase. A similar melatonin rhythm with high night-time values was also observed in the plasma. The pineal and retinal melatonin rhythms mirror oscillations in the activity of serotonin N-acetyltransferase (AANAT; the penultimate enzyme in the melatonin biosynthetic pathway). In contrast, in both the pineal gland and retina the activity of the enzyme hydroxyindole-O-methyltransferase (HIOMT) did not exhibit significant changes throughout the 24-h period. Acute exposure of turkeys to light at night dramatically decreased melatonin levels in the pineal gland, retina and plasma. The rhythms in AANAT activity and melatonin concentrations in the turkey pineal gland and retina were circadian in nature as they persisted under conditions of constant darkness (DD). Under DD, however, the amplitudes of AANAT and melatonin rhythms were significantly lower (by 50-80%) than those found under the LD cycle. The findings indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The rapid dampening of the rhythms under DD suggests that of these two regulatory factors, environmental light may be the primary stimulus in the maintenance of the high amplitude melatonin rhythms in the turkey.     

Starting the zebrafish pineal circadian clock with a single photic transition

The issue of what starts the circadian clock ticking was addressed by studying the developmental appearance of the daily rhythm in the expression of two genes in the zebrafish pineal gland that are part of the circadian clock system. One encodes the photopigment exorhodopsin and the other the melatonin synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT2). Significant daily rhythms in AANAT2 mRNA abundance were detectable for several days after fertilization in animals maintained in a normal or reversed lighting cycle providing 12 h of light and 12 h of dark. In contrast, these rhythms do not develop if animals are maintained in constant lighting or constant darkness from fertilization. In contrast to exorhodopsin, rhythmicity of AANAT2 can be initiated by a pulse of light against a background of constant darkness, by a pulse of darkness against a background of constant lighting, or by single light-to-dark or dark-to-light transitions. Accordingly, these studies indicate that circadian clock function in the zebrafish pineal gland can be initiated by minimal photic cues, and that single photic transitions can be used as an experimental tool to dissect the mechanism that starts the circadian clock in the pineal gland.     

Evidence of immune system melatonin production by two pineal melatonin deficient mice, C57BL/6 and Swiss strains

Abstract:  We evaluated two pineal melatonin deficient mice described in the literature, i.e., C57BL/6 and Swiss mice, as animal models for studying the immunomodulatory action of melatonin. Plasma melatonin levels in C57BL/6 and Swiss strains were detectable, but lower than levels in control C3H/HENHSD mice. Since these strains are suppose to be pineal melatonin deficient an extrapineal melatonin synthesis may contribute to plasma levels. Regarding cells and tissues from the immune system, all of them were found to synthesize melatonin although at low levels. N-acetyltransferase (AANAT) mRNA was also amplified in order to analyze the alternative splicing between exons 3–4 described for pineal C57BL/6 mice which generates an inclusion of a pseudoexon of 102 bp. For the pineal gland, both the wild type and the mutant isoforms were present in all mice strains although in different proportions. We observed a predominant wild type AANAT mature RNA in thymus, spleen and bone marrow cells. Peripheral blood mononuclear cells (PBMC) culture shown an evident AANAT amplification in all strains studied. Although the bands detected were less intense in melatonin deficient mice, the amplification almost reached the control cell intensity after stimulation with phytohemaglutinin (PHA). In summary, melatonin detection and AANAT mRNA expression in inbred and outbred mice clearly indicate that different cells and tissues from the immune system are able to synthesize melatonin. Thus, the pineal defect seems not to be generalized to all tissues, suggesting that other cells may compensate the low pineal melatonin production contributing to the measurable plasma melatonin level.     

Regulation of arylalkylamine N-acetyltransferase-2 (AANAT2, EC 2.3.1.87) in the fish pineal organ: evidence for a role of proteasomal proteolysis

In fish, individual photoreceptor cells in the pineal organ and retina contain complete melatonin rhythm generating systems. In the pike and seabream, this includes a photodetector, circadian clock, and melatonin synthesis machinery; the trout lacks a functional clock. The melatonin rhythm is due in part to a nocturnal increase in the activity of the arylalkylamine N-acetyltransferase (AANAT) which is inhibited by light. Two AANATs have been identified in fish: AANAT1, more closely related to AANATs found in higher vertebrates, is specifically expressed in the retina; AANAT2 is specifically expressed in the pineal organ. We show that there is a physiological day/night rhythm in pineal AANAT2 protein in the pike, and that light exposure at midnight decreases the abundance of AANAT2 protein and activity. In culture, this decrease is blocked by inhibitors of the proteasomal degradation pathway. If glands are maintained under light at night, treatment with these inhibitors increases AANAT2 activity and protein. Organ culture studies with the trout and seabream also indicate that the light-induced decrease of AANAT2 activity is prevented when proteasomal proteolysis is blocked. A cAMP-dependent pathway protects AANAT2 protein from degradation. These results provide a clue to understanding how light regulates the daily rhythm in melatonin secretion in fish photoreceptor cells and provides evidence that proteasomal proteolysis is a conserved element in the regulation of AANAT in vertebrates.     

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.     

Melatonin formation in mammals: In vivo perspectives

Melatonin is a hormone secreted from the pineal gland specifically at night and contributes to a wide array of physiological functions in mammals. Melatonin is one of the most well understood output of the circadian clock located in the suprachiasmatic nucleus. Melatonin synthesis is controlled distally via the circadian clock located in the suprachiasmatic nucleus and proximally regulated by norepinephrine released in response to the circadian clock signals. To understand melatonin synthesis in vivo, we have performed microdialysis analysis of the pineal gland, which monitors melatonin as well as the precursor (serotonin) and intermediate (N-acetylserotonin) of melatonin synthesis in freely moving animals in realtime at high resolution. Our data revealed a number of novel features of melatonin production undetected using conventional techniques, which include (1) large inter-individual variations of melatonin onset timing; (2) circadian regulation of serotonin synthesis and secretion in the pineal gland; and (3) a revised view on the rate-limiting step of melatonin formation in vivo. This article will summarize the main findings from our laboratory regarding melatonin formation in mammals.     

Diurnal variation of the adenylyl cyclase type 1 in the rat pineal gland.

Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:00-3:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calcium-calmodulin complex. These findings suggest (i) the involvement of AC1 acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of AC1 in the rat pineal gland.     

The rat pineal gland comprises an endocannabinoid system

In the mammalian pineal gland, the rhythm in melatonin biosynthesis depends on the norepinephrine (NE)-driven regulation of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme of melatonin biosynthesis. A recent study showed that phytocannabinoids like tetrahydrocannabinol reduce AANAT activity and attenuate NE-induced melatonin biosynthesis in rat pineal glands, raising the possibility that an endocannabinoid system is present in the pineal gland. To test this hypothesis, we analyzed cannabinoid (CB) receptors and specific enzymes for endocannabinoid biosynthesis or catabolism in rat pineal glands and cultured pinealocytes. Immunohistochemical and immunoblot analyses revealed the presence of CB1 and CB2 receptor proteins, of N-acyl phosphatidyl ethanolamine hydrolyzing phospholipase D (NAPE-PLD), an enzyme catalyzing endocannabinoid biosynthesis and of fatty acid amide hydrolase (FAAH), an endocannabinoid catabolizing enzyme, in pinealocytes, and in pineal sympathetic nerve fibers identified by double immunofluorescence with an antibody against tyrosine hydroxylase. The immunosignals for the CB2 receptor, NAPE-PLD, and FAAH found in pinealocytes did not vary under a 12 hr light:12 hr dark cycle. The CB1 receptor immunoreaction in pinealocytes was significantly reduced at the end of the light phase [zeitgeber time (ZT) 12]. The immunosignal for NAPE-PLD found in pineal sympathetic nerve fibers was reduced in the middle of the dark phase (ZT 18). Stimulation of cultured pinealocytes with NE affected neither the subcellular distribution nor the intensity of the immunosignals for the investigated CB receptors and enzymes. In summary, the pineal gland comprises indispensable compounds of the endocannabinoid system indicating that endocannabinoids may be involved in the control of pineal physiology.     

N‐terminal residues regulate proteasomal degradation of AANAT

Abstract: Serotonin N‐acetyltransferase (AANAT) catalyzes the conversion of serotonin to N‐acetylserotonin, which is the immediate precursor for formation of melatonin. Although it is known that AANAT is degraded via the proteasomal proteolysis, detailed mechanisms are not defined. In this paper, we tested the in vivo role of proteasome inhibition on AANAT activity and melatonin release and examined the amino acid residues in AANAT that contribute to the proteasome degradation. We have shown that inhibition of proteasome activities in vivo in the intact pineal gland fails to prevent the light‐induced suppression of melatonin secretion. Furthermore, in cell lines stably expressing AANAT, inhibition of proteasomal proteolysis, which resulted in a large accumulation of AANAT protein, similarly failed to increase AANAT enzyme activity proportional to the amount of proteins accumulated. Site‐directed mutagenesis analysis of AANAT revealed that the AANAT degradation is independent of lysine and the two surface cysteine residues. Deletion analysis of N‐terminus identified the second amino acid leucine (L2) as the key residue that contributes to the proteasomal proteolysis of AANAT protein. These results suggest that rat AANAT protein is degraded via the N‐end rule pathway of proteasomal proteolysis and the leucine at the N‐terminus appears to be the key residue recognized by N‐end rule pathway.     

Are cortisol and melatonin involved in the immune modulation by the light environment in pike perch Sander lucioperca?

The pineal gland is the main organ involved in the transduction process converting environmental light information into a melatonin response. Since light environment was described as an important factor that could affect physiology of teleosts, and because melatonin is a crucial hormone regulating numerous physiological processes, we hypothesized that environmental light may act on both stress and circadian axes which in turn could influence the immune status of pike perch. Therefore, we investigated the effects of two light spectra (red and white) and two light intensities (10 and 100 lx) with a constant photoperiod 12L_{(8:00‐20:00)}/12D on pike perch physiological and immune responses. Samples were collected at 04:00 and 16:00 at days 1 and 30 of the experiment. Stress markers, plasma melatonin levels, humoral innate immune markers, and expression of key immune genes in the head kidney were assessed. Light intensity clearly affected pike perch physiology. This included negative growth performances, increase in stress status, decrease in plasma melatonin levels, and immune depression. Light spectrum had only little influences. These results demonstrate that high stress status may have impacted melatonin production and secretion by the pineal organ. The drop in circulating melatonin and the increase in stress status may both be involved in the immune suppression.