Expression Of Arylalkylamine N-acetyltransferase Research Articles

Sleep deprivation during pregnancy leads to poor fetal outcomes in Sprague–Dawley rats

Sleep deprivation is a common problem during pregnancy, but its impact on the fetus remains unclear. We aimed to investigate the effect of sleep deprivation during pregnancy on fetal outcomes and its mechanism in Sprague–Dawley rats. Sleep deprivation was performed from gestational day(GD) 1–19 using a multiplatform method for 18 h/day. Rats were sacrificed on GD20, and their blood and placentas were collected. Fetal and placental parameters were ascertained. Melatonin, adrenocorticotropic hormone (ACTH) and corticosterone were also measured in serum. The levels of arylalkylamine N-acetyltransferase (AANAT) and two melatonin receptors MT1 and MT2, in placental tissues were detected by western blotting. The inflammatory status and oxidative stress in serum and placentas were investigated. Miscarriage and intrauterine growth restriction were observed in the sleep deprivation group. Sleep deprivation resulted in an increased fetal absorption rate, while fetal weight, crown-rump length and placental weight were reduced. Placental histopathology showed that the labyrinth ratio in the sleep deprivation group was significantly reduced, with hypoplastic villi and obviously decreased blood vessels. Sleep deprivation decreased melatonin in serum and the expression of AANAT, MT1 and MT2 in placental tissues, elevated the oxidative stress products 8-hydroxy-deoxyguanosine (8-OHdG) and malondialdehyde(MDA) in serum and 4-hydroxynonenal (4HNE) in the placenta, and decreased the antioxidants superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in serum. Serum proinflammatory cytokines including interleukin-1-beta (IL-1β), interleukin-6 (IL-6), necrotizing factor-alpha (TNF-α), and interleukin-8(IL-8), were all elevated by sleep deprivation, and the inflammatory regulatory factor nuclear factor-κB p65 (NF-κB p65) in the placenta was enhanced when examined by immunohistochemistry. Corticosterone levels were comparable between the two groups, although ACTH levels were elevated significantly in the sleep deprivation group. Our study revealed that sleep deprivation during pregnancy can adversely impact fetal outcomes. Melatonin may play an important role in this pathology through the oxido-inflammatory process.

Arylalkylamine N-acetyltransferase (AANAT): Blue light induction, nuclear translocation, and potential role in the survival of chicken retina neuronal cells.

In vertebrates, arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is the time-keeping and key regulatory enzyme in melatonin (Mel) biosynthesis. AANAT is present in the pineal gland, retina, and other regions where it is controlled by light, cyclic adenosine monophosphate (cAMP) levels, and the molecular clock. AANAT converts serotonin to N-acetyl serotonin (NAS) and the last enzyme in the pathway, hydroxy-o-methyltransferase (HIOMT), forms Mel by NAS methylation. We have previously shown that AANAT is expressed in chicken retinal ganglion cells (RGCs) during daytime at the level of mRNA and enzyme activity. Here we investigated the presence of AANAT protein and mRNA throughout development in the chicken embryonic retina as well as AANAT expression, phosphorylation, and its sub-cellular localization in primary cultures of retinal neurons from E10 embryonic retinas exposed to blue light (BL) and controls kept in the dark (D). From embryonic days 7-10 (E7-10) AANAT mRNA and protein were visualized mainly concentrated in the forming ganglion cell layer (GCL), while from E17 through postnatal days, expression was detectable all through the different retinal cell layers. At postnatal day 10 (PN10) when animals were subjected to a 12:12 h LD cycle, AANAT was mainly expressed in the GCL and inner nuclear layer cells at noon (Zeitgeber Time (ZT 6)) and in the photoreceptor cell layer at night (ZT 21). Primary cultures of retinal neurons exhibited an induction of AANAT protein when cells were exposed to BL for 1 h as compared with D controls. After BL exposure, AANAT showed a significant change in intracellular localization from the cytoplasm to the nucleus in the BL condition, remaining in the nucleus 1-2 h in the D after BL stimulation. BL induction of nuclear AANAT was substantially inhibited when cultures were treated with the protein synthesis inhibitor cycloheximide (CHD). Furthermore, the phosphorylated form of the enzyme (pAANAT) increased after BL in nuclear fractions obtained from primary cultures as compared with D controls. Finally, the knockdown of AANAT by sh-RNA in primary cultures affected cell viability regardless of the light condition. AANAT knockdown also affected the redox balance, sh-AANAT treated cultures showing higher levels of reactive oxygen species (ROS) than in the sh-control. Our results support the idea that AANAT is a BL-sensing enzyme in the inner retina of diurnal vertebrates, undergoing phosphorylation and nuclear importation in response to BL stimulation. Moreover, it can be inferred that AANAT plays a novel role in nuclear function, cell viability, and, likely, through redox balance regulation.

The melatonin system is expressed in the ovine uterus: effect of the day of the oestrous cycle and undernutrition.

Melatonin influences female reproduction, but expression of the melatonin system has not been characterised in the ovine uterus. We aimed to determine whether synthesising enzymes (arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin-O-methyltransferase (ASMT)), melatonin receptors 1 and 2 (MT1 and MT2), and catabolising enzymes (myeloperoxidase (MPO) and indoleamine 2,3-dioxygenase 1 and 2 (IDO1 and 2)), are expressed in the ovine uterus, and if they are influenced by the oestrous cycle (Experiment 1) or by undernutrition (Experiment 2). In Experiment 1, gene and protein expression was determined in sheep endometrium samples collected on days 0 (oestrus), 5, 10 and 14 of the oestrous cycle. In Experiment 2, we studied uterine samples from ewes fed either 1.5 or 0.5times their maintenance requirements. We have demonstrated the expression of AANAT and ASMT in the endometrium of sheep. AANAT and ASMT transcripts, and AANAT protein were more elevated at day 10, then decreased to day 14. A similar pattern was observed for MT2 , IDO1 , and MPO mRNA, which suggests that the endometrial melatonin system might be influenced by ovarian steroid hormones. Undernutrition increased AANAT mRNA expression, but seemed to decrease its protein expression, and increased MT2 and IDO2 transcripts, whereas ASMT expression was unaffected. The melatonin system is expressed in the ovine uterus and is affected by oestrous cycle and undernutrition. The results help explain the adverse effects of undernutrition on reproduction in sheep, and the success of exogenous melatonin treatments in improving reproductive outcomes.

Effects of different light conditions on growth, muscle nutrients content, and clock gene circadian expression of Takifugu rubripes

Basic knowledge about the effect of light on fish in needed to improve their development, survival, and growth to increase aquaculture efficiency. In this study, the effects of light photoperiods (light (L): dark (D) = 12:12 and 16:8) and spectrum (full spectrum (W), blue (B) and yellow (Y)) on Takifugu rubripes larvaes were assessed from 20 to 80 days after hatching (dah). At 50 dah, the body length of larvae exposed to Y16:8 and B16:8 was significantly higher than that in the W16:8 group (P < 0.05). The wet weight of larvae reared under B16:8 was significantly higher than that under B12:12 (P < 0.05). The wet weight of larvae exposed to B12:12 was significantly higher than that in the W12:12, and the wet weight of larvae exposed to B16:8 was significantly higher than that of larvae exposed to W16:8 and Y16:8 (P < 0.05). At 80 dah, the body length and wet weight of larvae reared under 12:12 were significantly higher than those of larvae reared under 16:8 in the white and yellow LEDs groups (P < 0.05). Additionally, the biochemical composition of muscle were affected, the levels of DHA and EPA+DHA were significantly higher in the W16:8 and B16:8 groups, compared to the W12:12 and B12:12 groups, respectively (P < 0.05). Since light is the predominant signal for the entrainment of circadian oscillators and controls growth as a zeitgeber, the expression of arylalkylamine N-acetyltransferase 2 (annat2), crytochrome 2 (cry2), peroid1 (per1) and per3 in the brain were also been measured. The long photoperiod 16:8 delayed the acrophases of annat2, and blue light decreased the expression of cry2 and per3. These results provide the theoretical basis for using LED lights in aquaculture production of T. rubripes larvae.

Melatonin relieves Th17/CD4−CD8− T cells inflammatory responses via nuclear-receptor dependent manner in peripheral blood of primary Sjögren’s syndrome

ObjectivesPrimary Sjögren’s syndrome (pSS) is a complex systemic autoimmune disease whose clinical spectrum extends from sicca syndrome to systemic involvement. T helper 17 cells (Th17) and CD4−CD8− (double negative, DN) T cells are actively involved in the pathogenesis of pSS. Melatonin shows important immunoregulatory functions in multiple T cell-mediated autoimmune diseases. However, the effects of melatonin on the immune cells of pSS patients are unclear. Hence, this study was aimed to evaluate the effects of melatonin on the immune responses of peripheral pathogenic Th17 and DN T cells from pSS patients, and explore the underlying receptor-related mechanism. MethodsThe concentration of serum and saliva melatonin of pSS patients and healthy controls (HCs) were detected using Enzyme-linked immunosorbent assays (ELISA). Expression of arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT) were conducted in labial glands samples by immunohistochemistry. The mechanism underlying the effects of melatonin on Th17 and DN T cells responses in peripheral blood from pSS was investigated by quantitative real-time polymerase chain reaction (RT-PCR), flow cytometry, ELISA, cell viability, and proliferation assays. ResultsSerum and saliva melatonin levels were lower in pSS patients than in HCs, which were negatively correlated with disease activity. The expression levels of melatonin's biosynthetic enzymes (AANAT, HIOMT) and nuclear receptors (RORα, RORγ) were significantly increased in peripheral blood mononuclear cells (PBMCs) from pSS patients. Furthermore, in vitro melatonin administration decreased the expression of melatonin effector/receptor system in peripheral blood of pSS patients. More importantly, Melatonin inhibited pathogenic responses of peripheral Th17 and DN T cells in PBMCs from pSS, which was independent of melatonin membrane receptors. However, melatonin nuclear receptor antagonist SR1001 enhanced the inhibitory ability of melatonin on Th17 and DN T cells production, and agonist SR1078 weakened the effects of melatonin. Additionally, overexpression of the melatonin effector/receptor system in pSS patients appeared to be involved in the disease, due to that melatonin effector/receptor system expression was correlated with the frequency of Th17 or DN T cells. ConclusionMelatonin relieved the inflammatory responses of Th17 and DN T cells in PBMCs from pSS patients in a nuclear receptors-dependent manner,suggesting that melatonin might be beneficial to pSS.

The effect of light regime and time of slaughter in broiler on broiler performance, liver antioxidant status, and expression of genes related to peptide absorption in the jejunum and melatonin synthesis in the brain.

This study aimed to assess the effects of light regime and time of slaughter on primal cut and organ weights, peptide transporter 1 (PEPT1) gene expression in the jejunum, arylalkylamine N-acetyltransferase (AANAT) gene expression in the brain, and liver oxidant/antioxidant status in broilers aged 37 days. The experiment was conducted in a factorial completely randomized design, with two light regimes (intermittent light varying according to bird age and continuous light under an 18 h light/6 h dark photoperiod) and four times of slaughter (2:00, 8:00, 14:00 and 20:00 h). There was an interaction effect on PEPT1 and AANAT expression, lipid and protein oxidation and superoxide dismutase (SOD) activity. In both light regimes, PEPT1 expression responded cubically to slaughter time. In the continuous light group, PEPT1 expression was highest in birds slaughtered at 2:00 and 14:00 h, whereas, in the intermittent light treatment, expression was highest at 8:00 h. In the continuous light regime, AANAT expression had a cubic relationship with time of slaughter, with the greatest values recorded at 20:00 h. In the intermittent light regime, slaughter time showed a cubic effect on lipid oxidation, which was highest at 8:00 h. In the continuous light group, there was a cubic effect on nitrite concentration, lipid oxidation, protein oxidation, and SOD activity; nitrite levels, lipid oxidation, and protein oxidation were highest and SOD activity was lowest in birds slaughtered at 14:00 h. Time of slaughter influenced catalase activity, which responded cubically; catalase activity was lowest at 8:00 and 14:00 h. This study is the first to demonstrate that PEPT1 expression in the jejunum of broilers follows a diurnal rhythm and varies according to light regime. The results also suggest that mainly continuous lighting and slaughter at 14:00 h when the animals are possibly more active may be more stressful to broilers.

Expression of arylalkylamine n-acetyltransferase (AANAT) and acetylserotonin o-methyltransferase (ASMT) in the corpus luteum of pregnant sows and synthesis of melatonin in luteal cells.

In vertebrates, melatonin is mainly synthesized from serotonin in the pineal gland. Many reports have documented that melatonin is also synthesized in the extra-pineal tissues, but the synthesis of melatonin in the corpus luteum (CL) of pregnant sows has never been studied. The objectives of this study were to evaluate the expression of melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT), in the CL of sows during pregnancy and to investigate the synthesis of melatonin in luteal cells. Results showed that AANAT and ASMT were both expressed in the CL of sows during pregnancy, higher levels were observed in the early- and mid-stage CL, and the lowest abundance was found in the regressing CL (later-stage). The immunostaining for AANAT and ASMT was predominantly localized in the large luteal cells of porcine CL during pregnancy. Furthermore, melatonin was synthesized in luteal cells from serotonin in a dose- and time-dependent manner. And the expressions of AANAT and ASMT were upregulated by serotonin in luteal cells. In addition, progesterone (P4) secretion and cell viability were promoted in luteal cells treated with serotonin, and the stimulatory effects were blocked by luzindole (a non-selective MT1 and MT2 antagonist). Finally, the expressions of MT1 and MT2 were augmented by serotonin in luteal cells. In conclusion, this study demonstrates for the first time the developmental expression of AANAT and ASMT in the CL and a local synthesis of melatonin in luteal cells of pregnant sows, and suggests a paracrine and/or autocrine role for melatonin in luteal function.

Pinealectomy or light exposure exacerbates biliary damage and liver fibrosis in cholestatic rats through decreased melatonin synthesis

Melatonin, a neuroendocrine hormone synthesized by the pineal gland and cholangiocytes, decreases biliary hyperplasia and liver fibrosis during cholestasis-induced biliary injury via melatonin-dependent autocrine signaling through increased biliary arylalkylamine N-acetyltransferase (AANAT) expression and melatonin secretion, downregulation of miR-200b and specific circadian clock genes. Melatonin synthesis is decreased by pinealectomy (PINX) or chronic exposure to light. We evaluated the effect of PINX or prolonged light exposure on melatonin-dependent modulation of biliary damage/ductular reaction/liver fibrosis. Studies were performed in male rats with/without BDL for 1 week with 12:12 h dark/light cycles, continuous light or after 1 week of PINX. The expression of AANAT and melatonin levels in serum and cholangiocyte supernatant were increased in BDL rats, while decreased in BDL rats following PINX or continuous light exposure. BDL-induced increase in serum chemistry, ductular reaction, liver fibrosis, inflammation, angiogenesis and ROS generation were significantly enhanced by PINX or light exposure. Concomitant with enhanced liver fibrosis, we observed increased biliary senescence and enhanced clock genes and miR-200b expression in total liver and cholangiocytes. In vitro, the expression of AANAT, clock genes and miR-200b was increased in PSC human cholangiocyte cell lines (hPSCL). The proliferation and activation of HHStecs (human hepatic stellate cell lines) were increased after stimulating with BDL cholangiocyte supernatant and further enhanced when stimulated with BDL rats following PINX or continuous light exposure cholangiocyte supernatant via intracellular ROS generation. Conclusion: Melatonin plays an important role in the protection of liver against cholestasis-induced damage and ductular reaction.

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.

Spontaneously Diabetic Torii (SDT) Fatty Rat, a Novel Animal Model of Type 2 Diabetes Mellitus, Shows Blunted Circadian Rhythms and Melatonin Secretion

In patients with diabetes mellitus (DM), impairments of circadian rhythms, including the sleep–wake cycle, blood pressure, and plasma melatonin concentrations, are frequently observed. Animal models of DM are also reported to show aberrant circadian rhythms. However, the changes in the circadian rhythms of plasma soluble substances, including melatonin, in diabetic animals are controversial. In the present study, we investigated the circadian rhythms of spontaneous locomotor activity, metabolic parameters (plasma glucose, triglyceride, and total cholesterol), and plasma melatonin concentrations in Spontaneously Diabetic Torii (SDT) fatty rats, a novel animal model of type 2 DM. Although SDT fatty rats exhibited low locomotor activity in the dark phase, no phase shifts were observed. The circadian variations of plasma metabolic parameters were more apparent in the SDT fatty rats compared with control Sprague–Dawley (SD) rats. The circadian rhythms of plasma melatonin concentrations were significantly impaired in SDT fatty rats. To get an insight into the mechanism underlying the impaired melatonin secretion in SDT fatty rats, the expression of arylalkylamine N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt) mRNA, which encode the rate-limiting enzymes for melatonin synthesis, was investigated in the pineal gland. There were no significant differences in Aanat and Asmt expression between the control SD and SDT fatty rats. These results suggest that SDT fatty rats show impaired circadian rhythms and dysregulated melatonin secretion.

AA-NAT, MT1 and MT2 Correlates with Cancer Stem-Like Cell Markers in Colorectal Cancer: Study of the Influence of Stage and p53 Status of Tumors.

The characterization of colon cancer stem cells (CSCs) may help to develop novel diagnostic and therapeutic procedures. p53 loss increases the pool of CSCs in colorectal cancer (CRC). Recent reports suggest that the oncostatic effects of melatonin could be related to its ability to kill CSCs. Although there are no data linking the loss of p53 function and melatonin synthesis or signaling in cancer, melatonin does activate the p53 tumor-suppressor pathway in this disease. In this work, we analyze whether the expression of melatonin synthesis and signaling genes are related to the expression of CSC markers and the implication of p53 status in samples from patients with CRC. Arylalkylamine N-acetyltransferase (AA-NAT), MT1, and MT2 expression decreased in tumor samples versus normal mucosa samples in mutated p53 (mtp53) tumors versus those with wild-type p53 (wtp53). Further, AA-NAT and MT2 expression were lower in advanced stages of the disease in wtp53 tumors. On the contrary, CD44 and CD66c expression was higher in tumor versus normal mucosa in wtp53 tumors. Additionally, CD44 expression was higher in advanced stages of the disease regardless of the p53 status. Patients with CD44highCD66chigh and wtp53 tumors in advanced stages showed low expression of AA-NAT and MT2 in wtp53 tumors. These results could indicate a possible interaction of these pathways in CRC.

Regulating the balance between the kynurenine and serotonin pathways of tryptophan metabolism.

Tryptophan is metabolized along the kynurenine and serotonin pathways, resulting in formation of kynurenine metabolites, neuroactive serotonin and melatonin. Each pathway is critical for maintaining healthy homeostasis. However, the two pathways are extremely unequal in their ability to degrade tryptophan, and little is known about the mechanisms maintaining the balance between them. Here, we demonstrated that in PC12 cells, a change of expression of key genes of one pathway resulted in a change of expression of key genes of the other. Melatonin, the end product of the serotonin pathway, played an important role in tryptophan metabolism by affecting both key enzymes of the two pathways. Melatonin treatment induced the expression of indole-2,3-dioxygenase 1 (IDO1) and enhanced the activity of the IDO1 promoter while decreasing the expression of arylalkylamine N-acetyl transferase. Melatonin treatment up-regulated the expression of forkhead box protein O1 (FoxO1) and enhanced the binding of FoxO1 to the IDO1 promoter. FoxO1 was shown to be a new regulator for IDO1 expression. Melatonin treatment decreased the phosphorylation of FoxO1 by extracellular signal-regulated kinases 1 and 2 and protein kinase B (Akt) and increased the phosphorylation of binding protein 14-3-3 by c-Jun N-terminal kinase (JNK), and thus the complex of FoxO1-14-3-3 in the cytoplasm was disassembled and FoxO1 was relocated to the nucleus to induce IDO1 expression. The JNK signaling pathway played an important role in melatonin-induced IDO1 up-regulation. In conclusion, this study suggests a link between melatonin, JNK, FoxO1 and IDO1 that acts as a potential balance regulator of tryptophan metabolism, and offers a new approach to treat diseases related to dysregulation of tryptophan metabolism.

Eight hours of nocturnal 915 MHz radiofrequency identification (RFID) exposure reduces urinary levels of melatonin and its metabolite via pineal arylalkylamine N-acetyltransferase activity in male rats

Purpose: We investigated the effects of whole-body exposure to the 915 MHz radiofrequency identification (RFID) on mela-tonin biosynthesis and the activity of rat pineal arylalkylamine N-acetyltransferase (AANAT).Materials and methods: Rats were exposed to RFID (whole-body specific absorption rate, 4 W/kg) for 8 h/day, 5 days/week, for weeks during the nighttime. Total volume of urine excreted during a 24-h period was collected after RFID exposure. Uri-nary melatonin and 6-hydroxymelatonin sulfate (6-OHMS) was measured by gas chromatography-mass spectrometry (GC-MS) and enzyme-linked immunosorbent assay (ELISA), respectively. AANAT enzyme activity was measured using liquid biphasic dif-13 fusion assay. Protein levels and mRNA expression of AANAT was 14 measured by Western blot and reverse transcription polymerase 15 chain reaction (RT-PCR) analysis, respectively.Results: Eight hours of nocturnal RFID exposure caused a sig-nificant reduction in both urinary melatonin (p = 0. 003) and 6-OHMS (p = 0. 026). Activity, protein levels, and mRNA expres-sion of AANAT were suppressed by exposure to RFID (p < 0. 05).Conclusions: Our results suggest that nocturnal RFID exposure can cause reductions in the levels of both urinary melatonin and 6-OHMS, possibly due to decreased melatonin biosynthesis via suppression of Aanat gene transcription in the rat pineal gland.

The adrenergic-regulated CRTC1 and CRTC2 phosphorylation and cellular distribution is independent of endogenous SIK1 in the male rat pinealocyte

Salt inducible kinase 1 (SIK1) has been reported to repress cAMP-response element binding protein (CREB)-mediated gene transcription by causing the nuclear export of CREB-regulated transcription coactivators (CRTCs) through phosphorylation. Although the repressor role of SIK1 in suppressing the expression of arylalkylamine N-acetyltransferase, the enzyme that controls the daily rhythm in melatonin production in the rat pineal gland, has been established, whether SIK1 regulates the phosphorylation and localization of CRTC1 and CRTC2 in this tissue remains unclear. The present study found that overexpressing SIK1 in NE-stimulated rat pinealocytes could increase the phosphorylation of CRTC1 and CRTC2, reduced selectively the nuclear level of CRTC2 (but not that of CRTC1), and elevated the cytosolic levels of both CRTC1 and CRTC2. In contrast, transient knockdown of endogenous SIK1 had no effect on the phosphorylation or distribution of CRTC1 and CRTC2 in norepinephrine (NE)-stimulated pinealocytes. Our results also showed that adrenergic blockade during NE stimulation led to a rapid rephosphorylation and decline in the nucleus levels of CRTC1 and CRTC2; however SIK1 knockdown had no effect on this rapid rephosphorylation. Moreover, studies with kinase inhibitors revealed that kinase(s) sensitive to KT5823 appeared to be involved in this rapid rephosphorylation. Together, these results indicate that although overexpressing SIK1 can phosphorylate CRTC1 and CRTC2 in the NE-stimulated pinealocyte, the endogenous SIK1, in spite of its induction by NE, does not appear to be the main regulator of the phosphorylation and intracellular localization of these two coactivators.

Melatonin reduces endothelin‐1 expression and secretion in colon cancer cells through the inactivation of FoxO‐1 and NF‐κβ

Melatonin is an indoleamine that is synthesised from tryptophan under the control of the enzymes arylalkylamine N-acetyltransferase (AA-NAT) and acetylserotonin methyltransferase (ASMT). Melatonin inhibits colon cancer growth in both in vivo and in vitro models; however, a precise mechanism responsible for inhibiting tumour growth has not been clearly described. Endothelin-1 (ET-1) is a peptide that acts as a survival factor in colon cancer, inducing cell proliferation, protecting carcinoma cells from apoptosis and promoting angiogenesis. The data presented show that melatonin inhibits edn-1 mRNA expression (the first step in ET-1 synthesis), ECE-1 protein expression and the release of ET-1 from colorectal cancer cells in vitro. ET-1 levels in cultured media present a similar inhibition pattern to that of edn-1 mRNA expression despite the inhibition of ECE-1 protein after melatonin treatment, which suggests that an endopeptidase other than ECE-1 could be mainly responsible for ET-1 synthesis. The inhibition of edn-1 expression is due to an inactivation of FoxO1 and NF-κβ transcription factors. FoxO1 inactivation is associated with an increased Src phosphorylation, due to elevated cAMP content and PKA activity, whereas NF-κβ inactivation is associated with the blockade of Akt and ERK phosphorylation due to the inhibition of PKC activity after melatonin treatment. Melatonin also inhibits edn-1 promoter activity regulated by FoxO1 and NF-κβ. Finally, a significant correlation was observed between AA-NAT and edn-1 expression downregulation in human colorectal cancer tissues. In conclusion, melatonin may be useful in treating colon carcinoma in which the activation of ET-1 plays a role in tumour growth and progression.

Effect of Monochromatic Light on Melatonin Secretion and Arylalkylamine N‐Acetyltransferase mRNA Expression in the Retina and Pineal Gland of Broilers

The goal of this study is to investigate the effects of various monochromatic lights on plasma melatonin (MT) levels and the expression of arylalkylamine N-acetyltransferase (AANAT) mRNA in the pineal gland and retina. A total of 160 newly hatched (posthatching day 1, P1) broilers, including intact, sham-operated, and pinealectomized groups were exposed to blue light (BL), green light (GL), red light (RL), and white light (WL) by light emitting diode (LED) system for short term (24 hr) or long term (2 weeks), separately. For intact and sham-operated birds, the plasma MT level exhibited marked circadian rhythms at P7 and P14 regardless of short-term and long-term exposure to four monochromatic lights. However, WL and BL showed a faint suppression of MT secretion in contrast to GL and RL at either light or dark time points, with the following rank order: GL < RL < WL < BL. Larger circadian amplitude of MT levels was observed in GL group versus BL group (at P14: 87.70 pg/mL vs. 19.85 pg/mL, respectively). Pinealectomy disturbed the MT rhythm under different light colors, especially in RL. Additionally, consistent with the alteration of plasma MT levels, we observed increased AANAT mRNA expression and immunoreactive cell numbers of proliferating cell nuclear antigen (PCNA) and c-Fos in the pineal gland or retina in GL than that of BL, whereas 5-HT immunoreactive cell number was significantly decreased in GL. These data suggested that GL enhanced chick pinealocytes and retinal cells to express AANAT mRNA and to secrete MT, which may be depended on promoting c-Fos expression and cell proliferation.

Modulation of the Biliary Expression of Arylalkylamine N-Acetyltransferase (AANAT, the Key Enzyme Regulating Melatonin Synthesis) Alters the Proliferative/Apoptotic Responses of Cholangiocytes to Liver Injury In Vivo and In Vitro

Modulation of the Biliary Expression of Arylalkylamine N-Acetyltransferase (AANAT, the Key Enzyme Regulating Melatonin Synthesis) Alters the Proliferative/Apoptotic Responses of Cholangiocytes to Liver Injury In Vivo and In Vitro

Expression and cellular localizaion of melatonin-synthesizing enzymes in rat and human salivary glands

Melatonin, discovered in 1958, is secreted by the pineal gland primarily during the night. Its secretion is controlled by the light/dark cycle of the environment. Melatonin is also produced in and secreted by various extrapineal organs, tissues and cells and its synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT) is expressed in various extrapineal organs, tissues and cells. Recently, it was reported that melatonin is present in saliva, but it is not certain where melatonin was synthesized and whether it was secreted into saliva and what function it may have in saliva. The present study was performed to investigate where melatonin was synthesized and whether it was secreted by salivary glands into saliva. We performed immunohistochemical analysis of the expression of AANAT in rat parotid, submandibular and sublingual glands and the expression of both AANAT and hydroxyindole-O-methyltransferase (HIOMT) in human submandibular glands. We evaluated the expression of AANAT and HIOMT mRNA in rat submandibular glands by quantitative reverse transcription-polymerase chain reaction. As a result, we observed expression of AANAT in epithelial cells of striated ducts in rat salivary glands and expression of AANAT, HIOMT and melatonin in epithelial cells of striated ducts in human submandibular glands. In addition, we also confirmed the expression of the most potent melatonin receptor, melatonin 1a receptor, in rat buccal mucosa. Our findings suggest that melatonin might be produced and secreted by salivary glands directly into saliva and that it might play some physiological role in the oral cavity.

Cloning and expression of arylalkylamine N-acetyltranferase-2 during early development and metamorphosis in the sole Solea senegalensis

The arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in the rhythmic production of melatonin. Two Aanats are expressed in teleost fish, one retinal specific, Aanat1, and the other one pineal specific, Aanat2, being the latter the main enzyme responsible of the plasma nocturnal melatonin increase in fish. In anurans melatonin has been involved in metamorphosis through antagonizing thyroid hormone function; however, no available data reports a relationship between melatonin system and metamorphosis in fish. In this study, we have cloned the AANAT2 ( SsAanat2) in a flatfish, Solea senegalensis, and studied its sites of expression and developmental expression pattern by in situ hybridization and Real Time PCR. These studies allowed us to demonstrate a specific signal in the pineal gland of sole larvae from 2 days post-fertilization (dpf), which was evident until post-metamorphosis. Immunohistochemical analysis on the hybridized slides showed that the sole pineal Aanat2 expressing cells corresponded to pineal photoreceptor cells. Real Time PCR was performed in animals kept under natural photoperiod and sampled at different stages from 0 to 21 dpf (including pre-, early-, middle- and late-metamorphic stages) and at midlight (ML) and middark (MD) daytimes. Sole Aanat2 expression was higher at MD than at ML from 2 dpf and at most developmental stages analyzed. The highest AANAT2 mRNA abundance was observed at 2 and 4 dpf. A significant 60-fold reduction in Aanat2 expression was seen just before metamorphosis demonstrating, for the first time in a vertebrate species, that the expression of pineal AANAT and thyroid hormones levels exhibit an inverse pattern during metamorphosis.

Daily Profile in Melanopsin Transcripts Depends on Seasonal Lighting Conditions in the Rat Retina

The retinal photopigment melanopsin (Opn4) mediates photoentrainment of the circadian system. In the present study, seasonal regulation of the melanopsin gene was investigated in comparison with the arylalkylamine N-acetyltransferase (AA-NAT) gene as an indicator of retinal pacemaker output. For this purpose, the daily profiles in the amount of melanopsin mRNA and AA-NAT mRNA were monitored under 8 : 16 h light/dark, 12 : 12 h light/dark and 16 : 8 h light/dark photoperiods using real-time polymerase chain reaction analysis. We found that, under all of the lighting regimes, melanopsin and AA-NAT expression oscillated with a peak around dark onset and the middle of the dark phase, respectively. The lighting regime influenced both genes, but in an opposing manner. Under long photoperiods, the duration of peak expression was prolonged for melanopsin, whereas it was shortened for AA-NAT. Under constant darkness, the rhythm of mRNA was abolished for melanopsin, but persisted for AA-NAT whereas, under constant light, the rhythm of mRNA was abolished for both genes. Our findings suggest that, in contrast to the AA-NAT gene, the daily and photoperiod-dependent regulation of the melanopsin gene does not rely on a circadian oscillator but is directly illumination-dependent.