The expression of MT1 and MT2 melatonin receptor mRNA in several rat tissues

The mechanisms that mediate the various effects of melatonin in mammalian tissues are not always known. Therefore, the aim of this study was to investigate whether MT(1) and MT(2) melatonin receptors are expressed in certain tissues of the rat. The expression of MT(1) and MT(2) melatonin receptor mRNA was determined using a real-time quantitative RT-PCR method. In addition, we examined whether mRNA for either subtype of receptor shows any difference in the expression between midnight and noon, similar to the changes in melatonin concentrations in plasma and tissue samples. MT(1) and MT(2) melatonin receptor mRNAs were found in the rat hypothalamus, retina and small intestine. We also showed a low expression of MT(2) mRNA in the rat liver and heart SA node. In the heart apex and the Harderian gland, no appearance of either of the receptor mRNAs was detectable. A significant difference in the expression of MT(1) mRNA between day and night was found in the hypothalamus. In conclusion, our findings suggest that at least some effects of melatonin are mediated through membrane MT(1) and MT(2) receptors in the hypothalamus, the retina and the small intestine. Down-regulation of receptors might be one reason for the difference in the hypothalamic MT(1) melatonin receptor mRNA expression between midnight and noon. In the liver and the heart SA node, the physiological significance of possible MT(2) receptors remains unclear. According to our negative midnight and noon results in the Harderian gland and heart apex melatonin may exert its effect on these tissues by a non-receptor mechanism.     

Refeeding after fasting elicits insulin-dependent regulation of Per2 and Rev-erbα with shifts in the liver clock

The mammalian circadian clock is known to be entrained by both a daily light-dark cycle and daily feeding cycle. However, the mechanisms of feeding-induced entrainment are not as fully understood as those of light entrainment. To elucidate the first step of entrainment of the liver clock, we identified the circadian clock gene(s) that show both phase advance and acute change of gene expression during the early term of the daytime refeeding schedule in mice. The expressions of liver Per2 and Rev-erbα genes were phase-advanced within 1 day of refeeding. Additionally, the upregulation of Per2 mRNA and down-regulation of Rev-erbα mRNA were induced within 2 hours, not only by food intake but also by insulin injection in intact mice. These expression changes by food intake were not revealed in streptozotocin-treated insulin-deficient mice, but insulin injection was able to recover the impairment of Per2 and Rev-erbα gene expression. Furthermore, we demonstrated using an ex vivo luciferase monitoring system that insulin injection during the daytime causes a phase advance of liver Per2 expression rhythm in Per2::luciferase knock-in mice. In embryonic fibroblasts from Per2::luciferase knock-in mice, insulin infusion caused an acute increase of Per2 gene expression and a similar phase advance of Per2 expression rhythm. Our results indicate that an acute change of Per2 and Rev-erbα gene expression mediated by refeeding-induced insulin secretion is a critical step mediating the early phase of feeding-induced entrainment of the liver clock.     

Diurnal and circadian regulations by three melatonin receptors in the brain and retina of olive flounder Paralichthys olivaceus: profiles following exogenous melatonin

To establish the molecular basis of circadian rhythm control by melatonin receptors (MTs), we investigated the mitochondrial ribonucleic acid (mRNA) expressions of three types of MTs in different tissues of the olive flounder (Paralichthys olivaceus). All three types of MT mRNAs were expressed in the neural tissues, while MT1 mRNA was expressed in the peripheral tissues and MT2 and MT3 mRNAs were weakly expressed or undetected in these tissues. We observed increased MT mRNA expression in the neural tissues at night under both light–dark (LD) and constant dark (DD) conditions. Although the melatonin-treated cultured pineal gland samples showed similar diurnal variations with high-MT mRNA expression levels at night compared to those of untreated cultured pineal gland samples, the expression levels were considerably higher in the melatonin-treated samples. The plasma melatonin level also significantly increased at night. Under DD conditions, the expression patterns of MT mRNAs were similar to those under the LD photocycle, but the peak was lower and the circadian change patterns were less clear. These findings reinforce the hypothesis that MTs are active in processing light information, and that these genes are regulated by the circadian clock and light, thus suggesting that MTs play an important role in daily and circadian variations in the brain and retina of olive flounders.     

Daily patterns of clock and cognition-related factors are modified in the hippocampus of vitamin A-deficient rats

The circadian expression of clock and clock-controlled cognition-related genes in the hippocampus would be essential to achieve an optimal daily cognitive performance. There is some evidence that retinoid nuclear receptors (RARs and RXRs) can regulate circadian gene expression in different tissues. In this study, Holtzman male rats from control and vitamin A-deficient groups were sacrificed throughout a 24-h period and hippocampus samples were isolated every 4 or 5 h. RARα and RXRβ expression level was quantified and daily expression patterns of clock BMAL1, PER1, RORα, and REVERB genes, RORα and REVERB proteins, as well as temporal expression of cognition-related RC3 and BDNF genes were determined in the hippocampus of the two groups of rats. Our results show significant daily variations of BMAL1, PER1, RORα, and REVERB genes, RORα and REVERB proteins and, consequently, daily oscillating expression of RC3 and BDNF genes in the rat hippocampus. Vitamin A deficiency reduced RXRβ mRNA level as well as the amplitude of PER1, REVERB gene, and REVERB protein rhythms, and phase-shifted the daily peaks of BMAL1 and RORα mRNA, RORα protein, and RC3 and BDNF mRNA levels. Thus, nutritional factors, such as vitamin A and its derivatives the retinoids, might modulate daily patterns of BDNF and RC3 expression in the hippocampus, and they could be essential to maintain an optimal daily performance at molecular level in this learning-and-memory-related brain area.     

The intrinsic circadian clock within the cardiomyocyte

Circadian clocks are intracellular molecular mechanisms that allow the cell to anticipate the time of day. We have previously reported that the intact rat heart expresses the major components of the circadian clock, of which its rhythmic expression in vivo is consistent with the operation of a fully functional clock mechanism. The present study exposes oscillations of circadian clock genes [brain and arylhydrocarbon receptor nuclear translocator-like protein 1 (bmal1), reverse strand of the c-erbaalpha gene (rev-erbaalpha), period 2 (per2), albumin D-element binding protein (dbp)] for isolated adult rat cardiomyocytes in culture. Acute (2 h) and/or chronic (continuous) treatment of cardiomyocytes with FCS (50% and 2.5%, respectively) results in rhythmic expression of circadian clock genes with periodicities of 20-24 h. In contrast, cardiomyocytes cultured in the absence of serum exhibit dramatically dampened oscillations in bmal1 and dbp only. Zeitgebers (timekeepers) are factors that influence the timing of the circadian clock. Glucose, which has been previously shown to reactivate circadian clock gene oscillations in fibroblasts, has no effect on the expression of circadian clock genes in adult rat cardiomyocytes, either in the absence or presence of serum. Exposure of adult rat cardiomyocytes to the sympathetic neurotransmitter norephinephrine (10 microM) for 2 h reinitiates rhythmic expression of circadian clock genes in a serum-independent manner. Oscillations in circadian clock genes were associated with 24-h oscillations in the metabolic genes pyruvate dehydrogenase kinase 4 (pdk4) and uncoupling protein 3 (ucp3). In conclusion, these data suggest that the circadian clock operates within the myocytes of the heart and that this molecular mechanism persists under standard cell culture conditions (i.e., 2.5% serum). Furthermore, our data suggest that norepinephrine, unlike glucose, influences the timing of the circadian clock within the heart and that the circadian clock may be a novel mechanism regulating myocardial metabolism.     

The circadian nuclear receptor RORα negatively regulates cerebral ischemia–reperfusion injury and mediates the neuroprotective effects of melatonin

Disruptions of the circadian rhythm and reduced circulating levels of the circadian hormone melatonin predispose to ischemic stroke. Although the nuclear receptor RORα is considered as a circadian rhythm regulator and a mediator of certain melatonin effects, its potential role in cerebral ischemia-reperfusion (CI/R) injury and in the neuroprotective effects of melatonin remain undefined. Here, we observed that CI/R injury in RORα-deficient mice was associated with greater cerebral infarct size, brain edema, and cerebral apoptosis compared with wild-type model. In contrast, transgenic mice with brain-specific overexpression of RORα versus non-transgenic controls exerted significantly reduced infarct volume, brain edema and apoptotic response induced by CI/R. Mechanistically, RORα deficiency was found to exacerbate apoptosis pathways mediated by endoplasmic-reticulum stress and mitochondria and aggravate oxidative/nitrative stress after CI/R. Further studies revealed that RORα deficiency intensified the activation of nuclear factor-κB signaling induced by CI/R. Given the emerging evidence of RORα as an essential melatonin activity mediator, we further investigated the RORα roles in melatonin-exerted neuroprotection against acute ischemic stroke. Melatonin treatment significantly decreased infarct volume and cerebral apoptosis; mitigated endoplasmic reticulum stress and mitochondrial dysfunction; and inhibited CI/R injury-induced oxidative/nitrative stress and nuclear factor-κB activation, which was eradicated in RORα-deficient mice. Collectively, current findings suggest that RORα is a novel endogenous neuroprotective receptor, and a pivotal mediator of melatonin's suppressive effects against CI/R injury.     

Sites and mechanisms of action of melatonin in mammals: the MT1 and MT2 receptors

The rhythmic secretion of melatonin by the pineal gland plays a key role in the synchronisation of circadian and seasonal functions with cyclic environmental variations. The biological effects of this neurohormone are relayed mainly by G-protein-coupled seven-transmembrane receptors. These receptors, known as MT1 and MT2, are present in a large number of central and peripheral structures in mammals, with considerable inter-species variations. However, only the suprachiasmatic nuclei of the hypothalamus, the site of the master circadian biological clock, and the pars tuberalis of the adenohypophysis contain melatonin receptors in the majority of species. Inhibition of the production of AMPc by a Gi/Go protein is one of the principal signalling pathways of the MT1 and MT2 receptors, although many other signal transduction pathways are also brought into play according to the cell type studied (PKC, Ca2+, K+ channels or GMPc in the case of MT2, etc.). Numerous factors or physiological stimuli are capable of influencing the number and functional status of the MT1 and MT2 receptors, such as melatonin, the photoperiod, the circadian clock or the phenomena of receptor dimerisation. Melatonin has numerous physiological effects for which the mechanisms of action and the specific role of the MT1 and MT2 receptors have not yet been clearly elucidated. However, selective pharmacological tools for each of the two receptor subtypes are currently being identified, notably in the Servier Group, for the purpose of furthering our knowledge of the functionality and physiological role of the MT1 and MT2 receptors in the central and peripheral structures.     

Activation of MT(2) melatonin receptors in rat suprachiasmatic nucleus phase advances the circadian clock

The aim of this study was to identify the melatonin receptor type(s) (MT(1) or MT(2)) mediating circadian clock resetting by melatonin in the mammalian suprachiasmatic nucleus (SCN). Quantitative receptor autoradiography with 2-[(125)I]iodomelatonin and in situ hybridization histochemistry, with either (33)P- or digoxigenin-labeled antisense MT(1) and MT(2) melatonin receptor mRNA oligonucleotide probes, revealed specific expression of both melatonin receptor types in the SCN of inbred Long-Evans rats. The melatonin receptor type mediating phase advances of the circadian rhythm of neuronal firing rate in the SCN slice was assessed using competitive melatonin receptor antagonists, the MT(1)/MT(2) nonselective luzindole and the MT(2)-selective 4-phenyl-2-propionamidotetraline (4P-PDOT). Luzindole and 4P-PDOT (1 nM-1 microM) did not affect circadian phase on their own; however, they blocked both the phase advances (approximately 4 h) in the neuronal firing rate induced by melatonin (3 pM) at temporally distinct times of day [i.e., subjective dusk, circadian time (CT) 10; and dawn, CT 23], as well as the associated increases in protein kinase C activity. We conclude that melatonin mediates phase advances of the SCN circadian clock at both dusk and dawn via activation of MT(2) melatonin receptor signaling.     

The circadian clock within the cardiomyocyte is essential for responsiveness of the heart to fatty acids

Cells/organs must respond both rapidly and appropriately to increased fatty acid availability; failure to do so is associated with the development of skeletal muscle and hepatic insulin resistance, pancreatic beta-cell dysfunction, and myocardial contractile dysfunction. Here we tested the hypothesis that the intrinsic circadian clock within the cardiomyocytes of the heart allows rapid and appropriate adaptation of this organ to fatty acids by investigating the following: 1) whether circadian rhythms in fatty acid responsiveness persist in isolated adult rat cardiomyocytes, and 2) whether manipulation of the circadian clock within the heart, either through light/dark (L/D) cycle or genetic disruptions, impairs responsiveness of the heart to fasting in vivo. We report that both the intramyocellular circadian clock and diurnal variations in fatty acid responsiveness observed in the intact rat heart in vivo persist in adult rat cardiomyocytes. Reversal of the 12-h/12-h L/D cycle was associated with a re-entrainment of the circadian clock within the rat heart, which required 5-8 days for completion. Fasting rats resulted in the induction of fatty acid-responsive genes, an effect that was dramatically attenuated 2 days after L/D cycle reversal. Similarly, a targeted disruption of the circadian clock within the heart, through overexpression of a dominant negative CLOCK mutant, severely attenuated induction of myocardial fatty acid-responsive genes during fasting. These studies expose a causal relationship between the circadian clock within the cardiomyocyte with responsiveness of the heart to fatty acids and myocardial triglyceride metabolism.     

A novel cardiac hypertrophic factor, neurotrophin-3, is paradoxically downregulated in cardiac hypertrophy

The neurotrophin family plays pivotal roles in the development of the nervous system. Recently, the role of the neurotrophin in non-neural tissue has been extensively investigated. Among them, neurotrophin-3 and its receptor TrkC are critical for embryonic heart development, though little is known about neurotrophin-3/TrkC function in adult heart. Moreover, the expressions of other neurotrophin and Trk families in the cardiovascular system have not been fully determined. In adult and neonatal rats, only TrkC mRNA was expressed more abundantly in heart than aorta among the neurotrophin receptors, while all neurotrophins were equally expressed in the cardiovascular system. Immunohistochemistry confirmed the protein expressions of neurotrophin-3/TrkC in rat ventricles. In primary-cultured rat cardiomyocytes, neurotrophin-3 strongly activated p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and Jun N-terminal kinase pathways in Western blot analysis. In Northern blot analysis, neurotrophin-3 strongly increased mRNA expressions of cardiac hypertrophic markers (skeletal alpha-actin and atrial natriuretic peptide) in cardiomocytes. [(3)H]-phenylalanine uptake into cardiomyocytes, myofilament reorganization, and cardiomyocyte size were also augmented with neurotrophin-3 stimulation, indicating that neurotrophin-3 is a novel cardiac hypertrophic factor. Unexpectedly, neurotrophin-3 was downregulated in cardiac hypertrophy induced by pressure overload (in vivo), and in cardiomyocyte hypertrophy evoked by endothelin-1 stimulation (in vitro). Interestingly, the cell size and BNP mRNA expression level (markers of hypertrophy) were greater in cardiomyocytes treated with both neurotrophin-3 and endothelin-1 than in those stimulated with endothelin-1 alone. These findings demonstrate that neurotrophin-3 is a unique hypertrophic factor, which is paradoxically downregulated in cardiac hypertrophy and might counteract hypertrophic change.     

Melatonin in the circadian system

In Mammals, the master circadian clock is located in the suprachiasmatic nuclei of the hypothalamus. This clock is synchronized with the astronomical time, essentially by the light/dark cycle. The different zeitgebers studied act on the Per1 and/or Per2 genes from the main molecular loop which initiates the circadian oscillations. Once synchronized with the environment, circadian oscillations are distributed through the organism by efferent signals, and the complex interaction of neural, hormonal and behavioural outputs from the circadian clock drive circadian expression of events, either directly or through coordination of the timing of peripheral oscillators. Melatonin, one of the endocrine output signals of the clock, provides the organism with circadian information, and can be considered as an endogenous synchronizer. Melatonin receptors are present in the suprachiasmatic nuclei which allows the hormone to feed back on the clock. To day, the physiological role of this peculiar feed-back has not yet been established. However, the presence of these receptors indicates that through an action on the circadian clock, exogenous melatonin can affect all levels of the circadian network and its capacity to entrain circadian rhythms to 24 h has been demonstrated. Melatonin is thus a zeitgeber. However, surprisingly, and different from the action mechanism of other zeitgebers on the clock, the chronobiotic effect of melatonin does not implicate Per1 and/or Per2. Rather, Rev-erb alpha could be the link between the physiological action of melatonin and the core of the molecular circadian clock.     

Effect of LED light spectra on exogenous prolactin-regulated circadian rhythm in goldfish,Carassius auratus

We investigated the effect of light spectra on circadian rhythm by exogenous prolactin (PRL) using light-emitting diodes (LEDs): red, green and purple. We injected PRL into live fish or treated cultured brain cells with PRL. We measured changes in the expressions of period 2 (Per2), cryptochrome 1 (Cry1), melatonin receptor 1 (MT1) mRNAs, and MT1 proteins, and in the plasma PRL, serotonin and melatonin levels. After PRL injection and exposure to green light, MT1 expression and plasma melatonin levels were significantly lower, but the expressions of Per2 and Cry1 were significantly higher than the others. Plasma serotonin after PRL injection and exposure to red light was significantly lower than others. These results indicate that injection of high concentration PRL inhibits melatonin, and inhibited melatonin regulates circadian rhythm via clock genes and serotonin. Thus, exogenous PRL regulates the circadian rhythm and light spectra influence the effect of PRL in goldfish.