Pinealectomy interferes with the circadian clock genes expression in white adipose tissue.

Melatonin, the main hormone produced by the pineal gland, is secreted in a circadian manner (24-hr period), and its oscillation influences several circadian biological rhythms, such as the regulation of clock genes expression (chronobiotic effect) and the modulation of several endocrine functions in peripheral tissues. Assuming that the circadian synchronization of clock genes can play a role in the regulation of energy metabolism and it is influenced by melatonin, our study was designed to assess possible alterations as a consequence of melatonin absence on the circadian expression of clock genes in the epididymal adipose tissue of male Wistar rats and the possible metabolic repercussions to this tissue. Our data show that pinealectomy indeed has impacts on molecular events: it abolishes the daily pattern of the expression of Clock, Per2, and Cry1 clock genes and Pparγ expression, significantly increases the amplitude of daily expression of Rev-erbα, and affects the pattern of and impairs adipokine production, leading to a decrease in leptin levels. However, regarding some metabolic aspects of adipocyte functions, such as its ability to synthesize triacylglycerols from glucose along 24 hr, was not compromised by pinealectomy, although the daily profile of the lipogenic enzymes expression (ATP-citrate lyase, malic enzyme, fatty acid synthase, and glucose-6-phosphate dehydrogenase) was abolished in pinealectomized animals.

[Monoamine oxidase activity in rat pineal gland: comparison with brain areas, alteration during aging].

Using benzylamine as a substrate, the amine oxidase activity was determined in the pineal gland of adult rats and compared with the same activity in brain areas and pituitary. Two groups of rats aged 6-8 and 14-15 months were also compared on the basis of this activity. Benzylamine deaminating activity in the pineal gland was significantly higher than in the area preoptica medialis, the corpus mamillare, the tuberculum olfactorium, and the hypophysis, and lower than in the eminentia mediana. The significant increase of the activity in the pineal gland in animals of age from 6-8 to 14-15-months was revealed. Benzylamine deaminating activity in the pineal gland was totally inhibited by 0,002 mM R deprenyl, indicating the B type monoamine oxidase (MAO B) activity. Age-associated increase of MAO B activity in the pineal gland accompanied by decrease of glutathione peroxidase activity, reported earlier, can promote the oxidative damage in the pineal gland during aging.

Pineal arylalkylamine N-acetyltransferase (Aanat) gene expression as a target of inflammatory mediators in the chicken.

Previously, we demonstrated that experimental peritonitis in chickens was attenuated by treatment with exogenous melatonin, while the developing inflammation decreased pineal AANAT activity. This suggested the existence of a bidirectional relationship between the activated immune system and pineal gland function. The aim of the present study was to identify the step(s) in the chicken pineal melatonin biosynthetic pathway that are affected by inflammation. Peritonitis was evoked by i.p. injection of thioglycollate solution, either 2h after the start, or 2h before the end of the light period, and the animals were sacrificed 4h later. The effect of inflammation on the expression of genes encoding enzymes participating in melatonin biosynthesis in the pineal gland, i.e. tryptophan hydroxylase 1 (Tph1), dopa decarboxylase (Ddc), arylalkylamine N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt), was evaluated by qPCR. The pineal and serum melatonin concentration as well as the content of its precursors in the pineal gland were measured, along with the activity of the relevant biosynthetic enzymes. Developing peritonitis caused an increase in the pineal levels of the Tph1 mRNA during the night and the Asmt mRNA during the day, while nocturnal Aanat transcription was reduced. Both the pineal and serum melatonin level and the pineal content of N-acetylserotonin (NAS) were decreased during the night in birds with peritonitis. The amount and activity of pineal AANAT were significantly reduced, while the activity of HIOMT was increased under these experimental conditions. These results indicate that the observed decrease in MEL biosynthesis in chickens with developing inflammation is a result of transcriptional downregulation of the Aanat gene, followed by reduced synthesis and activity of the encoded enzyme.

Evidence of melatonin secretion in cetaceans: plasma concentration and extrapineal HIOMT-like presence in the bottlenose dolphin Tursiops truncatus.

The pineal gland is generally believed to be absent in cetaceans, although few and subsequently unconfirmed reports described the organ in some species. The recent description of a complete and photographed pineal body in a bottlenose dolphin (Tursiops truncatus) prompted us to examine a series of 29 brains of the same species, but no gland was found. We then decided to investigate if the main product of the gland, melatonin, was nevertheless produced and present in the plasma of this species. We collected plasma and serum samples from a series of captive bottlenose dolphins for a period of 7 months spanning from winter to summer and we determined the indoleamine concentration by radio-immunoassay (RIA). The results demonstrated for the first time a quantitative assessment of melatonin production in the blood of a cetacean. Melatonin levels were comparable to those of terrestrial mammals (5.15-27.74 pg/ml daylight concentration), with indications of both seasonal and daily variation although the presence of a circadian rhythm remains uncertain. Immunohistochemical analyses using as a marker hydroxyindole-O-methyl-transferase (HIOMT, the key enzyme involved in the biosynthesis of the hormone), suggested extrapineal melatonin production by the retina, the Harderian gland and the gut. The enzyme was unequivocally localized in all the three tissues, and, specifically, ganglion cells in the retina showed a very strong HIOMT-immunoreactivity. Our results suggest that further research might reveal unexplored aspects of melatonin production in cetaceans and deserves special attention and further efforts.

The expression of Per1 and Aa-nat genes in the pineal gland of postnatal rats.

BACKGROUND: The circadian rhythm of melatonin synthesis is controlled by the master clock, suprachiasmatic nucleus (SCN). The level of melatonin changes throughout the aging process. The SCN's rhythm is driven by autoregulatory feedback loop composed of a set of clock genes families and their corresponding proteins. The Period (Per1), one of clock gene develops gradually during postnatal ontogenesis in the rat SCN and is also expressed in the pineal gland. OBJECTIVE: It is of interest to study the relationship between the postnatal development of Per1 and Aa-nat, genes that produce the rate-limiting enzyme in melatonin synthesis, in the pineal. MATERIAL AND METHOD: Daily profiles of mRNA expression of Per1 and Aa-nat were analyzed in the pineal gland of pups at postnatal ages 4 (P4), P8, P16 and P32, at puberty age of 6 weeks; and in 8 week-old adult rats by real-time PCR. RESULTS: As early as P4, Per1 and Aa-nat mRNAs were expressed and existed at relatively high levels during the nighttime. They gradually increased until puberty and decreased at 8 weeks of age. Additionally, the nocturnal changes of Per1 and Aa-nat mRNA levels in the rat pineal gland from P4 to adults were strongly correlated at r = 0.97 (p < 0.01). CONCLUSION: The present data indicate that there is a close relationship between the expression pattern of Per1 and that of melatonin synthesis during the development of postnatal rats.

Phosphodiesterase 10A in the rat pineal gland: localization, daily and seasonal regulation of expression and influence on signal transduction.

The cyclic nucleotide phosphodiesterase 10A (PDE10A) is highly expressed in striatal spiny projection neurons and represents a therapeutic target for the treatment of psychotic symptoms. As reported previously [J Biol Chem 2009; 284:7606-7622], in this study PDE10A was seen to be additionally expressed in the pineal gland where the levels of PDE10A transcript display daily changes. As with the transcript, the amount of PDE10A protein was found to be under daily and seasonal regulation. The observed cyclicity in the amount of PDE10A mRNA persists under constant darkness, is blocked by constant light and is modulated by the lighting regime. It therefore appears to be driven by the master clock in the suprachiasmatic nucleus (SCN). Since adrenergic agonists and dibutyryl-cAMP induce PDE10A mRNA, the in vitro clock-dependent control of Pde10a appears to be mediated via a norepinephrine → ß-adrenoceptor → cAMP/protein kinase A signaling pathway. With regard to the physiological role of PDE10A in the pineal gland, the specific PDE10A inhibitor papaverine was seen to enhance the adrenergic stimulation of the second messenger cAMP and cGMP. This indicates that PDE10A downregulates adrenergic cAMP and cGMP signaling by decreasing the half-life of both nucleotides. Consistent with its effect on cAMP, PDE10A inhibition also amplifies adrenergic induction of the cAMP-inducible gene arylalkylamine N-acetyltransferase (Aanat) which codes the rate-limiting enzyme in pineal melatonin formation. The findings of this study suggest that Pde10a expression is under circadian and seasonal regulation and plays a modulatory role in pineal signal transduction and gene expression.

Changes in plasma melatonin levels and pineal organ melatonin synthesis following acclimation of rainbow trout (Oncorhynchus mykiss) to different water salinities.

Melatonin has been suggested to play a role in fish osmoregulation, and in salmonids has been related to the timing of adaptive mechanisms during smolting. It has been described that acclimation to different environmental salinities alters levels of circulating melatonin in a number of fish species, including rainbow trout. However, nothing is known regarding salinity effects on melatonin synthesis in the pineal organ, which is the main source of rhythmically produced and secreted melatonin in blood. In the present study we have evaluated, in rainbow trout, the effects of acclimation to different salinities on day and night plasma melatonin values and pineal organ melatonin synthesis. Groups of freshwater (FW)-adapted rainbow trout were placed in tanks with four different levels of water salinity (FW, 6, 12, 18 p.p.t.; parts per thousand) and maintained for 6 h or 5 days. Melatonin content in plasma and pineal organs, as well as the pineal content of serotonin (5-HT) and its main oxidative metabolite (5-hydroxyindole-3-acetic acid; 5-HIAA) were measured by high performance liquid chromatography. In addition, day-night changes in pineal organ arylalkylamine N-acetyltransferase (AANAT2) activity and aanat2 gene expression were studied. Plasma osmolalities were found to be higher in rainbow trout exposed to all salinity levels compared with the control FW groups. A salinity-dependent increase in melatonin content was found in both plasma and pineal organs. This effect was observed during the night, and was related to an increase in aanat2 mRNA abundance and AANAT2 enzyme activity, both of which also occurred during the day. Also, the levels of indoles (5-HT, 5-HIAA) in the pineal organ were negatively affected by increasing water salinity, which seems to be related to the higher recruitment of 5-HT as a substrate for the increased melatonin synthesis. A stimulatory effect of salinity on pineal aanat2 mRNA expression was also identified. These results indicate that increased external salinity promotes melatonin synthesis in the pineal organ of rainbow trout by enhancing synthesis of AANAT protein independently of its regulation by light. The possibility that pineal melatonin is a target for hormones involved in the response of fish to osmotic challenge is discussed, as well as the potential role of melatonin in the timing of osmoregulatory processes.

Dynamics in enzymatic protein complexes offer a novel principle for the regulation of melatonin synthesis in the human pineal gland.

Time of day is communicated to the body through rhythmic cues, including pineal gland melatonin synthesis, which is restricted to nighttime. Whereas in most rodents transcriptional regulation of the arylalkylamine N-acetyltransferase (Aanat) gene is essential for rhythmic melatonin synthesis, investigations into nonrodent mammalian species have shown post-transcriptional regulation to be of central importance, with molecular mechanisms still elusive. Therefore, human pineal tissues, taken from routine autopsies were allocated to four time-of-death groups (night/dawn/day/dusk) and analyzed for daytime-dependent changes in phosphorylated AANAT (p31T-AANAT) and in acetyl-serotonin-methyltransferase (ASMT) expression and activity. Protein content, intracellular localization, and colocalization of p31T-AANAT and ASMT were assessed, using immunoblotting, immunofluorescence, and immunoprecipitation techniques. Fresh sheep pineal gland preparations were used for comparative purposes. The amount of p31T-AANAT and ASMT proteins as well as their intracellular localization showed no diurnal variation in autoptic human and fresh sheep pineal glands. Moreover, in human and sheep pineal extracts, AANAT could not be dephosphorylated, which was at variance to data derived from rat pineal extracts. P31T-AANAT and ASMT were often found to colocalize in cellular rod-like structures that were also partly immunoreactive for the pinealocyte process-specific marker S-antigen (arrestin) in both, human and sheep pinealocytes. Protein-protein interaction studies with p31T-AANAT, ASMT, and S-antigen demonstrated a direct association and formation of robust complexes, involving also 14-3-3. This work provides evidence for a regulation principle for AANAT activity in the human pineal gland, which may not be based on a p31T-AANAT phosphorylation/dephosphorylation switch, as described for other mammalian species.

Impaired light masking in dopamine D2 receptor-null mice.

Disruption of overt circadian rhythms can occur without influencing the endogenous pacemaker, the so-called 'masking' effect classically elicited by light. As the physiological pathways involved in light masking remain elusive, we analyzed mice lacking the dopamine D2 receptor. Although circadian rhythmicity was normal, D2R-null mice showed a markedly deficient light masking response, indicating that D2R-mediated signaling is an essential component of the neuronal pathways leading to light masking of circadian rhythms.

[Pineal gland glutathione peroxidase activity in rats and its age-associated change].

Glutathione peroxidase activity has been studied in the pineal gland (epiphysis) of young and aging female Wistar rats (2-4 and 17-19 month old). For comparison the same activity was studied in the pyramids of medulla oblongata and in the olfactory tubercle. These two brain structures represent white and gray matter respectively. The determination of the activity was performed with H2O2 as a substrate and with 5,5'-dithio-bis-(2-nitrobenzoic acid) for estimation of the decrease of restored form of glutathione concentration. The glutathione peroxidase activity was higher in the pineal gland than in the brain structures used. Pineal glutathione peroxidase activities (micromole of GSH per minute per milligram of protein, M +/- m) in young and old rats were 1,52 +/- 0,07 and 1,27 +/- 0,06 respectively (p<0,05). The potential reason for the declined enzymatic activity found in the aged rats is the age-associated decrease of the selenium content in the pineal gland. The decline found may be one of the reflections of the pineal gland functional involution.

In vitro effects of 5-hydroxytryptophan, indoleamines and leptin on arylalkylamine N-acetyltransferase (AA-NAT) activity in pineal organ of the fish, Clarias gariepinus (Burchell, 1822) during different phases of the breeding cycle.

Arylalkylamine N-acetyltransferase (AA-NAT) is the rate-limiting enzyme of melatonin biosynthetic pathway. In vitro effects of 5-hydroxytryptophan (5-HTP) and indoleamines (serotonin, N-acetylserotonin and melatonin) were studied on AA-NAT activity in the pineal organ of the fish, C. gariepinus during different phases of its annual breeding cycle. Further, in vitro effects of leptin on AA-NAT activity in the pineal organ were studied in fed and fasted fishes during summer and winter seasons. Treatments with 5-HTP and indoleamines invariably stimulated pineal AA-NAT activity in a dose-dependent manner during all the phases. However, leptin increased AA-NAT activity in a dose-dependent manner only in the pineal organ of the fed fishes, but not of the fasted fishes irrespective of the seasons.

Circadian rhythm in pineal tyrosine hydroxylase.

Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis. The rat pineal gland is richly innervated by sympathetic nerves from the superior cervical ganglia. The activity of tyrosine hydroxylase was measured in rat pineal gland at 4-hour intervals over a daily cycle of 12 hours of light (7 a.m. to 7 p.m.) and 12 hours of darkness. The results indicate a circadian rhythm with the maximum activity, at 11 p.m. to 3 a.m., about triple the low values observed at 3 p.m. The pattern is similar in phase to that previously reported for melatonin and hydroxyindole-O-methyl transferase activity.

Visual pathway mediating pineal response to environmental light.

Activity of the melatoninforming enzyme, hydroxyindole-O-methyltransferase, in rat pineal is increased when the animal is exposed to continuous darkness, and it is decreased by exposure to continuous light. Response to environmental light is initiated in the retina and transmitted to the pineal by way of the central nervous system and the cervical sympathetics. The central visual pathway essential for mediation of this response is the inferior accessory optic tract. Visual pathways to thalamus and tectum do not participate in this response.

Circadian clock in culture: N-acetyltransferase activity of chick pineal glands oscillates in vitro.

N-Acetyltransferase activity was measured in organ-cultured chick pineal glands. A circadian rhythm of enzyme activity persisted in cultured glands for up to 4 days. The phase of the rhythm in vitro closely approximates its phase in vivo. These observations demonstrate that the pineal gland of chicks contains (or is) a self-sustained circadian oscillator.

Circadian rhythm of serotonin N-acetyltransferase activity in organ culture of chicken pineal gland.

When chicken pineal glands were organ-cultured in darkness, serotonin N-acetyltransferase activity was low during daytime, increased at midnight, and decreased on the next morning. The autonomous increase of N-acetyltransferase activity was suppressed by illumination of the glands. When pineal glands were cultured under a light-dark cycle (LD 12:12), the change of N-acetyltransferase activity continued to oscillate in phase with the light-dark cycle for 3 days.

Timekeeping by the pineal gland.

N-Acetyltransferase, an enzyme involved in melatonin production in the pineal gland, exhibits a circadian rhythm in chickens with peak values in the dark-time and low values during the light-time, commencing at lights-on. When pineal glands of chickens killed during the dark-time (with high N-acetyltransferase activity) were organ-cultured, there was a decline in enzyme activity to light-time values. Regardless of the time of the dark at which the chickens were killed, the enzyme activity reached light-time levels at precisely the same time.

Harderian gland: influence on pineal hydroxyindole-O-methyltransferase activity in neonatal rats.

A circadian rhythm has been found in hydroxyindole-O-methyltransferase activity of the pineal gland of blinded 12-day-old rats. Five additional hours of lighting can partly prevent the nocturnal increase in pineal hydroxyindole-O-methyltransferase activity in such rats. Removal of the Harderian gland abolishes this response to light in 12-day-old blinded animals, giving further support to the suggestion that this gland may function as an extraretinal photosensitive organ influencing the pineal gland in blinded suckling rats.

Viral neoplastic transformation of hamster pineal cells in vitro: retention of enzymatic function.

Cultures of hamster pineal tissue infected with certain oncogenic DNA viruses undergo neoplastic transformation and produce tumors when injected into homologous hosts. Hydroxyindole-O-methyl transferase, an enzyme found exclusively in the pineal gland, is present in low concentrations in transformed pineal cells in vitro and in larger amounts in tumors produced by the injected cells. This enzyme is not present in several nonpineal tissues similarly transformed.

Enzymatic synthesis of melatonin in avian pineal body: extraretinal response to light.

In the chick pineal body, activity of the melatonin-forming enzyme hydroxyindole-O-methyl transferase is greater in the light than in darkness. Neither bilateral enucleation of the eyes nor sympathetic denervation prevented this light-induced elevation of enzyme activity. This fact indicates that in the bird, in contrast to mammals, neither the retinas nor sympathetic innervation of the pineal body are essential for environmental control of melatonin formation.

Hydroxyindole-O-methyl-transferase activity: effect of sympathetic nerve stimulation.

Stimulation of the preganglionic nerve trunk to the superior cervical ganglion causes a reduction in pineal hydroxyindole-O-methyl-transferase levels which is time-dependent. The results provide direct evidence for a role of afferent input in the control of pineal enzymatic activity.