Intermittent and rhythmic exposure to melatonin in primary cultured adipocytes enhances the insulin and dexamethasone effects on leptin expression

Considering the cyclic characteristic of production and secretion of pineal melatonin, it is reasonable to assume that this oscillation might be important in determining the variety of its circadian and seasonal effects. To simulate this physiological condition in vitro, isolated adipocytes were exposed to melatonin in a circadian-like pattern by adding the hormone to the incubating medium during 12 hr (mimicking the night), followed by an equal period without melatonin (mimicking the day). This intermittent procedure was interrupted when three cycles with melatonin were fulfilled (60-hr incubation). Here, we report the effects of melatonin (1 nM) added intermittently or continuously to the incubating medium alone or in combination with insulin (5 nM) and/or dexamethasone (7 nM) on leptin release and expression by rat adipocytes. After acute 12-hr incubation neither melatonin nor insulin alone affected leptin expression, but together they increased it by 105%. Dexamethasone increased leptin mRNA content and release (70%) but this effect was not enhanced by melatonin. Nevertheless, after 60 hr under intermittent melatonin, we observed a synergism between melatonin and dexamethasone. This interaction promoted an increment (75% compared with dexamethasone alone) in leptin release and expression. Our results suggest that circadian-like exposure to melatonin potentiates the dexamethasone action and is important to the effects promoted by insulin on leptin expression. Based on an in vitro approach, this work helps to clarify the physiological relevance and the repercussions of the in vivo circadian pattern of melatonin secretion.     

Maternal melatonin effects on clock gene expression in a nonhuman primate fetus

In the adult mammal the circadian system, which allows predictive adaptation to daily environmental changes, comprises peripheral oscillators in most tissues, commanded by the suprachiasmatic nucleus (SCN) of the hypothalamus. The external environment of the fetus is provided by its mother. In primates, maternal melatonin is a candidate to entrain fetal circadian rhythms, including the SCN rhythms of metabolic activity. We found in the 90% of gestation capuchin monkey fetus expression of the clock genes Bmal-1, Per-2, Cry-2, and Clock in the SCN, adrenal, pituitary, brown fat, and pineal. Bmal-1, Per-2, and the melatonin 1 receptor (MT1) showed a robust oscillatory expression in SCN and adrenal gland, whereas a circadian rhythm of dehydroepiandrosterone sulphate was found in plasma. Maternal melatonin suppression changed the expression of Bmal-1, Per-2, and MT1 in the fetal SCN. These effects were reversed by maternal melatonin replacement. In contrast, neither maternal melatonin suppression nor its replacement had effects on the expression of Per-2 and Bmal-1 or MT1 in the fetal adrenal gland or the circadian rhythm of fetal plasma dehydroepiandrosterone sulphate. Our data suggest that maternal melatonin is a Zeitgeber for the fetal SCN but probably not for the adrenal gland.     

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.     

A circadian clock entrained by melatonin is ticking in the rat fetal adrenal

The adrenal gland in the adult is a peripheral circadian clock involved in the coordination of energy intake and expenditure, required for adaptation to the external environment. During fetal life, a peripheral circadian clock is present in the nonhuman primate adrenal gland. Whether this extends to the fetal adrenal gland like the rat is unknown. Here we explored in vivo and in vitro whether the rat fetal adrenal is a peripheral circadian clock entrained by melatonin. We measured the 24-h changes in adrenal content of corticosterone and in the expression of clock genes Per-2 and Bmal-1 and of steroidogenic acute regulatory protein (StAR), Mt1 melatonin receptor, and early growth response protein 1 (Egr-1) expression. In culture, we explored whether oscillatory expression of these genes persisted during 48 h and the effect of a 4-h melatonin pulse on their expression. In vivo, the rat fetal adrenal gland showed circadian expression of Bmal-1 and Per-2 in antiphase (acrophases at 2200 and 1300 h, respectively) as well as of Mt1 and Egr-1. This was accompanied by circadian rhythms of corticosterone content and of StAR expression both peaking at 0600 h. The 24-h oscillatory expression of Bmal-1, Per-2, StAR, Mt1, and Egr-1 persisted during 48 h in culture; however, the antiphase between Per-2 and Bmal-1 was lost. The pulse of melatonin shifted the acrophases of all the genes studied and restored the antiphase between Per-2 and Bmal-1. Thus, in the rat, the fetal adrenal is a strong peripheral clock potentially amenable to regulation by maternal melatonin.     

Acute modifications in the levels of daytime melatonin do not influence leptin in postmenopausal women

Melatonin shows a clear circadian rhythm with peak values at night, and may act directly with fat cells. Leptin, the anorexic hormone synthesized mainly by adipocytes, is produced in a circadian fashion, similar to that of melatonin. Accordingly, in the present study, we investigated whether melatonin may contribute to the rise in circulating leptin. The study was performed in postmenopausal women with 2 months of treatment with placebo or estradiol (50 microg/day). Melatonin was administered in doses of 1 mg by mouth versus placebo. In experiment 1, melatonin was administered at 08:30 hr. In experiment 2, at 08:30 hr and 10:30 hr, and in experiment 3 at 15:30 hr. Three blood samples, one every 15 min, were collected prior to the administration of melatonin and 2 hr after the administration of the single melatonin dose or the second melatonin administration (experiment 2). Following its administration, circulating melatonin reached pharmacological levels. In the three experiments, levels of leptin were not modified by the daytime administration of melatonin. These data indicate that, at least in daytime hours, acute modifications in daytime melatonin levels do not influence levels of leptin of postmenopausal women either without or with estradiol replacement. Accordingly, the metabolic, endocrine, reproductive and biological modifications induced by acute daytime melatonin in women do not seem to be mediated by modifications in circulating leptin.     

Advancing human circadian rhythms with afternoon melatonin and morning intermittent bright light

CONTEXT: Both light and melatonin can be used to phase shift the human circadian clock, but the phase-advancing effect of the combination has not been extensively investigated. OBJECTIVE: The objective of the study was to determine whether phase advances induced by morning intermittent bright light and a gradually advancing sleep schedule could be increased with afternoon melatonin. PARTICIPANTS: Healthy adults (25 males, 19 females, between the ages of 19 and 45 yr) participated in the study. DESIGN: There were 3 d of a gradually advancing sleep/dark period (wake time 1 h earlier each morning), bright light on awakening [four 30-min bright-light pulses (approximately 5000 lux) alternating with 30 min room light < 60 lux] and afternoon melatonin, either 0.5 or 3.0 mg melatonin timed to induce maximal phase advances, or matching placebo. The dim light melatonin onset was measured before and after the treatment to determine the phase advance. RESULTS: There were significantly larger phase advances with 0.5 mg (2.5 h, n = 16) and 3.0 mg melatonin (2.6 h, n = 13), compared with placebo (1.7 h, n = 15), but there was no difference between the two melatonin doses. Subjects did not experience jet lag-type symptoms during the 3-d treatment CONCLUSIONS: Afternoon melatonin, morning intermittent bright light, and a gradually advancing sleep schedule advanced circadian rhythms almost 1 h/d and thus produced very little circadian misalignment. This treatment could be used in any situation in which people need to phase advance their circadian clock, such as before eastward jet travel or for delayed sleep phase syndrome.     

Melatonin resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells

Abstract: Prostate cancer (PCa) is a major age‐related malignancy as increasing age correlates with increased risk for developing this neoplasm. Similarly, alterations in circadian rhythms have also been associated with the aging population and cancer risk. The pineal hormone melatonin is known to regulate circadian rhythms, which is under the control of a core set of genes: Period 1, 2, 3 (Per 1–3); Cryptochrome 1, 2 (Cry 1, 2); Clock, and Bmal 1, 2. Melatonin levels have been shown to decrease in patients with cancer and exogenous melatonin exhibits antiproliferative effects against certain cancers. In this study, we challenged the hypothesis that melatonin imparts antiproliferative effects in prostate cancer via resynchronization of deregulated core clock circuitry. We found that Clock and Per2 protein levels were downregulated whereas Bmal1 protein levels were upregulated in PCa cells, compared to normal prostate cells. Additionally, employing automated quantitative analysis of a microarray containing human tissues, we found that compared to benign tissues, Clock and Per2 levels were downregulated, whereas Bmal1 levels were upregulated in PCa and other proliferative prostatic conditions. Overexpression of Per2 was found to result in a significant loss of PCa cell growth and viability. Interestingly, melatonin treatment resulted in an increase in Per2 and Clock and a reduction in Bmal1 in PCa cells. Further, melatonin treatment resulted in a resynchronization of oscillatory circadian rhythm genes (Dbp and Per2). Our data support our hypothesis and suggest that melatonin should be thoroughly investigated as an agent for the management of PCa and other age‐related malignancies.     

Development of the rhythmic melatonin secretion in the embryonic chicken pineal gland

In order to elucidate details on the development of the circadian clock, the effects of light on the in vitro melatonin (MT) release and the presence of mRNAs of several clock genes in the embryonic chicken pineal gland were investigated. Chicken embryos of various developmental stages were exposed to stimuli of light in vitro in dynamic, four day long bioassay (perifusion). MT secretion and mRNA levels of Cry1, Cry2, Clock and Bmal2 clock genes were determined. Our conclusions: (1) environmental illumination modified MT secretion from explanted embryonic pineal glands as early as on the 13th embryonic day, (2) daily rhythm of MT release develops between embryonic days 16 and 18 under periodic environmental illumination. (3) Chicken Cry1, Cry2, Clock and Bmal2 clock gene mRNAs were also detected in glands of animals of 15th embryonic day. Although both MT secretion and clock genes have been developed by then, the circadian MT rhythm appears first on the 17th embryonic day. Either the mechanisms coupling the clock with the melatonin output or the synchronization of the individual pinealocytes develop around this age. Rhythmic MT release in the embryonic chicken pineal gland evolves only if the egg is exposed to rhythmic environmental stimuli.     

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.     

Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine‐induced hepatocellular carcinoma

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long‐term diseases including hepatocellular carcinoma (HCC ). Melatonin has been reported to alleviate promotion and progression of HCC , but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN ) (35 mg/kg body weight ip) once a week for 8 weeks. Melatonin was given at 5 or 10 mg kg^?1d^?1 ip beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK 1ε, Rev‐erbα, and Rev‐erbβ decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT 1 receptor and reduced levels of the hypoxia‐inducible factors Hif‐1α and Hif‐2α. An increased expression of p21, p53, and PARP 1/2, a higher Bax/Bcl‐2 ratio, and a lower expression of Cyclin D1, CDK 6, HSP 70, HSP 90, and GRP 78 proteins were also observed in melatonin‐treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV ‐ERB agonist SR 9009 in human Hep3B cells, and BMAL 1 knocking down attenuated the pro‐apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.     

Effects of photoperiod and temperature on rhythmic melatonin secretion from the pineal organ of the white sucker (Catostomus commersoni) in vitro.

The secretion rate of melatonin from cultured pineal organs of the white sucker was examined for several days under either a 12:12-hr light:dark (LD) cycle or continuous darkness (DD) at either 10 degrees or 20 degrees. The incubation medium was changed at 3-hr intervals and secreted melatonin was measured by RIA. Under a 12:12-hr LD cycle (0800 light on, 2000 light off) melatonin secretion was suppressed during the day and highly active at night, with larger amplitudes at 20 than at 10 degrees. In DD at 10 degrees no circadian rhythmicity in secretion was found in October or January, whereas at 20 degrees a circadian-like pattern was detected in pineals which were derived from animals reared at either 10 degrees or 20 degrees for 1 week prior to the experiment in October or January. The pineals in the DD experiment still responded to an additional 24-hr LD cycle at both temperatures even after 6 or more days. These results clearly reveal the influence of photoperiod and temperature on melatonin secretion of organ-cultured pineal glands. The existence of a circadian oscillator for melatonin secretion in the pineal gland of the white sucker is suggested.     

Retinal melatonin is metabolized within the eye of xenopus laevis.

Retinal synthesis of melatonin, a potent modulator of rhythmic retinal processes, is elevated at night as a result of regulation by a circadian clock. Despite high nocturnal synthetic capacity, both melatonin content and release are low in the retina of the frog Xenopus laevis. We report here that cultured eyecups from Xenopus have the capacity for rapid metabolic breakdown of melatonin. Pharmacological analysis indicates that the initial step in this degradation pathway is deacetylation of melatonin by the enzyme aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13). This produces 5-methoxytryptamine, which is then deaminated by monoamine oxidase [amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4], producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. Inhibition of aryl acylamidase with eserine dramatically increases the release of endogenous melatonin by eyecups cultured at night, indicating that this pathway is the normal fate of retinal melatonin. Metabolism within the eye suggests a local neuromodulatory role for retinal melatonin, in contrast to the hormonal role of pineal melatonin.     

Reduced lipolysis and increased lipogenesis in adipose tissue from pinealectomized rats adapted to training

The current study investigated the effects of chronic training and pinealectomy on the lipogenic and lipolytic activity of adipose tissue. Pinealectomized and sham-operated adult male Wistar rats were distributed in to four subgroups: pinealectomized untrained, pinealectomized trained, control untrained and control trained. At the end of the training period (8 wk) the rats were killed. Blood samples were collected for glucose, insulin and leptin determinations. Peri-epididymal adipocytes were isolated for measurement of in vitro rates of lipolysis and incorporation of substrates (D-[U-14C]-glucose, L-[U-14C]-lactate, [2-14C]-acetate and [1-14C]-palmitate) into lipids, and samples of epididymal adipose tissue were homogenized for evaluation of glucose-6-phosphate dehydrogenase maximal activity. Pinealectomy resulted in a significantly increased lipolytic capacity in response to isoproterenol and a decrease in circulating leptin levels without affecting the rates of incorporation of different substrates into lipids. However, only in the intact control group did training promote a higher basal and isoproterenol-stimulated lipolysis, increase the incorporation of palmitate (esterification), decrease the incorporation of acetate (lipogenesis) into lipids and diminish circulating leptin levels. These effects of exercise training were not seen in pinealectomized rats. However, pinealectomized trained animals showed a marked reduction in lipolysis and an increased rate of acetate incorporation. In conclusion, we demonstrated for the first time that the pineal gland plays an important role in the regulation of lipid metabolism in such a way that its absence caused a severe alteration in the balance between lipogenesis and lipolysis, which becomes evident with the adaptation to exercise training.     

Temporal expression of seven clock genes in the suprachiasmatic nucleus and the pars tuberalis of the sheep: evidence for an internal coincidence timer

The 24-h expression of seven clock genes (Bmal1, Clock, Per1, Per2, Cry1, Cry2, and CK1 epsilon ) was assayed by in situ hybridization in the suprachiasmatic nucleus (SCN) and the pars tuberalis (PT) of the pituitary gland, collected every 4 h throughout 24 h, from female Soay sheep kept under long (16-h light/8-h dark) or short (8-h light/16-h dark) photoperiods. Locomotor activity was diurnal, inversely related to melatonin secretion, and prolactin levels were increased under long days. All clock genes were expressed in the ovine SCN and PT. In the SCN, there was a 24-h rhythm in Clock expression, in parallel with Bmal1, in antiphase with cycles in Per1 and Per2; there was low-amplitude oscillation of Cry1 and Cry2. The waveform of only Per1 and Per2 expression was affected by photoperiod, with extended elevated expression under long days. In the PT, the high-amplitude 24-h cycles in the expression of Bmal1, Clock, Per1, Per2, Cry1, and Cry2, but not CK1 epsilon, were influenced by photoperiod. Per1 and Per2 peaked during the day, whereas Cry1 and Cry2 peaked early in the night. Hence, photoperiod via melatonin had a marked effect on the phase relationship between Per/Cry genes in the PT. This supports the conclusion that an "external coincidence model" best explains the way photoperiod affects the waveform of clock gene expression in the SCN, the central pacemaker, whereas an "internal coincidence model" best explains the way melatonin affects the phasing of clock gene expression in the PT to mediate the photoperiodic control of a summer or winter physiology.     

Potential role of high serum leptin concentration in age-related decrease of fatty acid synthase gene expression in rat white adipose tissue

Ageing in rats is associated with significant reduction of lipogenic enzymes genes expression and the rate of fatty acids synthesis in white adipose tissue. Since resistance to insulin and/or triiodothyronine is not directly related to reduction in lipogenic enzymes activity in white adipose tissue of old rats, we have proposed recently that the age-related increase in leptin gene expression and high serum leptin concentration could, at least partly, account for the down regulation of lipogenic enzymes gene expression. To test this hypothesis, in this paper we experimentally (by single intraperitoneal injection of recombinant leptin) increased plasma leptin concentration in young rats to the level observed in old animals, and we examined its effect on fatty acids synthase (FAS) gene expression in white adipose tissue. The results presented in this paper indicate that leptin administration to young rats, in amount that cause the increase in serum leptin concentration to that found in old rats, significantly decreased the white adipose tissue FAS gene expression. We propose, therefore, that leptin could play a causative role in the down-regulation of lipogenic enzyme gene expression observed with ageing.