Melatonin-proficient Mice Research Articles

Disruption of Melatonin Signaling Leads to Lipids Accumulation in the Liver of Melatonin Proficient Mice.

Melatonin signaling via melatonin receptor type 1 (MT1) and type 2 (MT2) plays an important role in the regulation of several physiological functions. Studies in rodents and humans have demonstrated that disruption of melatonin signaling may affect glucose metabolism, insulin sensitivity, and leptin levels. Accumulating experimental evidence also indicates that in rodents the administration of exogenous melatonin has a beneficial effect on the blood lipid levels. However, the molecular mechanism by which melatonin signaling may regulate lipids is still unclear. In addition, most of the studies with mice have been performed in melatonin-deficient mice by administering exogenous melatonin at supraphysiological doses. Hence the results of these studies may be greatly affected by these two factors. In this study, we report the effects of melatonin signaling removal on the liver biology and transcriptome using melatonin-proficient mice (C3H-f+/f+) in which MT1 or MT2 have been genetically ablated. Our data indicate that the absence of MT1 or MT2 signaling leads to disruption of the blood lipids profile and an increase in lipids deposition in the liver. These effects were more pronounced in the mice lacking MT1 than MT2. The gene expression profiles obtained with RNA-seq from the livers of the three genotypes revealed that removal of MT1 affected the transcription of 4255 genes (i.e., 40.6%). Conversely, the removal of MT2 affected the transcription of 1864 transcripts (i.e., 17.2%). Finally, we identified a group of 13 genes involved in lipids biology that may play a key role in the accumulation of lipids in the liver when melatonin signaling is disrupted. In conclusion, our study indicates that melatonin signaling is an important modulator of liver physiology and metabolism. Our study also indicated that the removal of MT1 signaling is more deleterious than MT2 removal.

Selective MT 2 Melatonin Receptor Antagonist Modulates Circadian Activity via Inhibition of the Endogenous Melatonin Signal in the East Bound Jet Lag Model

Activation of melatonin receptors facilitates re-entrainment of circadian activity rhythms following an advance of the dark onset. In an eastbound jet lag model, administration of exogenous melatonin, at the new dark onset, accelerates re-entrainment via activation of the MT1 receptor (Dubocovich et al., 2005). Conversely, melatonin proficient mice (C3H/HeN) with a targeted deletion of the MT2 receptor (MT2KO), but not the MT1 receptor (MT1KO), experience a deceleration in re-entrainment, probably due to impairment of the endogenous melatonin facilitated signaling mediated by activation of the MT2 receptor (Pfeffer et al., 2012). To further elucidate the functional modulatory role of melatonin receptors on the synchronization of the circadian clock, we assessed locomotor activity rhythms in C3H/HeN mice in response to pharmacological intervention to determine the ability of the MT2 selective antagonist, ATBT-23 (hMT1/hMT2 Ki=190), to decelerate re-entrainment by blocking the MT2 receptor, thus mimicking the MT2KO. Male C3H/HeN mice in a 12/12 L/D cycle were subjected to an eastbound jet lag paradigm in which Vehicle (30% ethanol/saline, s.c) or ATBT-23 (1 mg/kg in Vehicle, s.c) were administered on three consecutive days at the new dark onset following an abrupt 6-hour advance of the L/D cycle. The number of days to re-entrain and re-entrainment rates (hrs/day) were analyzed by two-way ANOVA (Tukey's post hoc test) and a mixed effect two-way repeated measure ANOVA (Sidak's post hoc test), respectively. WT (7.54±0.33 days, n=28) and MT1KO (7.36±0.54 days, n=14) mice treated with vehicle did not show a significant difference in the number of days necessary to attain re-entrainment. However, MT2KO (11.92±0.40 days, n=13) mice, treated with vehicle, took significantly longer to re-entrain when compared to WT and MT1KO mice (p<0.0001). This deceleration of re-entrainment in the MT2KO was reproduced by ATBT-23, which decelerated the rate of re-entrainment in both WT (p<0.0001, n=25) and MT1KO (p<0.01, n=12) males, increasing the number of days to re-entrain to 9.76±0.46 (p<0.01) and 10.00±0.63 (p<0.05) days, respectively. ATBT-23 (n=14) did not significantly affect the number of days to re-entrain in MT2KO mice, suggesting deceleration of re-entrainment is not mediated by the MT1 receptor. The present study suggests that ATBT-23 decelerates re-entrainment of circadian activity rhythms by blocking the potent acceleration of re-entrainment by endogenous melatonin via action on the MT2 receptor. Therefore, selective MT2 melatonin receptor antagonists can potentially be used to delay advanced circadian rhythms, as observed in depression and advance sleep phase disorders, when administered at proper circadian times.

Removal of melatonin receptor type 1 signalling induces dyslipidaemia and hormonal changes in mice subjected to environmental circadian disruption.

BackgroundMelatonin is a hormone secreted by the pineal gland in a circadian rhythmic manner with peak synthesis at night. Melatonin signalling was suggested to play a critical role in metabolism during the circadian disruption.MethodsMelatonin‐proficient (C3H‐f+/+ or WT) and melatonin receptor type 1 knockout (MT1 KO) male and female mice were phase‐advanced (6 hours) once a week for 6 weeks. Every week, we measured weight, food intake and basal glucose levels. At the end of the experiment, we sacrificed the animals and measured the blood's plasma for lipids profile (total lipids, phospholipids, triglycerides and total cholesterol), metabolic hormones profiles (ghrelin, leptin, insulin, glucagon, glucagon‐like‐peptide and resistin) and the body composition.ResultsEnvironmental circadian disruption (ECD) did not produce any significant effects in C3H‐f+/+, while it increased lipids profile in MT1 KO with the significant increase observed in total lipids and triglycerides. For metabolic hormones profile, ECD decreased plasma ghrelin and increased plasma insulin in MT1 KO females. Under control condition, MT1 KO females have significantly different body weight, fat mass, total lipids and total cholesterol than the control C3H‐f+/+ females.ConclusionOur data show that melatonin‐proficient mice are not affected by ECD. When the MT1 receptors are removed, ECD induced dyslipidaemia in males and females with females experiencing the most adverse effect. Overall, our data demonstrate that MT1 signalling is an essential modulator of lipid and metabolic homeostasis during ECD.

Impact of melatonin receptor-signaling on Zeitgeber time-dependent changes in cell proliferation and apoptosis in the adult murine hippocampus.

The hippocampus is subjected to diurnal/circadian rhythms on both the morphological and molecular levels. Certain aspects of cell proliferation in the adult hippocampus are regulated by melatonin and accompanied by apoptosis to ensure proper tissue maintenance and function. The present study investigated Zeitgeber time (ZT)-dependent changes in cell proliferation and apoptosis in the adult murine hippocampus and their regulation by melatonin receptor type1 and type2 (MT1/2)-mediated signaling. Adult melatonin-proficient C3H/HeN mice and melatonin-proficient (C3H/HeN) mice with targeted deletion of MT1/2 were adapted to a 12-h light, 12-h dark photoperiod and were sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 and activated caspase-3 in combination with different markers for the diverse cell types residing in the hippocampus served to identify and quantify proliferating and apoptotic cells in the hippocampal subregions. ZT-dependent changes in cell proliferation and apoptosis were found exclusively in the subgranular zone (SGZ) and granule cell layer (GCL) of melatonin-proficient mice with functional MT1/2. Cell proliferation in the SGZ showed ZT-dependent changes indicated by an increase of proliferating immature neurons during the dark phase of the 24-h light-dark cycle. Apoptosis showed ZT-dependent changes in the SGZ and GCL indicated by an increase of apoptotic immature neurons at ZT06 (SGZ) and a decrease of immature and mature neurons at ZT18 (GCL). Our results indicate that ZT-dependent changes in proliferation of immature neurons in the SGZ are counterbalanced by ZT-dependent changes in apoptosis of immature and mature neurons in the SGZ and GCL exclusively in mice with functional MT1/2. Therefore, MT1/2-mediated signaling appears to be crucial for generation and timing of ZT-dependent changes in cell proliferation and apoptosis and for differentiation of proliferating cells into neurons in the SGZ. © 2017 Wiley Periodicals, Inc.

Circadian and light-driven regulation of rod dark adaptation.

Continuous visual perception and the dark adaptation of vertebrate photoreceptors after bright light exposure require recycling of their visual chromophore through a series of reactions in the retinal pigmented epithelium (RPE visual cycle). Light-driven chromophore consumption by photoreceptors is greater in daytime vs. nighttime, suggesting that correspondingly higher activity of the visual cycle may be required. However, as rod photoreceptors are saturated in bright light, the continuous turnover of their chromophore by the visual cycle throughout the day would not contribute to vision. Whether the recycling of chromophore that drives rod dark adaptation is regulated by the circadian clock and light exposure is unknown. Here, we demonstrate that mouse rod dark adaptation is slower during the day or after light pre-exposure. This surprising daytime suppression of the RPE visual cycle was accompanied by light-driven reduction in expression of Rpe65, a key enzyme of the RPE visual cycle. Notably, only rods in melatonin-proficient mice were affected by this daily visual cycle modulation. Our results demonstrate that the circadian clock and light exposure regulate the recycling of chromophore in the RPE visual cycle. This daily melatonin-driven modulation of rod dark adaptation could potentially protect the retina from light-induced damage during the day.

Impact of Melatonin on Zeitgeber Time-Dependent Changes in Cell Proliferation and Apoptosis in the Adult Murine Hypothalamic-Hypophyseal System

Background/Aims: Cell proliferation and apoptosis are known to adjust neuroendocrine circuits to the photoperiod. The latter is communicated by melatonin, the hormone secreted by the pineal organ. The present study investigated zeitgeber time (ZT)-dependent changes in cell proliferation and apoptosis in the adult murine neuroendocrine system and their regulation by melatonin. Methods: Adult melatonin-proficient (C3H/HeN) and melatonin-deficient (C57Bl/6J) mice, as well as melatonin-proficient (C3H/HeN) mice with targeted deletion of both melatonin receptor types (MT1 and MT2) were adapted to a 12-hour light, 12-hour dark photoperiod and were sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 and activated caspase-3 served to identify and quantify proliferating and apoptotic cells in the median eminence (ME), hypophyseal pars tuberalis, and pars distalis (PD). Results: ZT-dependent changes in cell proliferation and apoptosis were found exclusively in melatonin-proficient mice with functional MTs. Cell proliferation in the ME and PD showed ZT-dependent changes indicated by an increase at ZT12 (ME) and a decrease at ZT06 (PD). Apoptosis showed ZT-dependent changes in all regions analyzed, indicated by an increase at ZT06. Proliferating and apoptotic cells were found in nearly all cell types residing in the regions analyzed. Conclusions: Our results indicate that ZT-dependent changes in cell proliferation are counterbalanced by ZT-dependent changes in apoptosis exclusively in melatonin-proficient mice with functional MTs. Melatonin signaling appears to be crucial in both the generation and timing of proliferation and apoptosis that serve the high rate of physiological cell turnover in the adult neuroendocrine system.

Melatonin administration alters nicotine preference consumption via signaling through high-affinity melatonin receptors.

While it is known that tobacco use varies across the 24-h day, the time-of-day effects are poorly understood. Findings from several previous studies indicate a potential role for melatonin in these time-of-day effects; however, the specific underlying mechanisms have not been well characterized. Understanding of these mechanisms may lead to potential novel smoking cessation treatments. The objective of this study is examine the role of melatonin and melatonin receptors in nicotine free-choice consumption A two-bottle oral nicotine choice paradigm was utilized with melatonin supplementation in melatonin-deficient mice (C57BL/6J) or without melatonin supplementation in mice proficient at melatonin synthesis (C3H/Ibg) compared to melatonin-proficient mice lacking both or one of the high-affinity melatonin receptors (MT1 and MT2; double-null mutant DM, or MT1 or MT2). Preference for bitter and sweet tastants also was assessed in wild-type and MT1 and MT2 DM mice. Finally, home cage locomotor monitoring was performed to determine the effect of melatonin administration on activity patterns. Supplemental melatonin in drinking water significantly reduced free-choice nicotine consumption in C57BL/6J mice, which do not produce endogenous melatonin, while not altering activity patterns. Independently, genetic deletion of both MT1 and MT2 receptors in a melatonin-proficient mouse strain (C3H) resulted in significantly more nicotine consumption than controls. However, single genetic deletion of either the MT1 or MT2 receptor alone did not result in increased nicotine consumption. Deletion of MT1 and MT2 did not impact taste preference. This study demonstrates that nicotine consumption can be affected by exogenous or endogenous melatonin and requires at least one of the high-affinity melatonin receptors. The fact that expression of either the MT1 or MT2 melatonin receptor is sufficient to maintain lower nicotine consumption suggests functional overlap and potential mechanistic explanations.

Melatonin receptor 1-dependent gene expression in the mouse pars tuberalis as revealed by cDNA microarray analysis andin situhybridization

Melatonin is an important rhythmic endocrine signal within the circadian system of mammals. The hypophyseal pars tuberalis (PT) is a major target for melatonin and shows a high density of melatonin type 1 receptors (MT1). Melatonin affects expression of clock genes in PT cells which encode for transcriptional regulators of rhythmic gene expression. In this study, microarray analysis was performed to screen for genes coding for transcriptional regulators under the control of MT1 receptors in the mouse PT. Gene expression levels were compared between melatonin-proficient mice deficient for MT1 (MT1-/-) and the corresponding wild-type (WT) during mid-subjective day (CT06, low endogenous melatonin levels) and mid-subjective night (CT18, high endogenous melatonin levels). In situ hybridization was used to validate the data obtained by microarray analysis to analyze the acute effect of daytime melatonin application on gene expression in the wild-type PT. In the wild-type PT, expression of Tim was higher during day as compared to night. These day/night differences in gene expression were not observed in the PT of MT1-/- mice, demonstrating the impact of MT1-mediated signal transduction pathway. Day-time application of melatonin acutely affected Tim and Cry1 expression but not Neurod1 and Npas4 expression. We conclude that melatonin regulates expression of genes coding for transcriptional regulators in the PT through MT1 receptors by either acute or long-term mechanisms.

Immunocytochemical demonstration of day/night changes of clock gene protein levels in the murine adrenal gland: differences between melatonin‐proficient (C3H) and melatonin‐deficient (C57BL) mice

The circadian system comprises several peripheral oscillators and a central rhythm generator that, in mammals, is located in the suprachiasmatic nucleus of the hypothalamus. Expression of clock genes is a characteristic feature of the central rhythm generator and the peripheral oscillators. With regard to the rhythmic production of glucocorticoids, the adrenal gland can be considered as peripheral oscillator, but little is known about clock gene expression in this tissue. Therefore, the present study investigates the levels of three clock gene proteins PER1, BMAL1 and CRY2 in the murine adrenal cortex and medulla at seven different time points of a 12-hr light/12-hr dark cycle. To determine a potential role of melatonin we compared the patterns of clock gene proteins in the adrenal gland of melatonin-proficient mice (C3H) with those of melatonin-deficient mice (C57BL). In C3H mice, both, the adrenal cortex and medulla displayed day/night variation in PER1-, CRY2- and BMAL1-protein levels. PER1 and CRY2 peaked in the middle of the light phase, whereas BMAL1 peaked in the dark phase. This pattern was also observed in the adrenal medulla of C57BL, but in the adrenal cortex of C57BL clock gene protein levels did not change with time and were consistently lower than in C3H mice. These results support the hypothesis that the adrenal gland is a peripheral oscillator and raise the possibility that melatonin may be involved in the control of clock gene protein levels in the adrenal cortex of mice.