Melatonin in the mammalian retina: Synthesis, mechanisms of action and neuroprotection.

Melatonin is an important player in the regulation of many physiological functions within the body and in the retina. Melatonin synthesis in the retina primarily occurs during the night and its levels are low during the day. Retinal melatonin is primarily synthesized by the photoreceptors, but whether the synthesis occurs in the rods and/or cones is still unclear. Melatonin exerts its influence by binding to G protein-coupled receptors named melatonin receptor type 1 (MT1) and type 2 (MT2). MT1 and MT2 receptors activate a wide variety of signaling pathways and both receptors are present in the vertebrate photoreceptors where they may form MT1/MT2 heteromers (MT1/2h). Studies in rodents have shown that melatonin signaling plays an important role in the regulation of retinal dopamine levels, rod/cone coupling as well as the photopic and scotopic electroretinogram. In addition, melatonin may play an important role in protecting photoreceptors from oxidative stress and can protect photoreceptors from apoptosis. Critically, melatonin signaling is involved in the modulation of photoreceptor viability during aging and other studies have implicated melatonin in the pathogenesis of age-related macular degeneration. Hence melatonin may represent a useful tool in the fight to protect photoreceptors-and other retinal cells-against degeneration due to aging or diseases.

Environmental circadian disruption re-programs liver circadian gene expression.

Circadian gene expression is fundamental to the establishment and functions of the circadian clock, a cell-autonomous and evolutionary-conserved timing system. Yet, how it is affected by environmental-circadian disruption (ECD) such as shiftwork and jetlag, which impact millions of people worldwide, are ill-defined. Here, we provided the first comprehensive description of liver circadian gene expression under normal and after ECD conditions. We found that post-transcription and post-translation processes are dominant contributors to whole-cell or nuclear circadian proteome, respectively. Furthermore, rhythmicity of 64% transcriptome, 98% whole-cell proteome and 95% nuclear proteome is re-written by ECD. The re-writing, which is associated with changes of circadian cis-regulatory elements, RNA-processing and protein trafficking, diminishes circadian regulation of fat and carbohydrate metabolism and persists after one week of ECD-recovery.

The circadian clock mediates the response to oxidative stress in a cone photoreceptor‒like (661W) cell line via regulation of glutathione peroxidase activity.

Background: The mammalian retina contains an autonomous circadian clock that controls many physiological functions within this tissue. Our previous studies have indicated that disruption of this circadian clock by removing Bmal1 from the retina affects the visual function, retinal circuitry, and cone photoreceptor viability during aging. In the present study, we employed a mouse-derived cone photoreceptor‒like cell, 661W, to investigate which molecular mechanisms of the circadian clock may modulate cone photoreceptor viability during aging. Methods: Bmal1 knockout (BKO) cells were generated from 661W cells using the CRISPR/Cas9 gene editing tool. Deletion of Bmal1 from 661W was verified by western blot and monitoring Per2-luc bioluminescence circadian rhythms. To investigate the effect of Bmal1 removal on an oxidative stress challenge, cells were treated with hydrogen peroxide (H 2O 2,1 mM) for two hours and then cell viability was assessed. Cells were also cultured and harvested for gene expression analysis and antioxidant assay. Results: Our data indicated that 661W cells contain a functional circadian clock that mediates the response to an oxidative stress challenge in vitro and that such a response is no longer present in the BKO cell. We also hypothesized that the effect was due to the circadian regulation of the intracellular antioxidant defense mechanism. Our results revealed that in 661W cells, the antioxidant defense mechanism showed time dependent variation , whereas in BKO cells, there was an overall reduction in this antioxidant defense mechanism, and it no longer showed time dependent variation. Conclusions: Our work supported the notion that the presence of a functional circadian clock and its ability to modulate the response to an oxidative stress is the underlying mechanism that may protect cones during aging.

Real-Time Monitoring of Circadian Rhythms in the Eye.

The mammalian eye harbors a full circadian system that controls several physiologically relevant functions within this organ. During the last two decades a few laboratories have developed transgenic animal models in which circadian rhythms can be monitored in real time using luciferase activity. The most famous transgenic mouse to record bioluminescence rhythms from different tissues and organs is the PERIOD2::LUCIFERASE (PER2::LUC) mouse developed by the Takahashi laboratory in early 2000. Since then, several studies have used this mouse model to dissect the mammalian circadian system by monitoring the circadian rhythm in the brain, the eye, and in many other peripheral organs and tissues. This chapter describes the methodology to record and analyze bioluminescence rhythms from the retina, retinal pigment epithelium, and cornea of PER2::LUC mice.

Assessing the Role of Melatonin in the Modulation of Visual Functions in the Mouse.

The electroretinogram (ERG) is a noninvasive method to evaluate retinal function. It can be applied to patients to be diagnosed a variety of retinal pathologies such as photoreceptor dystrophy, diabetic retinopathy, macular degeneration, and glaucoma. ERG has also been a reliable tool to assess retinal functions in animal studies that range from fish to humans. Melatonin is a neurohormone that regulates several retinal functions within the retina, and previous studies have shown that melatonin plays an important role in the modulation of the ERG in humans and other vertebrates. This chapter describes experimental methods to evaluate retinal function using ERG in the mouse and how to assess the contribution of melatonin. An introduction is provided for materials, environmental settings, recording procedures, and analysis necessary for ERG measurements.

"The ubiquitin ligase SIAH2 is a female-specific regulator of circadian rhythms and metabolism".

Circadian clocks enable organisms to predict and align their behaviors and physiologies to constant daily day-night environmental cycle. Because the ubiquitin ligase Siah2 has been identified as a potential regulator of circadian clock function in cultured cells, we have used SIAH2-deficient mice to examine its function in vivo. Our experiments demonstrate a striking and unexpected sexually dimorphic effect of SIAH2-deficiency on the regulation of rhythmically expressed genes in the liver. The absence of SIAH2 in females, but not in males, altered the expression of core circadian clock genes and drastically remodeled the rhythmic transcriptome in the liver by increasing the number of day-time expressed genes, and flipping the rhythmic expression from nighttime expressed genes to the daytime. These effects are not readily explained by effects on known sexually dimorphic pathways in females. Moreover, loss of SIAH2 in females, not males, preferentially altered the expression of transcription factors and genes involved in regulating lipid and lipoprotein metabolism. Consequently, SIAH2-deficient females, but not males, displayed disrupted daily lipid and lipoprotein patterns, increased adiposity and impaired metabolic homeostasis. Overall, these data suggest that SIAH2 may be a key component of a female-specific circadian transcriptional output circuit that directs the circadian timing of gene expression to regulate physiological rhythms, at least in the liver. In turn, our findings imply that sex-specific transcriptional mechanisms may closely interact with the circadian clock to tailor overt rhythms for sex-specific needs.

The Circadian Clock in the Retinal Pigment Epithelium Controls the Diurnal Rhythm of Phagocytic Activity.

The diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control and believed that this process involves interactions from the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE. Thereby, the aim of this study was to determine whether the clock in the retina or RPE controls the diurnal phagocytic peak and whether disruption of the circadian clock in the RPE would affect cellular function and the viability during aging. To that, we generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then determined the daily rhythm in phagocytic activity by the RPE in mice lacking a functional circadian clock in the retina or RPE. Then, using electroretinography, spectral domain-optical coherence tomography, and optomotor response of visual function we determined the effect of Bmal1 removal in young (6 months) and old (18 months) mice. RPE morphology and lipofuscin accumulation was determined in young and old mice. Our data shows that the clock in the RPE, rather than the retina clock, controls the diurnal phagocytic peak. Surprisingly, absence of a functional RPE clock and phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the circadian clock in the RPE controls the daily peak of phagocytic activity. However, the absence of the clock in the RPE does not result in deterioration of photoreceptors or the RPE during aging.

Circadian Regulation of Retinal Pigment Epithelium Function.

The retinal pigment epithelium (RPE) is a single layer of cells located between the choriocapillaris vessels and the light-sensitive photoreceptors in the outer retina. The RPE performs physiological processes necessary for the maintenance and support of photoreceptors and visual function. Among the many functions performed by the RPE, the timing of the peak in phagocytic activity by the RPE of the photoreceptor outer segments that occurs 1-2 h. after the onset of light has captured the interest of many investigators and has thus been intensively studied. Several studies have shown that this burst in phagocytic activity by the RPE is under circadian control and is present in nocturnal and diurnal species and rod and cone photoreceptors. Previous investigations have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE cells. However, the anatomical location of the circadian controlling this activity is not clear. Experimental evidence indicates that the circadian clock, melatonin, dopamine, and integrin signaling play a key role in controlling this rhythm. A series of very recent studies report that the circadian clock in the RPE controls the daily peak in phagocytic activity. However, the loss of the burst in phagocytic activity after light onset does not result in photoreceptor or RPE deterioration during aging. In the current review, we summarized the current knowledge on the mechanism controlling this phenomenon and the physiological role of this peak.

Association between MTNR1B polymorphisms and obesity in African American: findings from the Jackson Heart Study.

BACKGROUND: Melatonin is a hormone that is secreted at night by the pineal gland. It exerts its function by binding to the MT1 and MT2 receptors, which are encoded by the MTNR1A and MTNR1B genes, respectively. Previous studies reveal that MTNR1B variants are associated with insulin secretion impairments and an increased body mass index (BMI) in individuals of European and Asian ancestries. Obesity is highly prevalent in the US and disproportionately affects African Americans. Here, we hypothesized that common single nucleotide polymorphisms (SNPs) imputed in 1000 Genomes in the MTNR1B gene are associated with adiposity in African American adult men and women and that the association is modified by insomnia. METHODS: We used an additive genetic model to describe the association between the adiposity traits (BMI and waist circumference) and selected MTNR1B variants in 3,029 Jackson Heart Study participants, with an average age of 55.13 ± 12.84 years, and 62% were women. We regressed the adiposity measures on the estimated allelic or genotypic dosage at every selected SNP and adjusted for age, sex, population stratification, and insomnia. Thirty common SNPs, spanning the MTNR1B gene, with a minor allele frequency ≥ 5%, a call rate ≥ 90%, a Hardy-Weinberg equilibrium p value > 10-6, were available for the analysis. RESULTS: The allele T of rs76371840 was associated with adiposity (OR = 1.47 [1.13-1.82]; PFDR-adjusted = 0.0499), and the allele A of rs8192552 showed a significant association with waist circumference (ß = 0.023 ± 0.007; PFDR-adjusted = 0.0077) after correcting for multiple testing. When insomnia was included in the adiposity analysis model, the following four variants became significantly associated with adiposity: rs6483208; rs4388843; rs4601728; and rs12804291. CONCLUSIONS: Our data indicate that polymorphisms in the MTNR1B gene are associated with obesity traits in African Americans. To the best of our knowledge, this is the first study to explore the effect of insomnia on the association between the circadian MTNR1B genetic variants and metabolic traits in an African American sample population. We observed that insomnia affected the association between the MTNR1B variants and adiposity.

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

Background: Melatonin 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. Methods: Melatonin-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. Results: Environmental 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. Conclusion: Our 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.

Photoreceptor Degeneration in Homozygous Male Per2luc Mice During Aging.

The Per2luc mouse model developed by Takahashi laboratory is one of the most powerful models to study circadian rhythms in real time. In this study, we report that photoreceptors degenerate in male Per2luc mice during aging. Young (2.5- to 5-month-old) and aged (11- to 13.5-month-old) homozygous male Per2luc mice and C57BL/6J mice were used for this study. Retina structure and function were investigated via spectral domain optical coherence tomography (SD-OCT), fundus imaging, and electroretinography (ERG). Zonula occludens-1 (ZO-1) immunofluorescence was used to analyze the retinal pigment epithelium (RPE) morphology. Fundus examination revealed no difference between young Per2luc and wild-type (WT) mice. However, the fundus of aged Per2luc mice showed white deposits, suggestive of age-related drusen-like formation or microglia, which were absent in age-matched WT mice. No differences in retinal structure and function were observed between young Per2luc and WT mice. However, with age, Per2luc mice showed a significant reduction in total retinal thickness with respect to C57BL/6J mice. The reduction was mostly confined to the photoreceptor layer. Consistent with these results, we observed a significant decrease in the amplitude of a- and b-waves of the ERG in aged Per2luc mice. Analysis of the RPE morphology revealed that in aged Per2luc mice there was an increase in compactness and eccentricity with a decrease in solidity with respect to the values observed in WT, pointing toward signs of aging in the RPE of Per2luc mice. Our data demonstrate that homozygous Per2luc mice show photoreceptor degeneration during aging and a premature aging of the RPE.

Circadian rhythms and obesity: Timekeeping governs lipid metabolism.

Almost all living organisms have evolved autoregulatory transcriptional-translational feedback loops that produce oscillations with a period of approximately 24-h. These endogenous time keeping mechanisms are called circadian clocks. The main function of these circadian clocks is to drive overt circadian rhythms in the physiology of the organisms to ensure that main physiological functions are in synchrony with the external environment. Disruption of circadian rhythms caused by genetic or environmental factors has long-term consequences for metabolic health. Of relevance, host circadian rhythmicity and lipid metabolism are increasingly recognized to cross-regulate and the circadian clock-lipid metabolism interplay may involve in the development of obesity. Multiple systemic and molecular mechanisms, such as hormones (ie, melatonin, leptin, and glucocorticoid), the gut microbiome, and energy metabolism, link the circadian clock and lipid metabolism, and predictably, the deregulation of circadian clock-lipid metabolism interplay can increase the risk of obesity, which in turn may exacerbate circadian disorganization. Feeding time and dietary nutrients are two of key environmental Zeitgebers affecting the circadian rhythm-lipid metabolism interplay, and the influencing mechanisms in obesity development are highlighted in this review. Together, the characterization of the clock machinery in lipid metabolism aimed at producing a healthy circadian lifestyle may improve obesity care.

Dopamine 2 Receptor Signaling Controls the Daily Burst in Phagocytic Activity in the Mouse Retinal Pigment Epithelium.

Purpose: A burst in phagocytosis of spent photoreceptor outer fragments by RPE is a rhythmic process occurring 1 to 2 hours after the onset of light. This phenomenon is considered crucial for the health of the photoreceptors and RPE. We have recently reported that dopamine, via dopamine 2 receptor (D2R), shifts the circadian rhythm in the RPE. Methods: Here, we first investigated the impact of the removal of D2R on the daily peak of phagocytosis by RPE and then we analyzed the function and morphology of retina and RPE in the absence of D2R. Results: D2R knockout (KO) mice do not show a daily burst of phagocytic activity after the onset of light. RNA sequencing revealed a total of 394 differentially expressed genes (DEGs) between ZT 23 and ZT 1 in the control mice, whereas in D2R KO mice, we detected 1054 DEGs. Pathway analysis of the gene expression data implicated integrin signaling to be one of the upregulated pathways in control but not in D2R KO mice. Consistent with the gene expression data, phosphorylation of focal adhesion kinase (FAK) did not increase significantly in KO mice at ZT 1. No difference in retinal thickness, visual function, or morphology of RPE cells was observed between wild-type (WT) and D2R KO mice at the age of 3 and 12 months. Conclusions: Our data suggest that removal of D2R prevents the burst of phagocytosis and a related increase in the phosphorylation of FAK after light onset. The pathway analysis points toward a putative role of D2R in controlling integrin signaling, which is known to play an important role in the control of the daily burst of phagocytosis by the RPE. Our data also indicate that the absence of the burst of phagocytic activity in the early morning does not produce any apparent deleterious effect on the retina or RPE up to 1 year of age.

Administration of Exogenous Melatonin Improves the Diurnal Rhythms of the Gut Microbiota in Mice Fed a High-Fat Diet.

Melatonin, a circadian hormone, has been reported to improve host lipid metabolism by reprogramming the gut microbiota, which also exhibits rhythmicity in a light/dark cycle. However, the effect of the administration of exogenous melatonin on the diurnal variation in the gut microbiota in mice fed a high-fat diet (HFD) is unclear. Here, we further confirmed the antiobesogenic effect of melatonin on mice fed an HFD for 2 weeks. Samples were collected every 4 h within a 24-h period, and diurnal rhythms of clock gene expression (Clock, Cry1, Cry2, Per1, and Per2) and serum lipid indexes varied with diurnal time. Notably, Clock and triglycerides (TG) showed a marked rhythm in the control in melatonin-treated mice but not in the HFD-fed mice. The rhythmicity of these parameters was similar between the control and melatonin-treated HFD-fed mice compared with that in the HFD group, indicating an improvement caused by melatonin in the diurnal clock of host metabolism in HFD-fed mice. Moreover, 16S rRNA gene sequencing showed that most microbes exhibited daily rhythmicity, and the trends were different for different groups and at different time points. We also identified several specific microbes that correlated with the circadian clock genes and serum lipid indexes, which might indicate the potential mechanism of action of melatonin in HFD-fed mice. In addition, effects of melatonin exposure during daytime or nighttime were compared, but a nonsignificant difference was noticed in response to HFD-induced lipid dysmetabolism. Interestingly, the responses of microbiota-transplanted mice to HFD feeding also varied at different transplantation times (8:00 and 16:00) and with different microbiota donors. In summary, the daily oscillations in the expression of circadian clock genes, serum lipid indexes, and the gut microbiota appeared to be driven by short-term feeding of an HFD, while administration of exogenous melatonin improved the composition and diurnal rhythmicity of some specific gut microbiota in HFD-fed mice.IMPORTANCE The gut microbiota is strongly shaped by a high-fat diet, and obese humans and animals are characterized by low gut microbial diversity and impaired gut microbiota compositions. Comprehensive data on mammalian gut metagenomes shows gut microbiota exhibit circadian rhythms, which is disturbed by a high-fat diet. On the other hand, melatonin is a natural and ubiquitous molecule showing multiple mechanisms of regulating the circadian clock and lipid metabolism, while the role of melatonin in the regulation of the diurnal patterns of gut microbial structure and function in obese animals is not yet known. This study delineates an intricate picture of melatonin-gut microbiota circadian rhythms and may provide insight for obesity intervention.

Circadian analysis of the mouse retinal pigment epithelium transcriptome.

The presence of a phagocytic peak of photoreceptor outer segments by the retinal pigment epithelium (RPE) one or 2 h after the onset of light has been reported for several diurnal and nocturnal species. This peak in phagocytic activity also persists under constant lighting conditions (i.e., constant light or dark) thus demonstrating that the timing of this peak is driven by a circadian clock. The aim of this study was to investigate the change in RPE whole transcriptome at two different circadian times (CT; 1 h before (CT23) and 1 h after (CT1) subjective light onset). C57BL/6J male mice were maintained in constant dark conditions for three days and euthanized under red light (<1 lux) at CT23 and CT1. RPE was isolated from whole eyes for RNA library preparation and sequencing on an Illumina HiSeq4000 platform. 14,083 mouse RPE transcripts were detected in common between CT23 and CT1. 12,005 were protein coding transcripts and 2078 were non-protein coding transcripts. 2421 protein coding transcripts were significantly upregulated whereas only 3 transcripts were significantly downregulated and 12 non-protein coding transcripts were significantly upregulated and 31 non-protein coding transcripts were significantly downregulated at CT1 when compared to CT23 (p < 0.05, fold change ≥ ±2.0). Of the protein coding transcripts, most of them were characterized as: enzymes, kinases, and transcriptional regulators with a large majority of activity in the cytoplasm, nucleus, and plasma membrane. Non-protein coding transcripts included biotypes such as long-non coding RNAs and pseudogenes. Gene ontology analysis and ingenuity pathway analysis revealed that differentially expressed transcripts were associated with integrin signaling, oxidative phosphorylation, protein phosphorylation, and actin cytoskeleton remodeling suggesting that these previously identified phagocytic pathways are under circadian control. Our analysis identified new pathways (e.g., increased mitochondrial respiration via increased oxidative phosphorylation) that may be involved in the circadian control of phagocytic activity. In addition, our dataset suggests a possible regulatory role for the identified non-protein coding transcripts in mediating the complex function of RPE phagocytosis. Finally, our results also indicate, as seen in other tissues, about 20% of the whole RPE transcriptome may be under circadian clock regulation.

Melatonin receptor heterodimerization in a photoreceptor-like cell line endogenously expressing melatonin receptors.

Purpose: Melatonin signaling plays an important role in the modulation of retinal physiology and photoreceptor viability during aging. In this study, we investigated whether 661W cells-a photoreceptor-like cell that endogenously expresses melatonin receptor type 1 (MT1) and melatonin receptor type 2 (MT2) receptors-represent a useful model for studying the biology of heterodimerization and signaling of MT1/2 receptors. Methods: 661W cells were cultured, and MT1/MT2 heterodimerization in 661W cells was assessed with proximity ligation assay. MT2 was removed from the 661W cells using the MT2-CRISPR/Cas9 system. Melatonin receptor signaling was investigated by measuring cAMP levels and activation of the AKT-FoxO1 pathway. Results: The results demonstrated that heterodimerization of MT1 and MT2 receptors occurs in 661W cells. The pathways activated by MT1/MT2 heterodimer (MT1/2h) in 661W cells are similar to those previously reported in mouse photoreceptors. Disruption of the heterodimer formation by genetically ablating MT2 from 661W cells abolished the activation of melatonin signaling in these cells. Conclusions: The data indicated that in 661W cells, MT1 and MT2 receptors are functional only when they are associated in a heteromeric complex, as occurs in mouse photoreceptors. 661W cells represent a useful model for studying the mechanism underlying MT1/MT2 heterodimerization.

Melatonin Signaling a Key Regulator of Glucose Homeostasis and Energy Metabolism.

Melatonin, a hormone synthesized by both the pineal gland and retina, functions as an important modulator of a number of physiological functions. In addition to its rather well-established roles in the regulation of circadian rhythms, sleep, and reproduction, melatonin has also been identified as an important regulator of glucose metabolism. Recent genomic studies have also shown that disruption of melatonin receptors signaling may contribute to the pathogenesis of type 2 diabetes, although the exact mechanisms underlying its action remain unclear. Additionally, a large number of animal studies have highlighted a role for melatonin in the regulation of both glucose metabolism and energy balance. This review summarizes the current knowledge on the role that melatonin and its associated receptors play in the regulation of metabolism.

Retinal Circadian Clocks are Major Players in the Modulation of Retinal Functions and Photoreceptor Viability.

Circadian rhythms control many biochemical and physiological functions within the body of an organism. These circadian rhythms are generated by a molecular clock that is located in almost every cell of the body. Accumulating data indicate that dysfunction of the circadian clock negatively affects the health status of the tissue in which the circadian clock has been disabled. The eye also contains a complex circadian system that regulates many important functions such as the processing of light information, the release of neurotransmitters, and phagocytic activity by the retinal pigment epithelium, to name just a few. Emerging experimental evidence indicates that dysfunction of the circadian clock within the retina has severe consequence for retinal function and photoreceptor viability. The aim of this review is to provide the reader with a summary of current knowledge about the eye circadian system and what effects emerge with a disruption of this system.

Removing melatonin receptor type 1 signaling leads to selective leptin resistance in the arcuate nucleus.

Recent studies have highlighted the involvement of melatonin in the regulation of energy homeostasis. In this study, we report that mice lacking melatonin receptor 1 (MT1 KO) gained more weight, had a higher cumulative food intake, and were more hyperphagic after fasting compared to controls (WT). In response to a leptin injection, MT1 KO mice showed a diminished reduction in body weight and food intake. To evaluate hypothalamic leptin signaling, we tested leptin-induced phosphorylation of the signal transducer and activator of transcription 3 (STAT3). Leptin failed to induce STAT3 phosphorylation in MT1 KO mice beyond levels observed in mice injected with phosphate-buffered saline (PBS). Furthermore, STAT3 phosphorylation within the arcuate nucleus (ARH) was decreased in MT1 KO mice. Leptin receptor mRNA levels in the hypothalamus of MT1 KO were significantly reduced (about 50%) compared to WT. This study shows that: (a) MT1 deficiency causes weight gain and increased food intake; (b) a lack of MT1 signaling induces leptin resistance; (c) leptin resistance is ARH region-specific; and (d) leptin resistance is likely due to down-regulation of the leptin receptor. Our data demonstrate that MT1 signaling is an important modulator of leptin signaling.