Antidepressant mechanism of traditional Chinese medicine: Involving regulation of circadian clock genes.

Numerous studies have demonstrated an intimate relationship between circadian rhythm disorders and the development and prevention of depression. The biological clock genes, which constitute the molecular basis of endogenous circadian rhythms, hold promising prospects for depression treatment. Based on an extensive review of recent domestic and international research, this article presents a comprehensive analysis of how traditional Chinese medicine (TCM) intervenes in depression by regulating circadian rhythms. The findings indicate that TCM exerts its antidepressant effects by targeting specific biological clock genes such as Bmal1, clock, Arntl, Per1, Per2, Per3, Nr1d1, Cry2, and Dbp, as well as regulating circadian rhythms of hormone secretion. However, most current research is still confined to basic experimental studies, lacking clinical double-blind control trials to further validate these viewpoints. Furthermore, there is insufficient research on the signal transduction pathway between biological clock genes and pathological changes in depression. Additionally, further clarification is needed regarding the specific targets of TCM on the biological clock genes.

Intense light-elicited alveolar type 2-specific circadian PER2 protects from bacterial lung injury via BPIFB1.

Circadian amplitude enhancement has the potential to be organ protective but has not been studied in acute lung injury (ALI). Consistent light and dark cycles are crucial for the amplitude regulation of the circadian rhythm protein Period2 (PER2). Housing mice under intense instead of ambient light for 1 wk (light: dark cycle:14h:10h), we demonstrated a robust increase of pulmonary PER2 trough and peak levels, which is consistent with circadian amplitude enhancement. A search for the affected lung cell type suggested alveolar type 2 (ATII) cells as strong candidates for light induction of PER2. A head-to-head comparison of mice with cell-type-specific deletion of Per2 in ATII, endothelial, or myeloid cells uncovered a dramatic phenotype in mice with an ATII-specific deletion of Per2. During Pseudomonas aeruginosa-induced ALI, mice with Per2 deletion in ATII cells showed 0% survival, whereas 85% of control mice survived. Subsequent studies demonstrated that intense light therapy dampened lung inflammation or improved the alveolar barrier function during P. aeruginosa-induced ALI, which was abolished in mice with an ATII-specific deletion of Per2. A genome-wide mRNA array uncovered bactericidal/permeability-increasing fold-containing family B member 1 (BPIFB1) as a downstream target of intense light-elicited ATII-PER2 mediated lung protection. Using the flavonoid and PER2 amplitude enhancer nobiletin, we recapitulated the lung-protective and anti-inflammatory effects of light and BPIFB1, respectively. Together, our studies demonstrate that light-elicited amplitude enhancement of ATII-specific PER2 is a critical control point of inflammatory pathways during bacterial ALI.

Period circadian regulator 2 suppresses drug resistance to cisplatin by PI3K/AKT pathway and improves chronochemotherapeutic efficacy in cervical cancer.

OBJECTIVE: Chronotherapy, a promising therapy, may build up the chemotherapy efficacy through thinking about timing of therapy. Here, we observed the roles of period circadian regulator 2 (PER2) on cervical cancer progression and the therapeutic efficacy of cisplatin (DDP) based on the circadian rhythm of PER2. METHODS: When Hela/DDP and SiHa/DDP transfected with pcDNA3.1-PER2 and/or treated with human epidermal growth factor (hEGF), viability, apoptosis, migration, and nuclear translocation of NF-κB p65 were detected by CCK-8, flow cytometry, transwell, immunofluorescence and western blot. Furthermore, the expression of circadian rhythm regulators, multidrug resistance, and epithelial-mesenchymal transition (EMT) proteins was detected by western blot. Hela/DDP cells-induced tumor formation in nude mice was constructed. The expression of PER2 was measured at different time point by RT-qPCR. Cisplatin was separately injected into mice with cervical cancer at the highest and lowest expression of PER2. After 5 weeks, tumor volume was measured and tumor proliferation was assessed by immunohistochemistry. RESULTS: Overexpression of PER2 significantly reduced proliferative and migrated capacities and nuclear translocation of NF-κB p65 as well as enhanced apoptosis in Hela/DDP and SiHa/DDP cells. Meanwhile, its overexpression elevated the expression of circadian rhythm regulators as well as lowered the expression of multidrug resistance proteins and EMT pathway activation by suppressing PI3K/AKT pathway. PER2 was rhythmically expressed in cervical cancer tissues. Compared to cisplatin treatment at the lowest expression of PER2, tumor growth and proliferation of tumor cells were distinctly suppressed in mice treated with cisplatin at the highest expression of PER2. CONCLUSION: Our findings confirmed the circadian rhythm of PER2 in cervical cancer and its overexpression restrained the resistance to cisplatin in cervical cancer by PI3K/AKT pathway. It may improve cisplatin efficacy through considering the circadian rhythm of PER2.

Light affects behavioral despair involving the clock gene Period 1.

Light at night has strong effects on physiology and behavior of mammals. It affects mood in humans, which is exploited as light therapy, and has been shown to reset the circadian clock in the suprachiasmatic nuclei (SCN). This resetting is paramount to align physiological and biochemical timing to the environmental light-dark cycle. Here we provide evidence that light at zeitgeber time (ZT) 22 affects mood-related behaviors also in mice by activating the clock gene Period1 (Per1) in the lateral habenula (LHb), a brain region known to modulate mood-related behaviors. We show that complete deletion of Per1 in mice led to depressive-like behavior and loss of the beneficial effects of light on this behavior. In contrast, specific deletion of Per1 in the region of the LHb did not affect mood-related behavior, but suppressed the beneficial effects of light. RNA sequence analysis in the mesolimbic dopaminergic system revealed profound changes of gene expression after a light pulse at ZT22. In the nucleus accumbens (NAc), sensory perception of smell and G-protein coupled receptor signaling were affected the most. Interestingly, most of these genes were not affected in Per1 knock-out animals, indicating that induction of Per1 by light serves as a filter for light-mediated gene expression in the brain. Taken together we show that light affects mood-related behavior in mice at least in part via induction of Per1 in the LHb with consequences on mood-related behavior and signaling mechanisms in the mesolimbic dopaminergic system.

GABAergic mechanisms in the suprachiasmatic nucleus that influence circadian rhythm.

The mammalian central circadian clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN contains multiple circadian oscillators which synchronize with each other via several neurotransmitters. Importantly, an inhibitory neurotransmitter, γ-amino butyric acid (GABA), is expressed in almost all SCN neurons. In this review, we discuss how GABA influences circadian rhythms in the SCN. Excitatory and inhibitory effects of GABA may depend on intracellular Cl- concentration, in which several factors such as day-length, time of day, development, and region in the SCN may be involved. GABA also mediates oscillatory coupling of the circadian rhythms in the SCN. Recent genetic approaches reveal that GABA refines circadian output rhythms, but not circadian oscillations in the SCN. Since several efferent projections of the SCN have been suggested, GABA might work downstream of neuronal pathways from the SCN which regulate the temporal order of physiology and behavior.

Modulation of cellular circadian clocks by triterpenoids.

Many living organisms on earth have clock systems in their body. It has increasingly become clear that a disturbance in the internal clocks has negative effects on our body. Terpenes are organic compounds found in various plants that are reported to have several pharmacological actions. In this study, we focused on commercially available 27 triterpenoids and evaluated their influence on the circadian rhythm of human U2OS cells and mouse NIH3T3 cells. The expression level of Per2, one of the core clock genes, was measured using luminescent reporters over the time period of a few days. We found that 8 triterpenoids reset the phase of the circadian clocks. Representative compounds were corosolic acid, cucurbitacin B, and celastrol; similar effects were also confirmed with some structural analogues of cucurbitacin B and celastrol. These compounds shifted the phase bilaterally depending on the stimulus timing and also acted as synchronizers in desynchronized cells. The effective concentrations of cucurbitacin B and celastrol were less than 0.5 µM. In addition, cucurbitacin B and celastrol were also found to be effective in tissue explants in mice. Furthermore, celastrol dose-dependently shortened the period length of NIH3T3 cells. Some of these compounds are found in edible and medicinal plants and may help regulate our circadian clocks in everyday life.

Intense light as anticoagulant therapy in humans.

Blood coagulation is central to myocardial ischemia and reperfusion (IR) injury. Studies on the light elicited circadian rhythm protein Period 2 (PER2) using whole body Per2-/- mice found deficient platelet function and reduced clotting which would be expected to protect from myocardial IR-injury. In contrast, intense light induction of PER2 protected from myocardial IR-injury while Per2 deficiency was detrimental. Based on these conflicting data, we sought to evaluate the role of platelet specific PER2 in coagulation and myocardial ischemia and reperfusion injury. We demonstrated that platelets from mice with tissue-specific deletion of Per2 in the megakaryocyte lineage (Per2loxP/loxP-PF4-CRE) significantly clot faster than platelets from control mice. We further found increases in infarct sizes or plasma troponin levels in Per2loxP/loxP-PF4-CRE mice when compared to controls. As intense light increases PER2 protein in human tissues, we also performed translational studies and tested the effects of intense light therapy on coagulation in healthy human subjects. Our human studies revealed that intense light therapy repressed procoagulant pathways in human plasma samples and significantly reduced the clot rate. Based on these results we conclude that intense light elicited PER2 has an inhibitory function on platelet aggregation in mice. Further, we suggest intense light as a novel therapy to prevent or treat clotting in a clinical setting.

Circadian Regulation of IOP Rhythm by Dual Pathways of Glucocorticoids and the Sympathetic Nervous System.

Purpose: Elevated IOP can cause the development of glaucoma. The circadian rhythm of IOP depends on the dynamics of the aqueous humor and is synchronized with the circadian rhythm pacemaker, that is, the suprachiasmatic nucleus. The suprachiasmatic nucleus resets peripheral clocks via sympathetic nerves or adrenal glucocorticoids. However, the detailed mechanisms underlying IOP rhythmicity remain unclear. The purpose of this study was to verify this regulatory pathway. Methods: Adrenalectomy and/or superior cervical ganglionectomy were performed in C57BL/6J mice. Their IOP rhythms were measured under light/dark cycle and constant dark conditions. Ocular administration of corticosterone or norepinephrine was also performed. Localization of adrenergic receptors, glucocorticoid receptors, and clock proteins Bmal1 and Per1 were analyzed using immunohistochemistry. Period2::luciferase rhythms in the cultured iris/ciliary bodies of adrenalectomized and/or superior cervical ganglionectomized mice were monitored to evaluate the effect of the procedures on the local clock. The IOP rhythm of retina and ciliary epithelium-specific Bmal1 knockout mice were measured to determine the significance of the local clock. Results: Adrenalectomy and superior cervical ganglionectomy disrupted IOP rhythms and the circadian clock in the iris/ciliary body cultures. Instillation of corticosterone and norepinephrine restored the IOP rhythm. ß2-Adrenergic receptors, glucocorticoid receptors, and clock proteins were strongly expressed within the nonpigmented epithelia of the ciliary body. However, tissue-specific Bmal1 knock-out mice maintained their IOP rhythm. Conclusions: These findings suggest direct driving of the IOP rhythm by the suprachiasmatic nucleus, via the dual corticosterone and norepinephrine pathway, but not the ciliary clock, which may be useful for chronotherapy of glaucoma.

Abnormal Photic Entrainment to Phase-Delaying Stimuli in the R6/2 Mouse Model of Huntington's Disease, despite Retinal Responsiveness to Light.

The circadian clock located in the suprachiasmatic nucleus (SCN) in mammals entrains to ambient light via the retinal photoreceptors. This allows behavioral rhythms to change in synchrony with seasonal and daily changes in light period. Circadian rhythmicity is progressively disrupted in Huntington's disease (HD) and in HD mouse models such as the transgenic R6/2 line. Although retinal afferent inputs to the SCN are disrupted in R6/2 mice at late stages, they can respond to changes in light/dark cycles, as seen in jet lag and 23 h/d paradigms. To investigate photic entrainment and SCN function in R6/2 mice at different stages of disease, we first assessed the effect on locomotor activity of exposure to a 15 min light pulse given at different times of the day. We then placed the mice under five non-standard light conditions. These were light cycle regimes (T-cycles) of T21 (10.5 h light/dark), T22 (11 h light/dark), T26 (13 h light/dark), constant light, or constant dark. We found a progressive impairment in photic synchronization in R6/2 mice when the stimuli required the SCN to lengthen rhythms (phase-delaying light pulse, T26, or constant light), but normal synchronization to stimuli that required the SCN to shorten rhythms (phase-advancing light pulse and T22). Despite the behavioral abnormalities, we found that Per1 and c-fos gene expression remained photo-inducible in SCN of R6/2 mice. Both the endogenous drift of the R6/2 mouse SCN to shorter periods and its inability to adapt to phase-delaying changes will contribute to the HD circadian dysfunction.

Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium.

Consistent daylight oscillations and abundant oxygen availability are fundamental to human health. Here, we investigate the intersection between light-sensing (Period 2 [PER2]) and oxygen-sensing (hypoxia-inducible factor [HIF1A]) pathways in cellular adaptation to myocardial ischemia. We demonstrate that intense light is cardioprotective via circadian PER2 amplitude enhancement, mimicking hypoxia-elicited adenosine- and HIF1A-metabolic adaptation to myocardial ischemia under normoxic conditions. Whole-genome array from intense light-exposed wild-type or Per2-/- mice and myocardial ischemia in endothelial-specific PER2-deficient mice uncover a critical role for intense light in maintaining endothelial barrier function via light-enhanced HIF1A transcription. A proteomics screen in human endothelia reveals a dominant role for PER2 in metabolic reprogramming to hypoxia via mitochondrial translocation, tricarboxylic acid (TCA) cycle enzyme activity regulation, and HIF1A transcriptional adaption to hypoxia. Translational investigation of intense light in human subjects identifies similar PER2 mechanisms, implicating the use of intense light for the treatment of cardiovascular disease.

Persistent Changes in Stress-Regulatory Genes in Pregnant Women or Children Exposed Prenatally to Alcohol.

BACKGROUND: We have recently shown that binge or heavy levels of alcohol drinking increase deoxyribonucleic acid (DNA) methylation and reduce gene expression of proopiomelanocortin (POMC) and period 2 (PER2) in adult human subjects (Gangisetty et al., Alcohol Clin Exp Res, 43, 2019, 212). One hypothesis would be that methylation of these 2 genes is consistently associated with alcohol exposure and could be used as biomarkers to predict risk of prenatal alcohol exposure (PAE). Results of the present study provided some support for this hypothesis. METHODS: We conducted a series of studies to determine DNA methylation changes in stress regulatory genes proopiomelanocortin (POMC) and period 2 (PER2) using biological samples from 3 separate cohorts of patients: (i) pregnant women who consumed moderate-to-high levels of alcohol or low/unexposed controls, (ii) children with PAE and non-alcohol-exposed controls, and (iii) children with PAE treated with or without choline. RESULTS: We found pregnant women who consumed moderate-to-high levels of alcohol and gave birth to PAE children had higher DNA methylation of POMC and PER2. PAE children also had increased methylation of POMC and PER2. The differences in the gene methylation of PER2 and POMC between PAE and controls did not differ by maternal smoking status. PAE children had increased levels of stress hormone cortisol and adrenocorticotropic hormone. Choline supplementation reduced DNA hypermethylation and increased expression of POMC and PER2 in children with PAE. CONCLUSIONS: These data suggest that PAE significantly elevates DNA methylation of POMC and PER2 and increases levels of stress hormones. Furthermore, these results suggest the possibility that measuring DNA methylation levels of PER2 and POMC in biological samples from pregnant women or from children may be useful for identification of a woman or a child with PAE.

Systems approach reveals photosensitivity and PER2 level as determinants of clock-modulator efficacy.

In mammals, the master circadian clock synchronizes daily rhythms of physiology and behavior with the day-night cycle. Failure of synchrony, which increases the risk for numerous chronic diseases, can be treated by phase adjustment of the circadian clock pharmacologically, for example, with melatonin, or a CK1δ/ε inhibitor. Here, using in silico experiments with a systems pharmacology model describing molecular interactions, and pharmacokinetic and behavioral experiments in cynomolgus monkeys, we find that the circadian phase delay caused by CK1δ/ε inhibition is more strongly attenuated by light in diurnal monkeys and humans than in nocturnal mice, which are common preclinical models. Furthermore, the effect of CK1δ/ε inhibition strongly depends on endogenous PER2 protein levels, which differs depending on both the molecular cause of the circadian disruption and the patient's lighting environment. To circumvent such large interindividual variations, we developed an adaptive chronotherapeutics to identify precise dosing regimens that could restore normal circadian phase under different conditions. Our results reveal the importance of photosensitivity in the clinical efficacy of clock-modulating drugs, and enable precision medicine for circadian disruption.

Effects of 50 Hz magnetic fields on circadian rhythm control in mice.

Artificial light and power frequency magnetic fields are ubiquitous in the built environment. Light is a potent zeitgeber but it is unclear whether power frequency magnetic fields can influence circadian rhythm control. To study this possibility, 8-12-week-old male C57BL/6J mice were exposed for 30 min starting at zeitgeber time 14 (ZT14, 2 h into the dark period of the day) to 50 Hz magnetic fields at 580 µT using a pair of Helmholtz coils and/or a blue LED light at 700 lux or neither. Our experiments revealed an acute adrenal response to blue light, in terms of increased adrenal per1 gene expression, increased serum corticosterone levels, increased time spent sleeping, and decreased locomotor activity (in all cases, P < 0.0001) compared to an unexposed control group. There appeared to be no modulating effect of the magnetic fields on the response to light, and there was also no effect of the magnetic fields alone (in both cases, P > 0.05) except for a decrease in locomotor activity (P < 0.03). Gene expression of the cryptochromes cry1 and cry2 in the adrenals, liver, and hippocampus was also not affected by exposures (in all cases, P > 0.05). In conclusion, these results suggest that 50 Hz magnetic fields do not significantly affect the acute light response to a degree that can be detected in the adrenal response. Bioelectromagnetics. 2019;9999:XX-XX. © 2019 Bioelectromagnetics Society.

Analyses of BMAL1 and PER2 Oscillations in a Model of Breast Cancer Progression Reveal Changes With Malignancy.

From an epidemiological standpoint, disruptions to circadian rhythms have been shown to contribute to the development of various disease pathologies, including breast cancer. However, it is unclear how altered circadian rhythms are related to malignant transformations at the molecular level. In this article, a series of isogenic breast cancer cells representing disease progression was used to investigate the expression patterns of core circadian clock proteins BMAL1 and PER2. Our model is indicative of 4 stages of breast cancer and includes the following cells: MCF10A (non-malignant), MCF10AT.Cl2 (pre-malignant), MCF10Ca1h (well-differentiated, malignant), and MCF10Ca1a (poorly differentiated, malignant). While studies of circadian rhythms in cancer typically use low-resolution reverse transcription polymerase chain reaction assays, we also employed luciferase reporters BMAL1:Luc and PER2:Luc in real-time luminometry experiments. We found that across all 4 cancer stages, PER2 showed relatively stable oscillations compared with BMAL1. Period estimation using both wavelet-based and damped-sine-fitting methods showed that the periods are distributed over a wide circadian range and there is no clear progression in mean period as cancer severity progresses. Additionally, we used the K-nearest neighbors algorithm to classify the recordings according to cancer line, and found that cancer stages were largely differentiated from one another. Taken together, our data support that there are circadian discrepancies between normal and malignant cells, but it is difficult and insufficient to singularly use period evaluations to differentiate them. Future studies should employ other progressive disease models to determine whether these findings are representative across cancer types or are specific to this series.

Therapeutic effects of curcumin on age-induced alterations in daily rhythms of clock genes and Sirt1 expression in the SCN of male Wistar rats.

The aging brain is linked to accumulation of oxidative stress and increase in damage to biomolecules which in turn may cause or promote circadian dysfunction by disruption of biological clock, the suprachiasmatic nucleus (SCN). Age associated alterations in clock gene expression in the SCN has been reported earlier. In the present study we have examined therapeutic effects of the antioxidant curcumin on age induced alterations in daily rhythms and levels of core clock genes in SCN of young [3 months (m)], middle (12 months) and old (24 months) male Wistar rats. Curcumin was administered orally at ZT-11, 1 hour (h) before the onset of darkness. The effect of curcumin administration on daily rhythms and levels of expression of clock genes such as rBmal1, rPer1, rPer2, rCry1, rCry2 and rRev-erbα as well as on the clock modulator rSirt1 were studied. There was restoration of phase of rPer1, rPer2, rCry1, rCry2 and daily pulse of rPer2 in middle aged animals. However, in old aged rats the phase and daily pulse of rPer1 were restored with curcumin treatment. rSirt1 did not show age related alterations in its transcript levels though the rhythms were abolished in old aged rat SCN. Pearson correlation analysis showed that curcumin administration to 12 and 24 months animals had resulted in restorations of several correlations among clock genes which were found to be altered/abolished in age matched control groups. In addition, strong interlocking interactions between rSirt1 and clock genes were observed in young age which were disrupted with aging and curcumin administration resulted in partial restoration.

Hypermethylation of Proopiomelanocortin and Period 2 Genes in Blood Are Associated with Greater Subjective and Behavioral Motivation for Alcohol in Humans.

BACKGROUND: Epigenetic modifications of a gene have been shown to play a role in maintaining a long-lasting change in gene expression. We hypothesize that alcohol's modulating effect on DNA methylation on certain genes in blood is evident in binge and heavy alcohol drinkers and is associated with alcohol motivation. METHODS: Methylation-specific polymerase chain reaction (PCR) assays were used to measure changes in gene methylation of period 2 (PER2) and proopiomelanocortin (POMC) genes in peripheral blood samples collected from nonsmoking moderate, nonbinging, binge, and heavy social drinkers who participated in a 3-day behavioral alcohol motivation experiment of imagery exposure to either stress, neutral, or alcohol-related cues, 1 per day, presented on consecutive days in counterbalanced order. Following imagery exposure on each day, subjects were exposed to discrete alcoholic beer cues followed by an alcohol taste test (ATT) to assess behavioral motivation. Quantitative real-time PCR was used to measure gene expression of PER2 and POMC gene levels in blood samples across samples. RESULTS: In the sample of moderate, binge, and heavy drinkers, we found increased methylation of the PER2 and POMC DNA, reduced expression of these genes in the blood samples of the binge and heavy drinkers relative to the moderate, nonbinge drinkers. Increased PER2 and POMC DNA methylation was also significantly predictive of both increased levels of subjective alcohol craving immediately following imagery (p < 0.0001), and with presentation of the alcohol (2 beers) (p < 0.0001) prior to the ATT, as well as with alcohol amount consumed during the ATT (p < 0.003). CONCLUSIONS: These data establish significant association between binge or heavy levels of alcohol drinking and elevated levels of methylation and reduced levels of expression of POMC and PER2 genes. Furthermore, elevated methylation of POMC and PER2 genes is associated with greater subjective and behavioral motivation for alcohol.

Bisphenol A Alters Bmal1, Per2, and Rev-Erba mRNA and Requires Bmal1 to Increase Neuropeptide Y Expression in Hypothalamic Neurons.

Bisphenol A (BPA), a ubiquitous environmental endocrine disruptor, is considered an obesogen. However, its role in the hypothalamic control of energy balance remains largely unexplored. Because disruption of the circadian clock is tightly associated with metabolic consequences, we explored how BPA affects the components of the molecular circadian clock in the feeding-related neurons of the hypothalamus. In immortalized POMC and NPY/AgRP-expressing hypothalamic cell lines and primary culture, we describe how BPA significantly alters mRNA expression of circadian clock genes Bmal1,Per2, and Rev-Erbα. Furthermore, we use newly generated Bmal1-knockout (KO) hypothalamic cell lines to link the BPA-induced neuropeptide dysregulation to the molecular clock. Specifically, BPA increased Npy, Agrp, and Pomc mRNA expression in wild type hypothalamic cells, whereas the increase in Npy, but not Agrp or Pomc, was abolished in cell lines lacking BMAL1. In line with this increase, BPA led to increased BMAL1 binding to the Npy promotor, potentially increasing Npy transcription. In conclusion, we show that BPA-mediated dysregulation of the circadian molecular clock is linked to the deleterious effects of BPA on neuropeptide expression. Furthermore, we describe hypothalamic Bmal1-KO cell lines to study the role of BMAL1 in hypothalamic responses to metabolic, hormonal, and environmental factors.

Time-Dependent Hypotensive Effect of Aspirin in Mice.

Objective- Evening but not morning administration of low-dose aspirin has been reported to lower blood pressure in hypertensive patients. The present study was designed to determine whether this phenomenon could be replicated in mice, and if so, whether a time-dependent effect of aspirin on blood pressure was because of alteration of circadian clock function. Approach and Results- We recapitulated the protective effect of aspirin (50 µg/d for 7 days) at zeitgeber time 0 (active-to-rest transit), but not at zeitgeber time 12, on a high-salt diet-induced increase of blood pressure. However, the time of aspirin administration did not influence expression of canonical clock genes or their acetylation. We used mouse Bmal1 and Per2-luciferase reporters expressed in U2OS cells to determine the real-time effect of aspirin on circadian function but found that the oscillation of bioluminescence was unaltered. Timing of aspirin administration also failed to alter urinary prostaglandin metabolites or catecholamines, or the acetylation of its COX-1 (cyclooxygenase-1) target in platelets. Conclusions- The time-dependent hypotensive effect of aspirin in humans has been recapitulated in hypertensive mice. However, this does not seem to reflect a direct impact of aspirin on circadian clocks or on acetylation of platelet COX-1.

Distinct control of PERIOD2 degradation and circadian rhythms by the oncoprotein and ubiquitin ligase MDM2.

The circadian clock relies on posttranslational modifications to set the timing for degradation of core regulatory components, which drives clock progression. Ubiquitin-modifying enzymes that target clock components for degradation mainly recognize phosphorylated substrates. Degradation of the circadian clock component PERIOD 2 (PER2) is mediated by its phospho-specific recognition by ß-transducin repeat-containing proteins (ß-TrCPs), which are F-box-containing proteins that function as substrate recognition subunits of the SCFß-TRCP ubiquitin ligase complex. However, this mode of regulating PER2 stability falls short of explaining the persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery. We identified PER2 as a previously uncharacterized substrate for the ubiquitin ligase mouse double minute 2 homolog (MDM2) and found that MDM2 targeted PER2 for degradation in a manner independent of PER2 phosphorylation. Deregulation of MDM2 plays a major role in oncogenesis by contributing to the accumulation of genomic and epigenomic alterations that favor tumor development. MDM2-mediated PER2 turnover was important for defining the circadian period length in mammalian cells, a finding that emphasizes the connection between the circadian clock and cancer. Our results not only broaden the range of specific substrates of MDM2 beyond the cell cycle to include circadian components but also identify a previously unknown regulator of the clock as a druggable node that is often found to be deregulated during tumorigenesis.

Gene expression profiling during hibernation in the European hamster.

Hibernation is an exceptional physiological response to a hostile environment, characterized by a seasonal period of torpor cycles involving dramatic reductions of body temperature and metabolism, and arousal back to normothermia. As the mechanisms regulating hibernation are still poorly understood, here we analysed the expression of genes involved in energy homeostasis, torpor regulation, and daily or seasonal timing using digital droplet PCR in various central and peripheral tissues sampled at different stages of torpor/arousal cycles in the European hamster. During torpor, the hypothalamus exhibited strongly down-regulated gene expression, suggesting that hypothalamic functions were reduced during this period of low metabolic activity. During both torpor and arousal, many structures (notably the brown adipose tissue) exhibited altered expression of deiodinases, potentially leading to reduced tissular triiodothyronine availability. During the arousal phase, all analysed tissues showed increased expression of the core clock genes Per1 and Per2. Overall, our data indicated that the hypothalamus and brown adipose tissue were the tissues most affected during the torpor/arousal cycle, and that clock genes may play critical roles in resetting the body's clocks at the beginning of the active period.