PER2 Research Articles

The Circadian Clock Is Sustained in the Thyroid Gland of VIP Receptor 2 Deficient Mice.

VIP/VPAC2-receptor signaling is crucial for functioning of the circadian clock in the suprachiasmatic nucleus (SCN) since the lack results in disrupted synchrony between SCN cells and altered locomotor activity, body temperature, hormone secretion and heart rhythm. Endocrine glands, including the thyroid, show daily oscillations in clock gene expression and hormone secretion, and SCN projections target neurosecretory hypothalamic thyroid-stimulating hormone (TSH)-releasing hormone cells. The aim of the study was to gain knowledge of mechanisms important for regulation of the thyroid clock by evaluating the impact of VIP/VPAC2-receptor signaling. Quantifications of mRNAs of three clock genes (Per1, Per2 and Bmal1) in thyroids of wild type (WT) and VPAC2-receptor deficient mice were done by qPCR. Tissues were taken every 4th h during 24-h 12:12 light-dark (LD) and constant darkness (DD) periods, both genders were used. PER1 immunoreactivity was visualized on sections of both WT and VPAC2 lacking mice during a LD cycle. Finally, TSH and the thyroid hormone T4 levels were measured in the sera by commercial ELISAs. During LD, rhythmic expression of all three mRNA was found in both the WT and knockout animals. In VPAC2-receptor knockout animals, the amplitudes were approximately halved compared to the ones in the WT mice. In the WT, Per1 mRNA peaked around “sunset”, Per2 mRNA followed with approximately 2 h, while Bmal1 mRNA was in antiphase with Per1. In the VPAC2 knockout mice, the phases of the mRNAs were advanced approximately 5 h compared to the WT. During DD, the phases of all the mRNAs were identical to the ones found during LD in both groups of mice. PER1 immunoreactivity was delayed compared to its mRNA and peaked during the night in follicular cells of both the thyroid and parathyroid glands in the WT animals. In WT animals, TSH was high around the transition to darkness compared to light-on, while T4 did not change during the 24 h cycle. In conclusion, sustained and identical rhythms (phases and amplitudes) of three clock genes were found in VPAC2 deficient mice during LD and DD suggesting high degree of independence of the thyroid clock from the master SCN clock.

Antisense-induced knockdown of cAMP response element-binding protein downregulates Per1 gene expression in the shell region of nucleus accumbens resulting in reduced alcohol consumption in mice.

We recently showed that circadian genes expressed in the shell region of nucleus accumbens (NAcSh) play a key role in alcohol consumption, though, the molecular mechanism of those effects is unclear. Because CREB-binding protein (CBP) promotes Per1 gene expression, we hypothesized that alcohol consumption would increase CBP expression in the NAcSh and antisense-induced knockdown of CBP would reduce Per1 expression and result in a reduction in alcohol consumption. To test our hypothesis, we performed two experiments. The Drinking-in-the-dark (DID) paradigm was used to evaluate alcohol consumption in male C57BL/6J mice. In Experiment 1 we examined the effects of alcohol consumption on CBP gene expression in the NAcSh. Control animals were exposed to, sucrose [10% (w/v) taste and calorie] and water (consummatory behavior). In Experiment 2 examined the effects of CBP gene silencing on the expression of the Per1 gene in the NAcSh and alcohol consumption in mice exposed to alcohol using the DID paradigm. CBP gene silencing was achieved by local infusion of two doses of either CBP antisense oligodeoxynucleotides (AS-ODNs; Antisense group) or nonsense ODNs (NS-ODNs; Nonsense group) bilaterally microinjected into the NAcSh within 24h before alcohol consumption on Day 4 of the DID paradigm. The microinfusion sites were verified by cresyl violet staining. Compared to sucrose, alcohol consumption, under the DID paradigm, significantly increased the expression of CBP in the NAcSh. Compared to Controls, bilateral infusion of CBP AS-ODNs significantly reduced the expression of Per1 in the NAcSh and alcohol consumption without affecting the amount of sucrose consumed. Our results suggest that CBP is an upstream regulator of Per1 expression in the NAcSh and may act via Per1 to modulate alcohol consumption.

Melatonin induces Nrf2-HO-1 reprogramming and corrections in hepatic core clock oscillations in Non-alcoholic fatty liver disease.

Melatonin pleiotropically regulates physiological events and has a putative regulatory role in the circadian clock desynchrony-mediated Non-alcoholic fatty liver disease (NAFLD). In this study, we investigated perturbations in the hepatic circadian clock gene, and Nrf2-HO-1 oscillations in conditions of high-fat high fructose (HFHF) diet and/or jet lag (JL)-mediated NAFLD. Melatonin treatment (100µM) to HepG2 cells led to an improvement in oscillatory pattern of clock genes (Clock, Bmal1, and Per) in oleic acid (OA)-induced circadian desynchrony, while Cry, Nrf2, and HO-1 remain oblivious of melatonin treatment that was also validated by circwave analysis. C57BL/6J mice subjected to HFHF and/or JL, and treated with melatonin showed an improvement in the profile of lipid regulatory genes (CPT-1, PPARa, and SREBP-1c), liver function (AST and ALT) and histomorphology of fatty liver. A detailed scrutiny revealed that hepatic mRNA and protein profiles of Bmal1 (at ZT6) and Clock (at ZT12) underwent corrective changes in oscillations, but moderate corrections were recorded in other components of clock genes (Per1, Per2, and Cry2). Melatonin induced changes in oscillations of anti-oxidant genes (Nrf2, HO-1, and Keap1) subtly contributed in the overall improvement in NAFLD recorded herein. Taken together, melatonin induced reprograming of hepatic core clock and Nrf2-HO-1 genes leads to an improvement in HFHF/JL-induced NAFLD.

Variants in clock genes could be associated with lower risk of type 2 diabetes in an elderly Greek population

Objectives: Recent evidence has linked circadian rhythm dysregulation to an increased risk of metabolic disorders. This study explores a potential association between variation in genes regulating the endogenous circadian timing system (clock genes) and the risk of type 2 diabetes (T2D) in a sample of Greek elderly people. Study design: Variants within and upstream or downstream of PPARA, PPARD, CLOCK/TMEM165, PER1, PER2 and PER3 genes were genotyped in 716 individuals with T2D (A) and 569 normoglycemic controls (B), and allele frequencies were compared between the groups in a case control study design. Main outcome measures: Samples were genotyped on Illumina Human PsychArray. Permutation test analysis was implemented to determine statistical significance. To avoid the possibility of subjects with prediabetes being included in the control group, people with HbA1c <5.7% and fasting glucose <100 mg/dl comprised group C (n = 393), for whom a separate analysis was performed (secondary analysis). Results: A protective role against T2D was identified for 14 variants in the PPARA gene. The rs7291444, rs36125344, rs6008384 in PKDREJ, located upstream of PPARA, and rs2859389 in UTS2, located upstream of PER3, demonstrated a protective role against T2D in both analyses. In contrast, rs6744132, located between HES6 and PER2, was positively correlated with T2D risk. Only in the secondary analysis, rs2278637 in VAMP2, located downstream of PER1, and rs11943456 in CLOCK/TMEM165 were found to confer protection against T2D. In a recessive model analysis of all groups, PPARD variants exhibited a protective role against disease. Conclusions: Our findings suggest a possible implication of clock genes in T2D susceptibility. Further studies are needed to clarify the mechanisms that connect circadian rhythm dysfunction and T2D pathogenesis.

Insulin Directly Regulates the Circadian Clock in Adipose Tissue.

Adipose tissue (AT) is a key metabolic organ which functions are rhythmically regulated by an endogenous circadian clock. Feeding is a "zeitgeber" aligning the clock in AT with the external time, but mechanisms of this regulation remain largely unclear. We tested the hypothesis that postprandial changes of the hormone insulin directly entrain circadian clocks in AT and investigated a transcriptional-dependent mechanism of this regulation. We analyzed gene expression in subcutaneous AT (SAT) of obese subjects collected before and after the hyperinsulinemic-euglycemic clamp or control saline infusion (SC). The expressions of core clock genes PER2, PER3, and NR1D1 in SAT were differentially changed upon insulin and saline infusion, suggesting insulin-dependent clock regulation. In human stem cell-derived adipocytes, mouse 3T3-L1 cells, and AT explants from mPer2Luc knockin mice, insulin induced a transient increase of the Per2 mRNA and protein expression, leading to the phase shift of circadian oscillations, with similar effects for Per1 Insulin effects were dependent on the region between -64 and -43 in the Per2 promoter but not on CRE and E-box elements. Our results demonstrate that insulin directly regulates circadian clocks in AT and isolated adipocytes, thus representing a primary mechanism of feeding-induced AT clock entrainment.

Norepinephrine influences the circadian clock in human dermal fibroblasts from study participants with a diagnosis of attention-deficit hyperactivity disorder

Attention-deficit hyperactivity disorder (ADHD) is characterized by changes to the circadian process. Many medications used to treat the condition, influence norepinephrine levels. Several studies have, in addition, reported that norepinephrine itself has an effect on circadian function. The aim of this study was to investigate the circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after norepinephrine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with an ADHD diagnosis. Circadian preference was evaluated with German Morningness–Eveningness Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different norepinephrine concentrations in HDF cultures, the rhythmicity of circadian gene expression was analyzed via qRT-PCR. The exposure of 1 µM norepinephrine to confluent cultures of human dermal fibroblasts from participants with a diagnosis of ADHD, was shown to dampen Per1 rhythmicity. The expression of Bmal1, Per1 and Per3 in control subjects was also influenced by incubation with 1 µM norepinephrine. Cultures from the ADHD group revealed no statistically significant overall differences in circadian gene expression, between cultures with and without norepinephrine incubation. Per3 expression showed a significant ZT × group interaction via mixed ANOVA. Per3 expression at ZT4 was significant higher in the group of control samples incubated with 1 µM norepinephrine, compared to the control group without norepinephrine. This effect was also shown in the control samples incubated with 1 µM norepinephrine and cultures from subjects with ADHD without norepinephrine incubation. Per3 expression differed between the healthy control group and the ADHD group without norepinephrine incubation at ZT28. The results of the present study illustrate that norepinephrine impacts on circadian function. In both groups, control group and cultures taken from subjects with ADHD, the expression of the periodic genes (Per1–3) was significantly influenced by incubation with norepinephrine.

Atomoxetine and circadian gene expression in human dermal fibroblasts from study participants with a diagnosis of attention-deficit hyperactivity disorder

Atomoxetine (ATO) is a second line medication for attention-deficit hyperactivity disorder (ADHD). We proposed that part of the therapeutic profile of ATO may be through circadian rhythm modulation. Thus, the aim of this study was to investigate the circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after ATO exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with a diagnosis of ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different ATO concentrations in HDF cultures, the rhythmicity of circadian gene expression was analyzed via qRT-PCR. No statistical significant effect of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, sleep WASO and total number of wake bouts was observed. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. ATO induced the rhythmicity of Clock in the ADHD group. This effect, however, was not observed in HDF cultures of healthy controls. Bmal1 and Per2 expression showed a significant ZT × group interaction via mixed ANOVA. Strong positive correlations for chronotype and circadian genes were observed for Bmal1, Cry1 and Per3 among the study participants. Statistical significant different Clock, Bmal1 and Per3 expressions were observed in HDFs exposed to ATO collected from ADHD participants exhibiting neutral and moderate evening preference, as well as healthy participants with morning preferences. The results of the present study illustrate that ATO impacts on circadian function, particularly on Clock, Bmal1 and Per2 gene expression.

Abstract LB244: The role of stromal Per2 in regulation of tumorigenesis

Abstract The tumor microenvironment (TME) comprised of multiple proteins, extracellular matrix(ECM) and various cell types that together promote tumor initiation, progression andhomeostasis in a cell-cell communication manner. In this study, we set to understand howthe stromal circadian clock supports the cancer cells, specifically the contribution of thecore clock gene Per2 to tumor initiation and progression. The circadian clock is anendogenous, evolutionally conserved and ubiquitously expressed pacemaker, consisting ofcell autonomous clocks and a central pacemaker located in the hypothalamussuperchiasmatic nucleus (SCN). Together these clocks synchronize numerous biologicalprocesses between the organism and its environment. Amongst these processes are DNAdamage repair, metabolism, and cell cycle. Several studies showed that disruption ofcircadian rhythms has been associated with various forms of cancer in humans and mice.While Per2 was previously suggested to be a tumor suppressor, in this study we found thatthe core clock gene Per2 in the TME is essential for tumor initiation and metastaticcolonization. Another core gene, Per1, is dispensable. We further showed that lossof Per2 in the TME leads to transcriptional rewiring at early stages of metastasesformation, and suppresses subsequent metastatic tumor progression. Thus, our resultsunravel an unexpected protumorigenic role for the core clock gene Per2 in the TME. Thesefindings may imply a non-circadian role for Per2, differentiating it from Per1 withpotential implications for therapeutic dosing strategies and treatment regimens. Citation Format: Shimrit Mayer, Lee Shaashua, Chen Lior, Hagar Lavon, Alexander Novoselsky, Ruth Scherz-Shouval. The role of stromal Per2 in regulation of tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB244.

Core circadian clock genes Per1 and Per2 regulate the rhythm in photoreceptor outer segment phagocytosis.

Retinal photoreceptors undergo daily renewal of their distal outer segments, a process indispensable for maintaining retinal health. Photoreceptor outer segment (POS) phagocytosis occurs as a daily peak, roughly about 1hour after light onset. However, the underlying cellular and molecular mechanisms which initiate this process are still unknown. Here we show that, under constant darkness, mice deficient for core circadian clock genes (Per1 and Per2) lack a daily peak in POS phagocytosis. By qPCR analysis, we found that core clock genes were rhythmic over 24hours in both WT and Per1, Per2 double mutant whole retinas. More precise transcriptomics analysis of laser capture microdissected WT photoreceptors revealed no differentially expressed genes between time points preceding and during the peak of POS phagocytosis. In contrast, we found that microdissected WT retinal pigment epithelium (RPE) had a number of genes that were differentially expressed at the peak phagocytic time point compared to adjacent ones. We also found a number of differentially expressed genes in Per1, Per2 double mutant RPE compared to WT ones at the peak phagocytic time point. Finally, based on STRING analysis, we found a group of interacting genes that potentially drive POS phagocytosis in the RPE. This potential pathway consists of genes such as: Pacsin1, Syp, Camk2b, and Camk2d among others. Our findings indicate that Per1 and Per2 are necessary clock components for driving POS phagocytosis and suggest that this process is transcriptionally driven by the RPE.

Circadian misalignment by environmental light/dark shifting causes circadian disruption in colon.

BackgroundPhysiological circadian rhythms (CRs) are complex processes with 24-hour oscillations that regulate diverse biological functions. Chronic weekly light/dark (LD) shifting (CR disruption; CRD) in mice results in colonic hyperpermeability. However, the mechanisms behind this phenomenon are incompletely understood. One potential innovative in vitro method to study colonic CRs are colon organoids. The goals of this study were to utilize circadian clock gene Per2 luciferase reporter (Per2::Luc) mice to measure the effects of chronic LD shifting on colonic tissue circadian rhythmicity ex vivo and to determine if organoids made from shifted mice colons recapitulate the in vivo phenotype.MethodsNon-shifted (NS) and shifted (S) BL6 Per2::Luc mice were compared after a 22-week experiment. NS mice had a standard 12h light/12h dark LD cycle throughout. S mice alternated 12h LD patterns weekly, with light from 6am-6pm one week followed by shifting light to 6pm-6am the next week for 22 weeks. Mice were tested for intestinal permeability while colon tissue and organoids were examined for CRs of bioluminescence and proteins of barrier function and cell fate.ResultsThere was no absolute difference in NS vs. S 24h circadian period or phase. However, chronic LD shifting caused Per2::Luc S mice colon tissue to exhibit significantly greater variability in both the period and phase of Per2::Luc rhythms than NS mice colon tissue and organoids. Chronic LD shifting also resulted in increased colonic permeability of the Per2::Luc mice as well as decreased protein markers of intestinal permeability in colonic tissue and organoids from shifted Per2:Luc mice.ConclusionsOur studies support a model in which chronic central circadian disruption by LD shifting alters the circadian phenotype of the colon tissue and results in colon leakiness and loss of colonic barrier function. These CRD-related changes are stably expressed in colon stem cell derived organoids from CRD mice.

Common genetic variation in circadian clock genes are associated with cardiovascular risk factors in an African American and Hispanic/Latino cohort.

Misalignment of the internal circadian time with external physical time due to environmental factors or due to genetic variantion in circadian clock genes has been associated with increased incidence of cardiovascular risk factors. Common genetic variation in circadian genes in the United States have been identified predominantly in European ancestry individuals. We therefore examined the association between circadian clock single nucleotide polymorphisms (SNPs) in Clock, Cry1, Cry2, Bmal1 and Per3 genes and cardiovascular risk factors in African Americans and Hispanic/Latinos. We analyzed 17 candidate circadian SNPs in 1,166 subjects who self-identified as African-American or Hispanic/Latino and were enrolled in the UIC Cohort of Patients, Family and Friends. We found significant differences in the minor allele frequencies between African American and Hispanic/Latino subjects. Our analyses also established ethnic-specific SNPs that are associated with cardiovascular risk factors. In Hispanic/Latinos, the rs6850524 in Clock was associated with increased risk for hypertension, meanwhile rs12649507, rs4864546, and rs4864548 reduced the risk, also rs8192440 (Cry1) reduced the risk for type 2 diabetes. In African Americans, the Clock rs1801260 and rs6850524 were negatively associated with the presence of obesity; Bmal1 rs11022775 reduced the risk for dyslipidemia; and the Cry2 rs2292912 increased the risk for dyslipidemia and diabetes. Genetic variations in candidate circadian-clock genes are associated with risk factors for cardiovascular disease in African-Americans and Hispanic/Latinos. Our findings may help to improve cardiovascular risk assessment as well as better understand how circadian misalignment impacts cardiovascular risk in diverse populations.

The NutriClock Study Protocol - Assessing the Impact of a Chrononutrition Intervention in Patients With Cardiometabolic Disturbances

Abstract Objectives The NutriClock Study aims to test the effect of a chronotype-based nutritional intervention in circadian homeostasis and in clinical and laboratory biomarkers of dysmetabolism. Methods The NutriClock Study is a randomised, controlled, parallel trial, conducted over 10 weeks (2 weeks of baseline assessments and 8 weeks of intervention), with a 3- and 6-months follow-up. The study includes 2 groups of patients with cardiometabolic disturbances: an experimental group with mealtimes defined according to their chronotype, without caloric restriction; and a control group benefitting only from treatment as usual, with no specific nutritional intervention. The primary endpoint is insulin sensitivity (HOMA Index), and secondary endpoints include cardiometabolic clinical and analytical features (body weight and composition, serum glucose, insulin, glycated haemoglobin, lipid profile, leptin, ghrelin), inflammatory biomarkers (CRP, IL-6, adiponectin) and chronobiological features (salivary clock gene Bmal1, Clock, Per1, Per2, Per3, Cry1, Cry2, Rev-erbα, Rev-erbβ, Dec1 expression, melatonin and cortisol). Circadian oscillations of target genes will be measured through bioinformatics and mathematical analysis of rhythmicity, period and amplitude. This method will be used to determine the participants’ chronotype along with the Munich Chronotype Questionnaire and salivary cortisol and melatonin and will assist in optimizing mealtimes based on the circadian clock phenotypes. A sample size of 60 patients (30 per group) is required for a pairwise comparison with overall power of 80% to detect a true between-group difference of 20% in the primary endpoint after 8 weeks of intervention. Results Not applicable. Conclusions Chrononutrition emerges as an innovative intervention to enhance the health of patients with cardiometabolic diseases. This study will characterize circadian rhythms patterns in this population to set up an algorithm that defines pinpointed feeding intervals based on chronotype assessment. Funding Sources M. Lages holds a PhD Scholarship FCT/UIDB/05,704/2020.

Sex Differences in Circadian Clock Genes and Myocardial Infarction Susceptibility.

The growing body of evidence shows a significant difference in the circadian rhythm of cardiovascular disease based on biological sex. The incidence of cardiovascular disease varies between women and men. Additionally, biological sex is vital for the timely application of therapy—chronotherapy, which benefits both sexes. This study aimed to examine the potential difference of single nucleotide polymorphisms (SNPs) of the circadian rhythm genes ARNTL, CLOCK, CRY2 and PER2 in women and men with myocardial infarction. A cross-sectional study was conducted, including 200 patients with myocardial infarction. Altogether, ten single nucleotide polymorphisms in the ARNTL, CLOCK, CRY2 and PER2 genes were analyzed. The Chi-square test yielded statistically significant differences in CLOCK gene rs11932595 polymorphism in a recessive genotype model between women and men with a p-value of 0.03 and an odds ratio 2.66, and a corresponding 95% confidence interval of 1.07 to 6.66. Other analyzed polymorphisms of the circadian rhythm genes ARNTL, CRY2, and PER2 did not significantly differ between the sexes. According to the study’s current results, the CLOCK gene’s genetic variability might affect myocardial infarction concerning biological sex.

Period1 gene expression in the olfactory bulb and liver of freely moving streptozotocin-treated diabetic mouse

Clock genes express circadian rhythms in most organs. These rhythms are organized throughout the whole body, regulated by the suprachiasmatic nucleus (SCN) in the brain. Disturbance of these clock gene expression rhythms is a risk factor for diseases such as obesity. In the present study, to explore the role of clock genes in developing diabetes, we examined the effect of streptozotocin (STZ)-induced high glucose on Period1 (Per1) gene expression rhythm in the liver and the olfactory bub (OB) in the brain. We found a drastic increase of Per1 expression in both tissues after STZ injection while blood glucose content was low. After a rapid expression peak, Per1 expression showed no rhythm. Associated with an increase of glucose content, behavior became arrhythmic. Finally, we succeeded in detecting an increase of Per1 expression in mice hair follicles on day 1 after STZ administration, before the onset of symptoms. These results show that elevated Per1 expression by STZ plays an important role in the aggravation of diabetes.

Photoperiod Alters Visceral Adiposity and Molecular Circadian Rhythms in White‐footed Mice at Thermoneutrality

The length of daylight (photoperiod) and environmental temperature fluctuate seasonally and act as cues for physiology. How photoperiod per se impacts adipose physiology is essential to consider as misalignment of circadian rhythms is suggested to promote obesity. Photoperiod alters fat deposition when temperature simultaneously acts as a cue. However, whether photoperiod regulates adipose physiology independent of temperature is unknown, as ambient temperatures below thermoneutrality are typically used. We compared the effects of 4 weeks of long (LD - 16hrs light:8hrs dark) and short (SD – 8hrs light:16hrs dark) photoperiod exposure at thermoneutrality (27°C) to isolate photoperiod from temperature cues in photosensitive F1 generation, male white-footed mice (Peromyscus leucopus). Short photoperiod selectively lowered visceral white adipose tissue (WAT) (vWAT) mass (p<0.05 vs LD), with no effect on subcutaneous WAT or interscapular brown adipose tissue mass or bodyweight. Calorie consumption and 24-hour feeding behaviour were not affected by photoperiod, although short photoperiod mice ate almost exclusively in the dark (SD ate 8.5% of daily intake in 8hr light phase vs. LD ate 26% of intake in 16hr light phase (p<0.0001)). In vWAT, short photoperiod increased Adrβ3, Lpl and Nr1d1 mRNA expression (p<0.05 vs LD), with no change in Ucp1, Pgc1a or Hsl. Circadian Per1 and Per2 gene expression aligned with the onset of dark and thus were misaligned between photoperiods (p<0.05), while Bmal1 and Clock were also misaligned. Thus, photoperiod alone is a strong enough cue to alter visceral fat accumulation and alter clock gene misalignment, without temperature-induced thermogenesis. These findings have important implications for understanding obesity in humans in whom day light is artificially extended while environmental temperatures are kept consistent.

Diet-altered body temperature rhythms are associated with altered rhythms of clock gene expression in peripheral tissues in vivo.

Temperature rhythms can act as potent signals for the modulation of the amplitude and phase of clock gene expression in peripheral organs in vitro, but the relevance of the circadian rhythm of core body temperature (Tc) as a modulating signal in vivo has not yet been investigated. Using calorie restriction and cafeteria feeding, we induced a larger and a dampened Tc amplitude, respectively, in male Wistar rats, and investigated the circadian expression profile of the core clock genes Bmal1, Per2, Cry1, and Rev-erbα, the heat-responsive genes heat shock protein 90 (Hsp90) and cold-inducible RNA binding protein (Cirbp), and Pgc1α, Pparα/γ/δ, Glut1/4, and Chop10 in the liver, skeletal muscle, white adipose tissue (WAT), and adrenal glands. Diet-altered Tc rhythms differentially affected the profiles of clock genes, Hsp90, and Cirbp expression in peripheral tissues. Greater Tc amplitudes elicited by calorie restriction were associated with large amplitudes of Hsp90 and Cirbp expression in the liver and WAT, in which larger amplitudes of clock gene expression were also observed. The amplitudes of metabolic gene expression were greater in the WAT, but not in the liver, in calorie-restricted rats. Conversely, diet-altered Tc rhythms were not translated to distinct changes in the amplitude of Hsp90, Cirbp, or clock or metabolic genes in the skeletal muscle or adrenal glands. While it was not possible to disentangle the effects of diet and temperature in this model, taken together with previous in vitro studies, our study presents novel data consistent with the notion that the circadian Tc rhythm can modulate the amplitude of circadian gene expression in vivo. The different responses of Hsp90 and Cirbp in peripheral tissues may be linked to the tissue-specific responses of peripheral clocks to diet and/or body temperature rhythms, but the association with the amplitude of metabolic gene expression is limited to the WAT.

Circadian rhythms in septic shock patients

BackgroundDespite the intensive efforts to improve the diagnosis and therapy of sepsis over the last decade, the mortality of septic shock remains high and causes substantial socioeconomical burden of disease. The function of immune cells is time-of-day-dependent and is regulated by several circadian clock genes. This study aims to investigate whether the rhythmicity of clock gene expression is altered in patients with septic shock.MethodsThis prospective pilot study was performed at the university hospital Charité–Universitätsmedizin Berlin, Department of Anesthesiology and Operative Intensive Care Medicine (CCM, CVK). We included 20 patients with septic shock between May 2014 and January 2018, from whom blood was drawn every 4 h over a 24-h period to isolate CD14-positive monocytes and to measure the expression of 17 clock and clock-associated genes. Of these patients, 3 whose samples expressed fewer than 8 clock genes were excluded from the final analysis. A rhythmicity score SP was calculated, which comprises values between -1 (arrhythmic) and 1 (rhythmic), and expression data were compared to data of a healthy study population additionally.Results77% of the measured clock genes showed inconclusive rhythms, i.e., neither rhythmic nor arrhythmic. The clock genes NR1D1, NR1D2 and CRY2 were the most rhythmic, while CLOCK and ARNTL were the least rhythmic. Overall, the rhythmicity scores for septic shock patients were significantly (p < 0.0001) lower (0.23 ± 0.26) compared to the control group (12 healthy young men, 0.70 ± 0.18). In addition, the expression of clock genes CRY1, NR1D1, NR1D2, DBP, and PER2 was suppressed in septic shock patients and CRY2 was significantly upregulated compared to controls.ConclusionMolecular rhythms in immune cells of septic shock patients were substantially altered and decreased compared to healthy young men. The decrease in rhythmicity was clock gene-dependent. The loss of rhythmicity and down-regulation of clock gene expression might be caused by sepsis and might further deteriorate immune responses and organ injury, but further studies are necessary to understand underlying pathophysiological mechanisms.Trail registration Clinical trial registered with www.ClinicalTrials.gov (NCT02044575) on 24 January 2014.

Glyphosate exposure attenuates testosterone synthesis via NR1D1 inhibition of StAR expression in mouse Leydig cells

Glyphosate is a broad-spectrum herbicide that impairs testosterone synthesis in mammals. Leydig cells (LCs), the primary producers of testosterone, demonstrate rhythmic expression of circadian clock genes both in vivo and in vitro. The nuclear receptor NR1D1 is an important clock component that constitutes the subsidiary transcriptional/translational loop in the circadian clock system. Nr1d1 deficiency resulted in diminished fertility in both male and female mice. However, whether NR1D1 is involved in the glyphosate-mediated inhibition of testosterone synthesis in LCs remains unclear. Here, the involvement of NR1D1 in glyphosate-mediated inhibition of testosterone synthesis was investigated both in vitro and in vivo. Glyphosate exposure of TM3 cells significantly increased Nr1d1 mRNA levels, but decreased Bmal1, Per2, StAR, Cyp11a1, and Cyp17a1 mRNA levels. Western blotting confirmed elevated NR1D1 and reduced StAR protein levels following glyphosate exposure. Glyphosate exposure also reduced testosterone production in TM3 cells. In primary LCs, glyphosate exposure also upregulated Nr1d1 mRNA levels and downregulated the mRNA levels of other clock genes (Bmal1 and Per2) and steroidogenic genes (StAR, Cyp17a1, Cyp11a1, and Hsd3b2), and inhibited testosterone synthesis. Moreover, glyphosate exposure significantly reduced the amplitude and shortened the period of PER2::LUCIFERASE oscillations in primary LCs isolated from mPer2Luciferase knock-in mice. Four weeks of oral glyphosate upregulated NR1D1 at both the mRNA and protein levels in mouse testes, and this was accompanied by a reduction in StAR expression. Notably, serum testosterone levels were also drastically reduced in mice treated with glyphosate. Moreover, dual-luciferase reporter and EMSA assays revealed that in TM3 cells NR1D1 inhibits the expression of StAR by binding to a canonical RORE element present within its promoter. Together, these data demonstrate that glyphosate perturbs testosterone synthesis via NR1D1 mediated inhibition of StAR expression in mouse LCs. These findings extend our understanding of how glyphosate impairs male fertility.

Multi-omics analysis of the prognosis and therapeutic significance of circadian clock in ovarian cancer.

Ovarian cancer (OV) is one of the most common female malignancies with high morbidity and mortality, but its mechanism is not fully understood. The circadian clock is involved in the regulation of the immune system and the tumor microenvironment, regulating biological processes and behaviors in multiple ways. Circadian rhythm disorders are considered a risk factor for tumorigenesis. Multi-omics analysis was performed to comprehensively illustrate the roles of circadian clock genes in OV, we found that most of circadian clock genes undergo epigenetic alterations in OV and are strongly correlated with overall and progression-free patient survival. These clock genes are mainly involved in the inhibition of Apoptosis pathway, Cell Cycle pathway and DNA Damage Response pathway, as well as the activation of RAS/MAPK pathway and RTK pathway. Drug sensitivity model indicate that the expression of core clock genes may associate with drug resistance. Further, immune infiltrates analysis shows that different mutant forms of core genes can not only suppress immune infiltration, but also affect clinical outcome of ovarian cancer patients. Overall, our results may provide novel insights for the potential selection of immunotherapeutic targets.

Adenosine integrates light and sleep signalling for the regulation of circadian timing in mice

The accumulation of adenosine is strongly correlated with the need for sleep and the detection of sleep pressure is antagonised by caffeine. Caffeine also affects the circadian timing system directly and independently of sleep physiology, but how caffeine mediates these effects upon the circadian clock is unclear. Here we identify an adenosine-based regulatory mechanism that allows sleep and circadian processes to interact for the optimisation of sleep/wake timing in mice. Adenosine encodes sleep history and this signal modulates circadian entrainment by light. Pharmacological and genetic approaches demonstrate that adenosine acts upon the circadian clockwork via adenosine A1/A2A receptor signalling through the activation of the Ca2+ -ERK-AP-1 and CREB/CRTC1-CRE pathways to regulate the clock genes Per1 and Per2. We show that these signalling pathways converge upon and inhibit the same pathways activated by light. Thus, circadian entrainment by light is systematically modulated on a daily basis by sleep history. These findings contribute to our understanding of how adenosine integrates signalling from both light and sleep to regulate circadian timing in mice.