Expression Of Core Clock Genes Research Articles

Intermittent feeding and circadian rhythm in critical illness.

Purpose of reviewCircadian rhythms, i.e., periodic oscillations in internal biological processes, modulate metabolic processes such as hormonal signalling, nutrient absorption, and xenobiotic detoxification. Meal timing is a strong entraining cue for peripheral clocks in various organs, and eating out of circadian phases can impair glucose, gastrointestinal, and muscle metabolism. Sleep/wake cycles and circadian rhythms are extremely disrupted during critical illness. Timing of nutritional support may help preserve circadian rhythms and improve post-Intensive Care Unit (ICU) recovery. This review summarises circadian disruptors during ICU admission and evaluates the potential benefits of intermittent feeding on metabolism and circadian rhythms.Recent findingsRhythmic expression of core clock genes becomes rapidly disturbed during critical illness and remains disturbed for weeks. Intermittent, bolus, and cyclic enteral feeding have been directly compared to routine continuous feeding, yet no benefits on glycaemic control, gastrointestinal tolerance, and muscle mass have been observed and impacts of circadian clocks remain untested.SummaryAligning timing of nutritional intake, physical activity, and/or medication with circadian rhythms are potential strategies to reset peripheral circadian rhythms and may enhance ICU recovery but is not proven beneficial yet. Therefore, selecting intermittent feeding over continuous feeding must be balanced against the pros and cons of clinical practice.

Racing and Pacing in the Reward System: A Multi-Clock Circadian Control Over Dopaminergic Signalling.

Level of motivation, responsiveness to rewards and punishment, invigoration of exploratory behaviours, and motor performance are subject to daily fluctuations that emerge from circadian rhythms in neuronal activity of the midbrain’s dopaminergic system. While endogenous circadian rhythms are weak in the ventral tegmental area and substantia nigra pars compacta, daily changes in expression of core clock genes, ion channels, neurotransmitter receptors, dopamine-synthesising enzymes, and dopamine transporters, accompanied by changes in electrical activity, are readily observed in these nuclei. These processes cause dopamine levels released in structures innervated by midbrain dopaminergic neurons (e.g., the striatum) to oscillate in a circadian fashion. Additionally, growing evidence show that the master circadian clock located in the suprachiasmatic nucleus of the hypothalamus (SCN) rhythmically influences the activity of the dopaminergic system through various intermediate targets. Thus, circadian changes in the activity of the dopaminergic system and concomitant dopamine release observed on a daily scale are likely to be generated both intrinsically and entrained by the master clock. Previous studies have shown that the information about the value and salience of stimuli perceived by the animal is encoded in the neuronal activity of brain structures innervating midbrain dopaminergic centres. Some of these structures themselves are relatively autonomous oscillators, while others exhibit a weak endogenous circadian rhythm synchronised by the SCN. Here, we place the dopaminergic system as a hub in the extensive network of extra-SCN circadian oscillators and discuss the possible consequences of its daily entrainment for animal physiology and behaviour.

Derailed peripheral circadian genes in polycystic ovary syndrome patients alters peripheral conversion of androgens synthesis.

Do circadian genes exhibit an altered profile in peripheral blood mononuclear cells (PBMCs) of polycystic ovary syndrome (PCOS) patients and do they have a potential role in androgen excess? Our findings revealed that an impaired circadian clock could hamper the regulation of peripheral steroid metabolism in PCOS women. PCOS patients exhibit features of metabolic syndrome. Circadian rhythm disruption is involved in the development of metabolic diseases and subfertility. An association between shift work and the incidence of PCOS in females was recently reported. This is a retrospective case-referent study in which peripheral blood samples were obtained from 101 control and 101 PCOS subjects. PCOS diagnoses were based on Rotterdam Consensus criteria. This study comprised 101 women with PCOS and 101 control volunteers, as well as Swiss albino mice treated with dehydroepiandrosterone (DHEA) to induce PCOS development. Gene expression analyses of circadian and steroidogenesis genes in human PBMC and mice ovaries and blood were executed by quantitative real-time PCR. We observed aberrant expression of peripheral circadian clock genes in PCOS, with a significant reduction in the core clock genes, circadian locomotor output cycles kaput (CLOCK) (P ≤ 0.00001), brain and muscle ARNT-like 1 (BMAL1) (P ≤ 0.00001) and NPAS2 (P ≤ 0.001), and upregulation of their negative feedback loop genes, CRY1 (P ≤ 0.00003), CRY2 (P ≤ 0.00006), PER1 (P ≤ 0.003), PER2 (P ≤ 0.002), DEC1 (P ≤ 0.0001) and DEC2 (P ≤ 0.00005). Transcript levels of an additional feedback loop regulating BMAL1 showed varied expression, with reduced RORA (P ≤ 0.008) and increased NR1D1 (P ≤ 0.02) in PCOS patients in comparison with the control group. We also demonstrated the expression pattern of clock genes in PBMCs of PCOS women at three different time points. PCOS patients also exhibited increased mRNA levels of steroidogenic enzymes like StAR (P ≤ 0.0005), CYP17A1 (P ≤ 0.005), SRD5A1 (P ≤ 0.00006) and SRD5A2 (P ≤ 0.009). Knockdown of CLOCK/BMAL1 in PBMCs resulted in a significant reduction in estradiol production, by reducing CYP19A1 and a significant increase in dihydrotestosterone production, by upregulating SRD5A1 and SRD5A2 in PBMCs. Our data also showed that CYP17A1 as a direct CLOCK-BMAL1 target in PBMCs. Phenotypic classification of PCOS subgroups showed a higher variation in expression of clock genes and steroidogenesis genes with phenotype A of PCOS. In alignment with the above results, altered expression of ovarian core clock genes (Clock, Bmal1 and Per2) was found in DHEA-treated PCOS mice. The expression of peripheral blood core clock genes in DHEA-induced PCOS mice was less robust and showed a loss of periodicity in comparison with that of control mice. N/A. We could not evaluate the circadian oscillation of clock genes and clock-controlled genes over a 24-h period in the peripheral blood of control versus PCOS subjects. Additionally, circadian genes in the ovaries of PCOS women could not be evaluated due to limitations in sample availability, hence we employed the androgen excess mouse model of PCOS for ovarian circadian assessment. Clock genes were assessed in the whole ovary of the androgen excess mouse model of PCOS rather than in granulosa cells, which is another limitation of the present work. Our observations suggest that the biological clock is one of the contributing factors in androgen excess in PCOS, owing to its potential role in modulating peripheral androgen metabolism. Considering the increasing prevalence of PCOS and the rising frequency of delayed circadian rhythms and insufficient sleep among women, our study emphasizes the potential in modulating circadian rhythm as an important strategy in PCOS management, and further research on this aspect is highly warranted. This work was supported by the RGCB-DBT Core Funds and a grant (#BT/PR29996/MED/97/472/2020) from the Department of Biotechnology (DBT), India, to M.L. B.S.J. was supported by a DST/INSPIRE Fellowship/2015/IF150361 and M.B.K. was supported by the Research Fellowship from Council of Scientific & Industrial Research (CSIR) (10.2(5)/2007(ii).E.U.II). The authors declare no competing interests. N/A.

Review on circadian rhythm the role of core clock gene expression during sleep in epileptic syndrome

Circadian rhythm is a 24 hour or a day’s rhythmic oscillations with physiological and behavioural changes that is internally governed by the brain. This activity is generally anticipated by the internal and external cues from the environment. In circadian rhythm of our body is regulated by a transcriptional –translational Auto-regulatory loop which leads to the molecular oscillations of our “core clock genes” at a specific cellular level. In the many mammalian and animal models, the master pacemaker is in the hypothalamus at the suprachiasmatic nucleus (SCN) that can regulate downstream oscillations in the peripheral tissues of the brain. The core clock genes are self- sustained and cell autonomous not only on the pace maker but also on the other dissociated cultured cells and peripheral tissues. Here we can review the general mechanisms involved in the clock genes that is responsible for the circadian rhythmicity in epileptic syndromes and these findings can elucidate the cell specific expression of the clock genes in different syndromes. In the recent past there has been considerable amount of progress in elucidating the molecular and cellular mechanisms that is involved in understand the core clock gene expression and the circadian rhythms effects on epileptic syndromes. However, there is very little understanding on how these two mechanisms interact to cause a core clock genes disruptions plays an important role. Recent studies in several animal models have shown the effects of time of the day on the neurophysiology and path-physiology in the epileptic syndrome. Together, these evidences from the past suggest that there can be a common mechanism underlying the circadian rhythmicity and epileptogenesis mechanism.

Comprehensive profiling of clock genes expression in colorectal cancer (CRC).

3129 Background: Disruption of the circadian clock has been linked to cancer risk, development and progression. Core clock proteins are emerging as novel therapeutic targets in cancer. We previously showed that polymorphisms in clock genes were associated with anti-VEGF treatment outcome in metastatic CRC. Here we further evaluated the molecular landscape of clock pathway alterations in CRC. Methods: 7591 CRC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Clock gene Score (CS) was determined using expression of core clock genes Z scores (positives of CLOCK, ARNTL, RORA/B/C and negatives of repressors CRY1/2, PER1/2/3, REVERBA/B) stratified by quartiles. xCell was used to quantify cell infiltration in the tumor microenvironment (TME). Consensus molecular subtypes (CMS) were assessed by RNAseq. Significance was determined as P-values adjusted for multiple testing ( q) of <.05. Real world survival was obtained from insurance claims data and Kaplan-Meier estimates were calculated for comparison. Results: CS was higher in primary tumors than metastases and in right- than left-sided CRC ( P <.001). Liver metastases were associated with lower CS (23% Q1 vs 19% Q4, P <.001). CS was positively associated with CMS1 and 3 (21 vs 11% and 23 vs 9%, respectively, Q4 vs Q1) and negatively correlated with CMS2 and 4 (22 vs 32% and 34 vs 48%) (all P <.001). These associations were confirmed in mismatch repair proficient (pMMR) tumors. Overall, TMB-H and dMMR/MSI-H were positively associated with CS (11 vs 6% and 8 vs 4%, Q4 vs Q1, q <.0001) and PD-L1 showed a similar trend ( P <.01, q =.06); the association with TMB-H was not significant in pMMR. High CS was associated with alterations of genes in WNT signaling, RAS, PI3K, TGF-β, and NOTCH pathways, while negatively associated with TP53 mutations, HER2 expression and CDX2 copy numbers, confirmed in pMMR (all q <.05). CS negatively correlated with the angiogenesis pathway signature (Q1 vs Q4 Z score: 6.6 vs -4.6, P <.001). B cells, M1 and M2 macrophages, neutrophils, NK, Tregs, CD4+ and CD8+ T cells, and myeloid dendritic cells were more abundant in the TME of tumors with high CS while cancer associated fibroblasts were lower, regardless of MMR status (all q <.001). Individually, ARNTL tumor expression below median was associated with better OS (overall: HR 0.88, 95% CI [0.82-0.94]; pMMR: HR 0.88 [0.81-0.94]) and longer time on treatment of bevacizumab (overall: HR 0.91 [0.83-0.99]; pMMR: HR 0.91 [0.83-0.99]). Conclusions: This is the most extensive profiling study to investigate the expression of clock genes in CRC. Our data show that clock genes expression is strongly associated with distinct molecular features, immune cell infiltration, angiogenesis pathway enrichment and patient outcomes. These findings support the clock pathway as a therapeutic target in CRC, with a major role in CRC biology and TME modulation.

Chronic Cold Exposure Leads to Daytime Preference in the Circadian Expression of Hepatic Metabolic Genes.

Circadian control allows organisms to anticipate and adapt to environmental changes through changes in physiology and behavior. The circadian system timing is entrained by cues, such as light, food, and temperature. An ambient temperature dramatically impacts the sleep–wake cycle and metabolic rhythmicity. As endotherms, mammals rely on tissues such as the liver to provide fuel for thermogenesis to maintain body temperature. The adaptive response of the circadian rhythm of liver metabolism to chronic cold exposure remains largely unexplored. Here, we investigated the circadian rhythm adaptation of hepatic metabolism in response to environmental cold stress using a mouse model of chronic cold exposure. We analyzed metabolites and transcripts of mouse livers at 24 h and found that long-term low-temperature exposure resulted in a synergistic and phase synchronization of transcriptional rhythms of many genes associated with metabolic pathways. Notably, transcription peaked in the early light phase when the body temperature was relatively low. Our results suggest that chronic cold does not alter the rhythmic expression of essential core clock genes in the liver, so the rewiring of clock control gene expression is another mechanism that optimizes the circadian rhythm of liver metabolism to meet the energy requirements of animal thermogenesis.

The hepatocyte insulin receptor is required to program the liver clock and rhythmic gene expression.

Liver physiology is circadian and sensitive to feeding and insulin. Food intake regulates insulin secretion and is a dominant signal for the liver clock. However, how much insulin contributes to the effect of feeding on the liver clock and rhythmic gene expression remains to be investigated. Insulin action partly depends on changes in insulin receptor (IR)-dependent gene expression. Here, we use hepatocyte-restricted gene deletion of IR to evaluate its role in the regulation and oscillation of gene expression as well as in the programming of the circadian clock in the adult mouse liver. We find that, in the absence of IR, the rhythmicity of core-clock gene expression is altered in response to day-restricted feeding. This change in core-clock gene expression is associated with defective reprogramming of liver gene expression. Our data show that an intact hepatocyte insulin receptor is required to program the liver clock and associated rhythmic gene expression.

Role of Sleep Restriction in Daily Rhythms of Expression of Hypothalamic Core Clock Genes in Mice.

Lack of sleep time is a menace to modern people, and it leads to chronic diseases and mental illnesses. Circadian processes control sleep, but little is known about how sleep affects the circadian system. Therefore, we performed a 28-day sleep restriction (SR) treatment in mice. Sleep restriction disrupted the clock genes’ circadian rhythm. The circadian rhythms of the Cry1 and Per1/2/3 genes disappeared. The acrophase of the clock genes (Bmal1, Clock, Rev-erbα, and Rorβ) that still had a circadian rhythm was advanced, while the acrophase of negative clock gene Cry2 was delayed. Clock genes’ upstream signals ERK and EIFs also had circadian rhythm disorders. Accompanied by changes in the central oscillator, the plasma output signal (melatonin, corticosterone, IL-6, and TNF-α) had an advanced acrophase. While the melatonin mesor was decreased, the corticosterone, IL-6, and TNF-α mesor was increased. Our results indicated that chronic sleep loss could disrupt the circadian rhythm of the central clock through ERK and EIFs and affect the output signal downstream of the core biological clock.

Bone adaptation to mechanical loading in mice is affected by circadian rhythms.

Physical forces are critical for successful function of many organs including bone. Interestingly, the timing of exercise during the day alters physiology and gene expression in many organs due to circadian rhythms. Circadian clocks in tissues, such as bone, express circadian clock genes that target tissue-specific genes, resulting in tissue-specific rhythmic gene expression (clock-controlled genes). We hypothesized that the adaptive response of bone to mechanical loading is regulated by circadian rhythms. First, mice were sham loaded and sacrificed 8h later, which amounted to tissues being collected at zeitgeber time (ZT)2, 6, 10, 14, 18, and 22. Cortical bone of the tibiae collected from these mice displayed diurnal expression of core clock genes and key osteocyte and osteoblast-related genes, such as the Wnt-signaling inhibitors Sost and Dkk1, indicating these are clock-controlled genes. Serum bone turnover markers did not display rhythmicity. Second, mice underwent a single bout of in vivo loading at either ZT2 or ZT14 and were sacrificed 1, 8, or 24h after loading. Loading at ZT2 resulted in Sost upregulation, while loading at ZT14 led to Sost and Dkk1 downregulation. Third, mice underwent daily in vivo tibial loading over 2weeks administered either in the morning, (ZT2, resting phase) or evening (ZT14, active phase). In vivo microCT was performed at days 0, 5, 10, and 15 and conventional histomorphometry was performed at day 15. All outcome measures indicated a robust response to loading, but only microCT-based time-lapse morphometry showed that loading at ZT14 resulted in a greater endocortical bone formation response compared to mice loaded at ZT2. The decreased Sost and Dkk1 expression coincident with the modest, but significant time-of-day specific increase in adaptive bone formation, suggests that circadian clocks influence bone mechanoresponse.

NF-κB modifies the mammalian circadian clock through interaction with the core clock protein BMAL1.

In mammals, the circadian clock coordinates cell physiological processes including inflammation. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how inflammation affects the clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-κB in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-κB subunit RELA in the human U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude, its inhibition lengthened period length and caused amplitude phenotypes. NF-κB perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity behavior under different light/dark conditions. We show that RELA, like the clock repressor CRY1, repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA binds to the transactivation domain of BMAL1. These data support a model in which NF-kB competes with CRY1 and coactivator CBP/p300 for BMAL1 binding to affect circadian transcription. This is further supported by chromatin immunoprecipitation analysis showing that binding of RELA, BMAL1 and CLOCK converges on the E-boxes of clock genes. Taken together, these data support a significant role for NF-κB in directly regulating the circadian clock and highlight mutual regulation between the circadian and inflammatory pathways.

Intrinsic circadian timekeeping properties of the thalamic lateral geniculate nucleus.

Circadian rhythmicity in mammals is sustained by the central brain clock-the suprachiasmatic nucleus of the hypothalamus (SCN), entrained to the ambient light-dark conditions through a dense retinal input. However, recent discoveries of autonomous clock gene expression cast doubt on the supremacy of the SCN and suggest circadian timekeeping mechanisms devolve to local brain clocks. Here, we use a combination of molecular, electrophysiological, and optogenetic tools to evaluate intrinsic clock properties of the main retinorecipient thalamic center-the lateral geniculate nucleus (LGN) in male rats and mice. We identify the dorsolateral geniculate nucleus as a slave oscillator, which exhibits core clock gene expression exclusively in vivo. Additionally, we provide compelling evidence for intrinsic clock gene expression accompanied by circadian variation in neuronal activity in the intergeniculate leaflet and ventrolateral geniculate nucleus (VLG). Finally, our optogenetic experiments propose the VLG as a light-entrainable oscillator, whose phase may be advanced by retinal input at the beginning of the projected night. Altogether, this study for the first time demonstrates autonomous timekeeping mechanisms shaping circadian physiology of the LGN.

Circadian Synchrony: Sleep, Nutrition, and Physical Activity

The circadian clock in mammals regulates the sleep/wake cycle and many associated behavioral and physiological processes. The cellular clock mechanism involves a transcriptional negative feedback loop that gives rise to circadian rhythms in gene expression with an approximately 24-h periodicity. To maintain system robustness, clocks throughout the body must be synchronized and their functions coordinated. In mammals, the master clock is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN is entrained to the light/dark cycle through photic signal transduction and subsequent induction of core clock gene expression. The SCN in turn relays the time-of-day information to clocks in peripheral tissues. While the SCN is highly responsive to photic cues, peripheral clocks are more sensitive to non-photic resetting cues such as nutrients, body temperature, and neuroendocrine hormones. For example, feeding/fasting and physical activity can entrain peripheral clocks through signaling pathways and subsequent regulation of core clock genes and proteins. As such, timing of food intake and physical activity matters. In an ideal world, the sleep/wake and feeding/fasting cycles are synchronized to the light/dark cycle. However, asynchronous environmental cues, such as those experienced by shift workers and frequent travelers, often lead to misalignment between the master and peripheral clocks. Emerging evidence suggests that the resulting circadian disruption is associated with various diseases and chronic conditions that cause further circadian desynchrony and accelerate disease progression. In this review, we discuss how sleep, nutrition, and physical activity synchronize circadian clocks and how chronomedicine may offer novel strategies for disease intervention.

Binge alcohol disrupts skeletal muscle core molecular clock independent of glucocorticoids.

Circadian rhythms are central to optimal physiological function, as disruption contributes to the development of several chronic diseases. Alcohol (EtOH) intoxication disrupts circadian rhythms within liver, brain, and intestines, but it is unknown whether alcohol also disrupts components of the core clock in skeletal muscle. Female C57BL/6Hsd mice were randomized to receive either saline (control) or alcohol (EtOH) (5 g/kg) via intraperitoneal injection at the start of the dark cycle [Zeitgeber time (ZT12)], and gastrocnemius was collected every 4 h from control and EtOH-treated mice for the next 48 h following isoflurane anesthetization. In addition, metyrapone was administered before alcohol intoxication in separate mice to determine whether the alcohol-induced increase in serum corticosterone contributed to circadian gene regulation. Finally, synchronized C2C12 myotubes were treated with alcohol (100 mM) to assess the influence of centrally or peripherally mediated effects of alcohol on the muscle clock. Alcohol significantly disrupted mRNA expression of Bmal1, Per1/2, and Cry1/2 in addition to perturbing the circadian pattern of clock-controlled genes, Myod1, Dbp, Tef, and Bhlhe40 (P < 0.05), in muscle. Alcohol increased serum corticosterone levels and glucocorticoid target gene, Redd1, in muscle. Metyrapone prevented the EtOH-mediated increase in serum corticosterone but did not normalize the EtOH-induced change in Per1, Cry1 and Cry2, and Myod1 mRNA expression. Core clock gene expression (Bmal, Per1/2, and Cry1/2) was not changed following 4, 8, or 12 h of alcohol treatment on synchronized C2C12 myotubes. Therefore, binge alcohol disrupted genes of the core molecular clock independently of elevated serum corticosterone or direct effects of EtOH on the muscle.NEW & NOTEWORTHY Alcohol is a myotoxin that impairs skeletal muscle metabolism and function following either chronic consumption or acute binge drinking; however, mechanisms underlying alcohol-related myotoxicity have not been fully elucidated. Herein, we demonstrate that alcohol acutely interrupts oscillation of skeletal muscle core clock genes, and this is neither a direct effect of ethanol on the skeletal muscle, nor an effect of elevated serum corticosterone, a major clock regulator.

Effects of the Clock Modulator Nobiletin on Circadian Rhythms and Pathophysiology in Female Mice of an Alzheimer's Disease Model.

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder and the most common cause of dementia. Various pathogenic mechanisms have been proposed to contribute to disease progression, and recent research provided evidence linking dysregulated circadian rhythms/sleep and energy metabolism with AD. Previously, we found that the natural compound Nobiletin (NOB) can directly activate circadian cellular oscillators to promote metabolic health in disease models and healthy aging in naturally aged mice. In the current study, using the amyloid-β AD model APP/PS1, we investigated circadian, metabolic and amyloid characteristics of female mice and the effects of NOB. Female APP/PS1 mice showed reduced sleep bout duration, and NOB treatment exhibited a trend to improve it. While glucose tolerance was unchanged, female APP/PS1 mice displayed exaggerated oxygen consumption and CO2 production, which was mitigated by NOB. Likewise, cold tolerance in APP/PS1 was impaired relative to WT, and interestingly was markedly enhanced in NOB-treated APP/PS1 mice. Although circadian behavioral rhythms were largely unchanged, real-time qPCR analysis revealed altered expression of several core clock genes by NOB in the cerebral cortex, notably Bmal1, Npas2, and Rora. Moreover, NOB was also able to activate various clock-controlled metabolic genes involved in insulin signaling and mitochondrial function, including Igf1, Glut1, Insr, Irs1, Ucp2, and Ucp4. Finally, we observed that NOB attenuated the expression of several AD related genes including App, Bace1, and ApoE, reduced APP protein levels, and strongly ameliorated Aβ pathology in the cortex. Collectively, these results reveal novel genotype differences and importantly beneficial effects of a natural clock-enhancing compound in biological rhythms and related pathophysiology, suggesting the circadian clock as a modifiable target for AD.

MicroRNA-21 Controls Circadian Regulation of Apoptosis in Atherosclerotic Lesions.

The necrotic core partly formed by ineffective efferocytosis increases the risk of an atherosclerotic plaque rupture. Microribonucleic acids contribute to necrotic core formation by regulating efferocytosis and macrophage apoptosis. Atherosclerotic plaque rupture occurs at increased frequency in the early morning, indicating diurnal changes in plaque vulnerability. Although circadian rhythms play a role in atherosclerosis, the molecular clock output pathways that control plaque composition and rupture susceptibility are unclear. Circadian gene expression, necrotic core size, apoptosis, and efferocytosis in aortic lesions were investigated at different times of the day in Apoe-/-Mir21+/+ mice and Apoe-/-Mir21-/- mice after consumption of a high-fat diet for 12 weeks. Genome-wide gene expression and lesion formation were analyzed in bone marrow-transplanted mice. Diurnal changes in apoptosis and clock gene expression were determined in human atherosclerotic lesions. The expression of molecular clock genes, lesional apoptosis, and necrotic core size were diurnally regulated in Apoe-/- mice. Efferocytosis did not match the diurnal increase in apoptosis at the beginning of the active phase. However, in parallel with apoptosis, expression levels of oscillating Mir21 strands decreased in the mouse atherosclerotic aorta. Mir21 knockout abolished circadian regulation of apoptosis and reduced necrotic core size but did not affect core clock gene expression. Further, Mir21 knockout upregulated expression of proapoptotic Xaf1 (XIAP-associated factor 1) in the atherosclerotic aorta, which abolished circadian expression of Xaf1. The antiapoptotic effect of Mir21 was mediated by noncanonical targeting of Xaf1 through both Mir21 strands. Mir21 knockout in bone marrow cells also reduced atherosclerosis and necrotic core size. Circadian regulation of clock gene expression was confirmed in human atherosclerotic lesions. Apoptosis oscillated diurnally in phase with XAF1 expression, demonstrating an early morning peak antiphase to that of the Mir21 strands. Our findings suggest that the molecular clock in atherosclerotic lesions induces a diurnal rhythm of apoptosis regulated by circadian Mir21 expression in macrophages that is not matched by efferocytosis, thus increasing the size of the necrotic core.

Disruptions of Circadian Rhythms and Thrombolytic Therapy During Ischemic Stroke Intervention.

Several endogenous and exogenous factors interact to influence stroke occurrence, in turn contributing to discernable daily distribution patterns in the frequency and severity of cerebrovascular events. Specifically, strokes that occur during the morning tend to be more severe and are associated with elevated diastolic blood pressure, increased hospital stay, and worse outcomes, including mortality, compared to strokes that occur later in the day. Furthermore, disrupted circadian rhythms are linked to higher risk for stroke and play a role in stroke outcome. In this review, we discuss the interrelation among core clock genes and several factors contributing to ischemic outcomes, sources of disrupted circadian rhythms, the implications of disrupted circadian rhythms in foundational stroke scientific literature, followed by a review of clinical implications. In addition to highlighting the distinct daily pattern of onset, several aspects of physiology including immune response, endothelial/vascular and blood brain barrier function, and fibrinolysis are under circadian clock regulation; disrupted core clock gene expression patterns can adversely affect these physiological processes, leading to a prothrombotic state. Lastly, we discuss how the timing of ischemic onset increases morning resistance to thrombolytic therapy and the risk of hemorrhagic transformation.

Restructuring of the male mice peripheral circadian network after bariatric surgery.

Bariatric surgery is still the most effective long-term weight-loss therapy. Recent data indicate that surgical outcomes may be affected by diurnal food intake patterns. In this study, we aimed to investigate how surgery-induced metabolic adaptations (i.e. weight loss) interact with circadian clock function. For that reason, vertical sleeve gastrectomy (VSG) was performed in obese mice and rhythms in behavior, tissue rhythmicity, and white adipose tissue transcriptome were evaluated. VSG under constant darkness conditions led to a maximum weight loss of 18% compared to a loss of 3% after sham surgery. Post-surgical weight development was characterized by two distinct intervals of catabolic and subsequent anabolic metabolic state. Locomotor activity was not affected. However, VSG significantly increased active phase meal frequency in the anabolic state. No significant effects on clock gene rhythmicity were detected in adrenal and white adipose tissue (WAT) explant cultures. Transcriptome rhythm analyses of subcutaneous WAT revealed a reduction of cycling genes after VSG (sham: 2493 vs VSG: 1013) independent of sustained rhythms in core clock gene expression. This may be a consequence of weight loss-induced morphological reconstruction of WAT that overwrites the direct influence of the local clock machinery on the transcriptome. However, VSG altered rhythmic transcriptional regulation of WAT lipid metabolism pathways. Thus, our data suggest a reorganization of diurnal metabolic rhythms after VSG downstream of the molecular clock machinery.

Drinking Off(s) the Clock: Chronic Alcohol Suppresses Skeletal Muscle Molecular Clock

Circadian rhythms generated both centrally in the suprachiasmatic nucleus (SCN) and peripherally at the tissue level assist in the maintenance of physiological homeostasis, while circadian disruption is a characteristic of several diseases. The intrinsic skeletal muscle core clock has emerged as a key feature of metabolic control and influences several aspects of muscle physiology including contractile strength and mitochondrial content. Alcoholic muscle disease or myopathy is characterized by skeletal muscle weakness and myofiber atrophy resulting from the prolonged consumption of alcohol. Despite alcoholic myopathy being more prevalent than alcoholic liver disease, the entirety of mechanisms leading to its onset remains unknown. Alcohol disrupts normal cyclic expression of several core clock genes within the brain and liver, and we previously reported that acute alcohol intoxication disrupts the skeletal muscle core clock. Thus, the goal of this work was to determine whether the chronic consumption of alcohol dysregulates the skeletal muscle core molecular clock and clock-controlled genes (CCGs) in concert with alcoholic myopathy. Methods: C57BL/6Hsd female mice (12wks old) were pair fed a control liquid diet (CON) or an isocaloric ethanol (EtOH) containing liquid diet of increasing concentration (up to 32% kcal EtOH) for 7 weeks. Gastrocnemius muscles were collected from CON (n=4) and EtOH (n=3-4) mice every 4-hours for 24-hours. RNA was isolated, cDNA synthesized, and RT-PCR was performed. Results: Body weight and fat mass between groups was similar at sacrifice while liver mass was increased by alcohol. Lean mass assessed by MRI tended to be decreased by alcohol (p=0.06). Chronic alcohol consumption disrupted genes of the core clock including suppressing the rhythmic peak in expression of brain muscle ARNT like-1 (Bmal1), period (Per) 1 and 2, and cryptochrome 2 (Cry2). Genes involved in the regulation of Bmal1 also exhibited lower rhythmic peaks including Rev-erb (NR1D1) and myoblast determination protein 1 (MyoD), with significant reductions apparent at several of the 4-hour time points. The CCG D-box binding PAR BZIP transcription factor (DBP) was also suppressed at the end of the light phase and beginning of the dark active phase (ZT8 and ZT12). Skeletal muscle atrophy related genes forkhead box O1 (Foxo1), regulated in development and DNA damage response 1 (Redd1) and muscle RING-finger protein 1 (Murf1) were also altered by alcohol at the start of the dark (active) cycle. These data contrasted our previously described acute model in which subtle rhythmic shifts and large changes in the expression of several genes were observed specifically when blood alcohol levels (~4-12hr) were elevated. Therefore, there is a differential response of the skeletal muscle to acute and chronic alcohol consumption with prolonged intake leading to a lower amplitude of change over the 24-hour cycle of several core clock related genes. Future work will focus on determining the contribution of these disruptions to metabolic and functional perturbations characteristic of chronic alcoholic myopathy and the impact of these disruptions on the development of alcoholic myopathy.

Pinealectomy and gonadectomy modulate amplitude, but not photoperiodic modulation of Clock gene expression in the Syrian hamster suprachiasmatic nuclei.

The duration of daytime light phase (photoperiod) controls reproduction in seasonal mammals. Syrian hamsters are sexually active when exposed to long photoperiod, while gonadal atrophy is observed after exposure to short photoperiod. The photorefractory period, or photorefractoriness, is a particular state of spontaneous recrudescence of sexual activity that occurs after a long-term exposure to short photoperiod. Expression of core clock genes in the master circadian clock contained in the suprachiasmatic nuclei depends on photoperiodic conditions. Interestingly, the expression of the Clock gene is also modified in photorefractory Syrian hamsters. Since melatonin and testosterone levels in seasonal species are dependent on photoperiod, photoperiodic variations of Clock mRNA levels in the suprachiasmatic clock could be a consequence of these hormonal changes. To test this hypothesis, we analysed the effects of pinealectomy on Clock mRNA changes due to long to short photoperiod transition and of gonadectomy on Clock mRNA levels in photorefractory period. Our data show that the suprachiasmatic integration of the short photoperiod (assessed by a rhythmic expression profile of Clock) is independent of the presence of melatonin. Furthermore, constitutively low expression of Clock observed during the photorefractory period does not require the presence of either melatonin or testosterone. However, we show that both hormones provide positive feedback on average levels of Clock expression. Thus, our data support the hypothesis that daily variations of Clock levels in the suprachiasmatic nuclei are influenced by photoperiodic changes and the time spent in short photoperiod, independently of seasonal modifications of melatonin or testosterone levels.

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.