Genes Bmal1 Research Articles

Clock And BMAL1 genes expression in the hypothalamus of sleep-deprived pregnant wistar rats and their offspring

Clock And BMAL1 genes expression in the hypothalamus of sleep-deprived pregnant wistar rats and their offspring

Leptin Receptor Mediates Bmal1 Regulation of Estrogen Synthesis in Granulosa Cells.

Simple SummaryThere is increased interest in determining the effect of the biological clock system on reproduction, but how this biological system affects mammalian fertility and the regulation by clock genes on key genes of reproduction is poorly understood. This study examined the function of Leptin on reproduction through interaction with the Leptin receptor (Lepr) and the regulation of the key clock gene brain and muscle ARNT-like 1 (Bmal1) on Lepr. The results suggested that estrogen (E2) synthesis is regulated by Bmal1 through the Leptin–Lepr pathway as part of the regulatory mechanism of the circadian system on the fertility of female mammals.Chronobiology affects female fertility in mammals. Lepr is required for leptin regulation of female reproduction. The presence of E-box elements in the Lepr promoter that are recognized and bound by clock genes to initiate gene transcription suggested that circadian systems might regulate fertility through Lepr. However, it is unclear whether Bmal1, a key oscillator controlling other clock genes, is involved in leptin regulation in hormone synthesis through Lepr. In this study, serum estradiol (E2) concentration and the expressions of Bmal1, Lepr, Cyp19a1, and Cyp11a1 genes were found to display well-synchronized circadian rhythms. Knockdown of Bmal1 significantly reduced expression levels of Lepr, Fshr, and Cyp19a1 genes; protein production of Bmal1, Lepr, and Cyp19a1; and the E2 concentration in granulosa cells. Knockdown of Lepr reduced the expression levels of Cyp19a1 and Cyp11a1 genes and Cyp19a1 protein, and also reduced E2 concentration. Addition of leptin affected the expression of Cyp19a1, Cyp11a1, and Fshr genes. Bmal1 deficiency counteracted leptin-stimulated upregulation of the genes encoding E2 synthesis in granulosa cells. These results demonstrated that Bmal1 participates in the process by which leptin acts on Lepr to regulate E2 synthesis.

The protective effects of hydrogen sulfide on the myocardial ischemia via regulating Bmal1

BackgroundTo investigate the effect of hydrogen peroxide (H2S) on myocardial clock gene Bmal1 in ischemic cardiomyocytes. Materials & methodsQuantitative PCR (qPCR) was used to detect the expression of Bmal1 at the mRNA level in H9C2 rat cardiomyocytes. The protein expressions of Bax and Bcl-2, PI3K/Akt, caspase-3 were measured by western blotting. The levels of reactive oxygen species (ROS) were determined by ELISA. ResultsThe expression level of clock gene Bmal1 demonstrated a clock rhythm of periodic oscillation within 24 h. Compared with the control group, H2S treatment maintained the rhythm of the clock gene in ischemic cardiomyocytes and increased the transcription and expression levels of Bmal1. H2S increased cell survival by activating PI3K/Akt signaling pathway, inhibiting mitochondrial apoptosis signaling, and reducing intracellular oxidative stress. PI3K/Akt and Bmal1 were demonstrated to be involved in H2S protection of cardiomyocyte ischemia. Knockout of Bmal1 gene affects the degree of phosphorylation of Akt and Erk proteins, and the level of ROS production, resulting in a decrease in the protective effects of H2S. ConclusionThe expression level of Bmal1 has effects on the function of cardiomyocytes such as ROS production. The potential mechanism by which H2S regulates clock genes may be related to the effect of clock genes on protein phosphorylation levels in ischemic cardiomyocytes.

Association of insulin resistance with polymorphic variants of Clock and Bmal1 genes: A case–control study

ABSTRACTBackground: Little information is available in the literature for the correlation of insulin resistance (IR) and CLOCK gene polymorphism in Chinese population. This study aimed to investigate the relationship of HOMA-IR (homeostasis model assessment of insulin resistance) to polymorphic variants of Clock and Bmal1 genes in Chinese patients with essential hypertension.Methods: A total of 334 outpatients with essential hypertension (103 patients of HOMA-IR positive and 231 patients of HOMA-IR negative) were recruited to analyze Clock T3111C and Bmal1 A1420G genotypes with DNA sequencing approach.Results: Waist circumference, body mass index, glycated hemoglobin, total cholesterol, triglyceride, and plasminogen activator inhibitor-1 were significantly increased, while high-density lipoprotein cholesterol was significantly decreased in patients with HOMA-IR positive (P < .05–0.001 vs. patients with HOMA-IR negative). Twenty-four-hour ambulatory blood pressure monitoring showed that 24-h mean systolic blood pressure (SBP), especially nightime SBP, was higher in patients with HOMA-IR positive (P < .05 vs. patients with HOMA-IR negative). Notably, compared with the negative group, the distribution frequency of C allele of Clock T3111C and GG genotype of Bmal1 A1420G were significantly higher in the HOMA-IR positive group (29.1 vs. 10.8% P < .000 and 43.7 vs. 27.7% P = .007, respectively). Logistic regression analysis showed that C allele of Clock T3111C (OR = 4.128, CI 95% 2.313–7.368, p = .000) and GG genotype of Bmal1 A1420G (OR = 1.983, CI 95% 1.117–3.521, p = .019) were independent risk factors for potential HOMA-IR in Chinese patients with essential hypertension.Conclusion: Our results indicated that Chinese hypertensive patients with C allele of Clock T3111C or GG genotype of Bmal1 A1420G might be susceptible to IR and are more likely to develop high nighttime SBP.

Disruption of the Molecular Circadian Clock and Cancer: An Epigenetic Link.

The circadian clock is regulated at the molecular level by feedback circuits of a group of genes known as "clock genes", which establish a mechanism that controls circadian cellular physiology to maintain the balance between cell proliferation, response to DNA damage and apoptosis. Alterations in the expression of clock genes due to genetic or epigenetic mechanisms have been associated with multiple diseases including cancer. Even some clock genes such as the Per1, Per2, Bmal1 genes have been proposed as tumor suppressor genes, with a relevant role during carcinogenesis. At the molecular level, multiple mechanisms of molecular control have been described to link circadian transcription, cell cycle control, and tumorigenesis. In addition, recent findings describe an epigenetic control of circadian transcription, at the level of DNA methylation as well as in the modifications of histones. However, the link between the circadian epigenome and cancer remains unclear. In this article, we review the evidence that suggests a relationship between alterations in the expression of clock genes, with the development of cancer, from the epigenetic landscape.

Clock Genes Disruption in the Intensive Care Unit.

Intensive care unit (ICU) environment disrupts the circadian rhythms due to environmental and other nonphotic synchronizers. The main purpose of this article is to establish whether critically patients have desynchronization at the molecular level after 1 week of stay in the ICU. The rhythm of Clock, Bmal1, Cry1, and Per2 genes in neuro-ICU patients (n = 11) on the first day after admission in the unit (1 day) and 1 week later (1 week) was studied, 4 time points throughout the day, at 6, 12, 18, and 24 hours. Human whole blood samples were obtained from neuro-ICU patients. The total RNA was isolated and each sample was reverse transcribed to complementary DNA and quantitative polymerase chain reaction (PCRq) was performed. The possible rhythm was studied using Fourier Series. After 1 week, the clock gene rhythmicity completely disappeared. Messenger RNA (mRNA) expression for the 4 clock genes was shown rhythmicity at the first day after admission in the ICU. Circadian rhythmicity for none of them was observed but rather, ultradian rhythmicity was found. The expression of Clock, Bmal1, and Per2 mRNA after 1 week was similar in the 4-time point studies without significant fluctuation among the 4 time points analyzed. Rhythmic mRNA expression is present at the first day after admission in the ICU. However, ICU stay during 1 week affects the molecular machinery of the biological clock generating chronodisruption. Circadian disruption is associated with the risk of several pathologies, thus, it seems to be clear that ICU stay in constant conditions could adversely affect patient evolution and probably, circadian resynchronization restoring clock gene expression could lead to a better clinical evolution of the patient. Clock genes disruption is observed in neuro-ICU patients. Light therapy as well as melatonin treatment could reduce the impact of ICU stay period in biological clock, thereby improving patients' recovery.

Circadian oscillations persist in low malignancy breast cancer cells

ABSTRACT Epidemiological studies have shown that humans with altered circadian rhythms have higher cancer incidence, with breast cancer being one of the most cited examples. To uncover how circadian disruptions may be correlated with breast cancer and its development, prior studies have assessed the expression of BMAL1 and PER2 core clock genes via RT-qPCR and western blot analyses. These and our own low-resolution data show that BMAL1 and PER2 expression are suppressed and arrhythmic. We hypothesized that oscillations persist in breast cancer cells, but due to limitations of protocols utilized, cannot be observed. This is especially true where dynamic changes may be subtle. In the present work, we generated luciferase reporter cell lines representing high- and low-grade breast cancers to assess circadian rhythms. We tracked signals for BMAL1 and PER2 to determine whether and to what extent oscillations exist and provide initial correlations of circadian rhythm alterations with breast cancer aggression. In contrast to previous studies, where no oscillations were apparent in any breast cancer cell line, our luminometry data reveal that circadian oscillations of BMAL1 and PER2 in fact exist in the low-grade, luminal A MCF7 cells but are not present in high-grade, basal MDA-MB-231 cells. To our knowledge, this is the first evidence of core circadian clock oscillations in breast cancer cells. This work also suggests that circadian rhythms are further disrupted in more aggressive/high tumor grades of breast cancer, and that use of real time luminometry to study additional representatives of breast and other cancer subtypes is merited.

The protective effect of cycloastragenol on aging mouse circadian rhythmic disorder induced by d‐galactose

Aging process in mammals is associated with a decline in amplitude and a long period of circadian behaviors which are regulated by a central circadian regulator in the suprachiasmatic nucleus (SCN) and local oscillators in peripheral tissues. It is unclear whether enhancing clock function can retard aging. Using fibroblasts expressing per2::lucSV and senescent cells, we revealed cycloastragenol (CAG), a natural aglycone derivative from astragaloside IV, as a clock amplitude enhancing small molecule. CAG could activate telomerase to antiaging, but no reports focused on its effects on circadian rhythm disorders in aging mice. Here we analyze the potential effects of CAG on d-galactose-induced aging mice on the circadian behavior and expression of clock genes. For this purpose, CAG (20 mg/kg orally), was administered daily to d-galactose (150 mg/kg, subcutaneous) mice model of aging for 6 weeks. An actogram analysis of free-running activity of these mice showed that CAG significantly enhances the locomotor activity. We further found that CAG increase expressions of per2 and bmal1 genes in liver and kidney of aging mouse. Furthermore, CAG enhanced clock protein BMAL1 and PER2 levels in aging mouse liver and SCN. Our results indicated that the CAG could restore the behavior of circadian rhythm in aging mice induced by d-galactose. These data of present study suggested that CAG could be used as a novel therapeutic strategy for the treatment of age-related circadian rhythm disruption.

Abstract 1729: Investigating deregulated circadian clock machinery in cancer cells

Abstract The circadian clock plays an integral role in cellular functioning by temporally controlling gene expression, and there is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study we aimed to investigate circadian clock gene expression and oscillation patterns in cervical and oesophageal cancers, in order to determine whether disruptions in circadian clock functioning occur in these cancer types. Microarray gene expression analysis revealed the circadian clock gene, Per2, to be one of the most significantly downregulated genes in cervical cancer patient tissue compared to normal epithelium. In addition, Oncomine data-mining revealed significant downregulation of not only Per2, but multiple members of the circadian clock gene family (Clock, Bmal1, Per1, Per2, Cry1, Rev-erbα and RORα) in cervical cancer tissue compared to normal. Real-time RT-PCR analysis of circadian clock genes in oesophageal cancer patient tissue compared to matched normal epithelium revealed significant downregulation of Clock, Per2, Cry1 and RORα in oesophageal tumour tissue. Significant positive correlations were observed where circadian clock genes were simultaneously dysregulated in tumour tissue specimens. In cell line models, Clock, Bmal1, Per2, Cry1 and RORα circadian clock genes were significantly downregulated in transformed cells, compared to their untransformed counterparts, as well as in most cervical and oesophageal cancer cell lines compared to non-cancer epithelial cells. Patterns of protein expression did not accurately match mRNA expression, likely due to extensive post-translational processing, but Clock and Cry1 protein levels were considerably reduced in the cancer cell lines compared to normal. Overexpression of circadian clock genes in cancer cell lines negatively affected cell proliferation, highlighting the tumour suppressor properties of these genes in cervical and oesophageal cancer cells. Despite downreglated expression of circadian clock genes, cervical and oesophageal cancer cells maintain functional circadian oscillations, after synchronisation with Dexamethasone, as shown using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are still intact. Together, this study is a first to describe deregulated circadian clock machinery in cervical and oesophageal cancer cells, although the cells maintain a functional circadian rhythm. The study has relevance to the field of chronotherapy, where elucidating differences in circadian clock functioning in normal and cancer cells could yield better insights into the timing of administration of chemotherapy, ultimately ensuring a better patient response. Citation Format: Pauline J. van der Watt, Kate Davis, Virna Leaner. Investigating deregulated circadian clock machinery in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1729.

The role of Bmal1 in neuronal radial migration and axonal projection of the embryonic mouse cerebral cortex

Normal development of the cerebral cortex is a basis for the formation and function of mammalian brains. During this process, the radial migration of cortical neurons, as well as the axon projection into specific layers, are the most important steps regulated by some transcription factors, but the underlying molecular mechanisms are still obscure. BMAL1 (brain and muscle Arnt-like protein 1) is a newly identified transcription factor that plays important roles in the circadian rhythms. It was recently found to regulate the proliferation of hippocampal neuronal progenitor/precursor cells (NPCs), implicating Bmal1 in the brain development. Here we employed both RT-RCR and real-time PCR to explore the expression pattern of the Bmal1 gene in the developing brain. We found BMAl1 is enriched in the brain cortex during the perinatal stages and peaked in P3 mouse brains. Combined with in utero electroporation and interference with RNAi, we found that reducing the expression level of Bmal1 in neurons, the radial migration of embryonic cortical neurons was largely delayed, in a gene dose-effect pattern. Moreover, reducing the level of Bmal1 expression in mouse brains, the axonal projection in the corpus callosum was also disrupted from ipsilateral to the lateral cerebral hemisphere. These findings indicate that BMAL1 is essential for the radial migration of neurons in the cerebral cortex and the axonal projection of the corpus callosum, providing insights into the molecular mechanisms of cerebral cortex development.

Evaluation of Naturally Occurring Compounds Regulating Brown/Beige Adipocyte Differentiation

Brown adipose tissue is a critical regulator of metabolic health, and contributes to thermogenesis by uncoupling oxidative phosphorylation through the action of mitochondrial uncoupling protein 1 (Ucp1). Recent studies have shown that cold exposure and the stimulation of β3-adrenergic receptors induce the development of brown cell-like "beige" adipocytes in white adipose tissue. Brown and/or beige adipocyte-mediated thermogenesis suppresses high-fat diet-associated obesity. Therefore, the development of brown/beige adipocytes may prevent obesity and metabolic diseases. In the present study, we elucidated whether naturally occurring compounds contribute to regulating the cellular differentiation of brown/beige adipocytes. We screened for the up-regulated expression of Ucp1 during beige adipogenesis using extracts of crude herbal drugs frequently used in Kampo prescriptions (therapeutic drugs in Japanese traditional medicine). This screening revealed that the extract prepared from Citri Unshiu Pericarpium [the peel of Citrus unshiu (Swingle) Marcov.] increased the expression of Ucp1 in beige adipocytes. We also focused on the function of clock genes in regulating brown/beige adipogenesis. Therefore, another aim of the present study was to evaluate naturally occurring compounds that regulate brain and muscle Arnt-like 1 (Bmal1) gene expression. In this review, we focus on naturally occurring compounds that affect regulatory processes in brown/beige adipogenesis, and discuss better preventive strategies for the management of obesity and other metabolic disorders.

The Absence of Pineal Melatonin Abolishes the Daily Rhythm of Tph1 (Tryptophan Hydroxylase 1), Asmt (Acetylserotonin O-Methyltransferase), and Aanat (Aralkylamine N-Acetyltransferase) mRNA Expressions in Rat Testes.

This study examined the effects of pinealectomy in Wistar rats and melatonin replacement therapy on the daily mRNA expression of melatonin (Tph1, Aanat, Asmt, Mt1, Mt2, and Rorα), and steroidogenic (Star, 17βhsd3, and Lhr) related genes as well as clock genes (Rev-erbα, Bmal1, Per1, Per2, Cry1, and Cry2) in testes. The testes of control animals express the Tph1, Aanat, and Asmt and Per2 genes with 24-h rhythms in mRNA, reaching the maximal values during the dark phase. Pinealectomy abolished and melatonin treatment restored the 24-h rhythmicity. Daytime differences in mRNA expression were significant for Star, Lhr, Mt1, Mt2, Rorα, Rev-erbα, Bmal1, Cry1, and Cry2 genes in testes of control rats. Conversely, 17βhsd3 and Per1 mRNA expression did not show a daytime difference in testes of control animals. Pinealectomy abolished the peak time of Mt1 and Mt2 mRNA expression, phase shifted the peak time of Star, Rorα, Rev-erbα, Bmal1, and Cry2 mRNA expression, downregulated the 24-h Lhr mRNA expression, and inverted the peak time of Per1, Per2, and Cry1 mRNA expression to the light phase. The melatonin replacement therapy completely restored the control levels of Lhr, Rev-erbα, and Per1 mRNA expression patterns, partially restored the daily control of Star, Mt2, Rorα, Bmal1, Cry1, and Cry2 mRNA expression but did not re-establish the daily control of Mt1 mRNA expression. This suggests that the daily mRNA expression of these genes is probably driven by pineal melatonin and melatonin treatment restores (partially or completely) the daily control of gene expression patterns.

Effects of dynamic feeding low- and high-methionine diets on the variation of glucose and lipid metabolism-related genes in the liver of laying hens

We examined the effects of dynamic feeding low- and high-methionine diets on the transcriptional level of glucose and lipid metabolism-related genes in the liver of laying hens. A total of 180 laying hens (Brown Hy-line, 41 wk old) were exposed to 16 h of light and 8 h of darkness with lights on at 06:00h local time (Zeitgeber time [ZT] 0) and allocated into 3 equal groups with 6 replicates. The control group was fed a 0.30% methionine diet. The low-high (LH) group was fed 0.27% methionine diet at ZT1.5 and 0.33% methionine diet at ZT8.5. The feeding regime in high-low (HL) group was opposite to that of the LH group. After 10 wk, a total of 108 hens (6 hens/group/time point) were randomly and equally selected for further analysis. Blood and liver tissues were collected at ZT21.5, ZT1.5, ZT5.5, ZT9.5, ZT13.5, and ZT17.5. The results showed that the serum ALP activity in the HL group was the highest at ZT9.5 and ZT13.5, and that the serum glucose content in the HL group was lowest at ZT21.5 compared to the other 2 groups (P < 0.05). The mesors, amplitudes, and acrophases of the cosine curves for the CLOCK, BMAL1, CRY1, PER2, and PER3 genes were altered in the LH and HL groups. G6PC3, PCK2, GPAT, INSIG2, FASN, ACACA, ACOX1, HMGCR, LDLR, and PPARA expression in the liver were affected by the feeding regime at some time point (P < 0.05). Two-way analysis of variance comparisons showed some significant effects of time on the mRNA expression of G6PC3, G6PC2, PCK2, FBP1, FBP2, GCK, GYS2, FASN, GPAT, HMGCR, LDLR, ACC, SREBP2, and INSIG1 (P < 0.05). Moreover, different feeding regimes significantly affected the expression of FASN, GPAT, HMGCR, LDLR, ACACA, SREBP1, and INSIG1 (P < 0.05). In conclusion, dynamical feeding low- and high-methionine diets affected the variation of serum ALP and glucose levels, as well as the mRNA expression of circadian clock- and glucose and lipid metabolism-related genes in the liver of laying hens.

BMAL1 Links Bevacizumab Resistance in Colorectal Cancer to Circadian Rhythm and Heme Receptor REVERBA

Heme sensor Nuclear Receptor Subfamily 1 Group D Member 1 (NR1D1/REVERBA) and its target gene Aryl Hydrocarbon Receptor Nuclear Translocator-Like (ARNTL/BMAL1) regulate circadian rhythm and angiogenesis. Given their involvement in the interplay between clock disruptions (e.g. by mutations or sleep deprival), cancer risk and vascular biology, we hypothesized that these transcription factors contribute to resistance against anti-angiogenic therapy with vascular endothelial growth factor (VEGFA) neutralizing antibody (bevacizumab, abbrev. Beva) in colorectal cancer (CRC). REVERBA bound to a predicted -672 bp Retinoic Acid Receptor-Related Orphan Receptor Alpha (RORA)-responsive element (RORE) adjacent to a BMAL1 DNA-binding motif (E-box) in the proximal promoter of the human VEGFA gene. REVERBA, indirectly and together with BMAL1, increased VEGFA mRNA and protein expression. Conversely, REVERBA siRNA and REVERBA-antagonist hemin decreased VEGFA synthesis. Beva treatment inhibited CRC growth in a C57BL/6J Apc min/+ genetically modified mouse model (GEMM) and BALB/c nu/nu mouse HCT116 xenografts, without attenuating BMAL1 levels or microvessel density (MVD). In CRC patient tumors (n=74), high BMAL1 protein expression was associated with clinical non-response to combination chemotherapy with Beva (*p=0.0061) and reduced progression-free survival (PFS) [*p=0.0223, Hazard Ratio (HR)=1.69]. In addition, single nucleotide polymorphisms (SNPs) in the BMAL1 (ARNTL) gene correlated with shorter overall survival (OS) [rs11022780, *p=0.014, HR=1.61] and PFS (rs7396943, rs7938307, rs2279287) in patients undergoing combination chemotherapy with Beva. Thus, BMAL1 is associated with Beva resistance in CRC. Since the REVERBA-BMAL1-VEGFA axis can be pharmacologically targeted, interference with this signaling pathway could circumvent resistance to anti-angiogenic therapy in CRC. Funding Statement: This study was supported by grants to EB from the Deutsche Krebshilfe (#108287 and #111086). FE received a fellowship from the “Studienstiftung des deutschen Volkes”. JB was supported by the Translational Physician Scientist (TraPS) Program of the Medical Faculty Mannheim of the Heidelberg University. WW was awarded a fellowship from the Chinese Scholarship Council (#201408080116). ME was supported by the State of Baden-Wurttemberg for “Center of Geriatric Biology and Oncology (ZOBEL) – Perspektivforderung” and “Biology of Frailty - Sonderlinie Medizin”. ATB and ME received funds from the European Commission Seventh Framework Programme (Contract No. 278981 [ANGIOPREDICT]) and are supported by the European Commission Horizon 2020 Programme (Contract No. 754923 [COLOSSUS]). ATB is further supported by Science Foundation Ireland under grant 13/CDA/2183 ”Coloforetell”. HJL was partly funded by the National Cancer Institute (grant number P30CA014089), the Gloria Borges WunderGlo Foundation -The Wunder Project, the Dhont Family Foundation, the San Pedro Peninsula Cancer Guild, the Daniel Butler Research Fund, and the Call to Cure Research Fund Declaration of Interests: The authors declare no conflicts of interest. The reagent mBeva was obtained on the basis of a material transfer agreement (MTA#10082012) from Roche Diagnostics GmbH (Penzberg, Germany). HJL has received clinical trial financial support from Merck Serono and Roche and honoraria for advisory board membership and lectures from Bayer, Boehringer Ingelheim, Genentech, Merck Serono and Roche Ethics Approval Statement: Xenograft studies conformed to the 2010/63/EU guidelines and were approved by the Dept. of Health and Children, Dublin, Ireland (B100/3654) and University College Dublin Animal Research Ethics Committee (P12-27). Therapy studies were conducted in accordance with ethical guidelines (Declaration of Helsinki) and approved by the local ethics committees for each participating site. All patients provided written informed consent for the analysis of molecular correlates.

Circadian Clock Gene Expression in Regions of Interest to Respiratory Control

Circadian rhythms are endogenous, ~24‐hour cycles that synchronize biological functions to external cues known as zeitgebers. Circadian rhythms emerge from self‐sustaining oscillations in a transcription‐translation feedback loop of core clock genes (Bmal1, Clock, Per1 and Per2). The central clock (suprachiasmatic nucleus) and peripheral tissues throughout the body utilize this same circadian machinery. Core clock components also exert tissue‐specific effects, including tissue‐appropriate gene regulation. Although clock gene networks have been established throughout the body, little is known concerning clock genes in the neuromuscular system regulating breathing. We tested the hypothesis that clock genes oscillate in key regions of the respiratory circuit by characterizing mRNA expression patterns of core clock genes in the medulla, ventral spinal cord and diaphragm throughout the day via qPCR. Samples were collected from Sprague Dawley rats entrained to a 12‐hour light/dark cycle at 4 zeitgeber times (ZT), including (n = 8 per group): ZT5 (11am), ZT11 (5pm), ZT17 (11pm) and ZT23 (5am); ZT0 corresponds to beginning of light cycle. Cosinor analysis revealed that Bmal1, Clock, Per1 and Per2 clock genes were rhythmic in all 3 regions (all p&lt;0.01). The expression pattern of Per1 and Per2 was antiphase to Bmal1 and Clock in all regions. We hypothesize that clock genes may influence the potential to elicit serotonin‐dependent respiratory motor plasticity, such as intermittent‐hypoxia‐induced phrenic long‐term facilitation. Thus, we tested the specific hypotheses that BMAL1 protein is expressed within phrenic motor neurons and molecules necessary for phrenic long‐term facilitation (5‐HT2A receptors, BDNF and TrkB) exhibit rhythmic gene expression in brainstem and spinal regions involved in this form of plasticity. Using immunofluorescence, we demonstrate that the clock protein BMAL1 is expressed within cholera toxin B labelled phrenic motor neurons. The plasticity‐related gene Bdnf was expressed rhythmically in medulla and in ventral spinal (C3–C5) homogenates (p&lt;0.001), peaking in the active phase. TrkB mRNA (Ntrk2) was expressed rhythmically within the medulla (p&lt;0.01), but not the ventral spinal cord. 5‐HT2A receptor mRNA (Htr2a) did not exhibit rhythmic expression in either region. In conclusion, daily rhythms are observed in transcripts from clock genes and molecules necessary and sufficient for phrenic long‐term facilitation (e.g. BDNF). Although we did not uncouple cellular clocks from zeitgebers (light/dark) in this initial study, we confirm the potential for local circadian regulation in regions of interest to respiratory motor control.Support or Funding InformationUniversity of Florida Graduate School Preeminence Award (MNK), NIH T32 HD043730 (MDS) and the McKnight Brain Institute (GSM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Male Kidney‐specific Bmal1 Knockout Mice Show a Similar, Exaggerated Active‐Inactive Period Difference in Ammonia Excretion to Controls after Potassium Deprivation

The influence of the circadian clock is manifested in the time‐of‐day variation in commonly recognized renal function parameters such as blood pressure and sodium excretion. Acid‐base balance is another important component of renal function, however the effect of the circadian clock on the diurnal variation of acid excretion, specifically the excretion of ammonia (NH3), has not yet been determined. We used a kidney‐specific Bmal1 knockout mouse model (KS‐Bmal1KO) created by crossing a distal nephron‐specific Ksp‐cadherin cre mouse with mice containing two loxP sites flanking exon 8 of the Bmal1 gene. Based on differences our lab has seen in the expression of genes related to acid‐base homeostasis between KS‐Bmal1KO mice and their littermate Cre‐ controls (CNTL), we hypothesized that there would be genotype differences in urinary NH3 excretion. Male control and KS‐Bmal1KO mice were placed in metabolic cages and had their urine collected in 12‐hour intervals, spanning the active (6pm‐6am) or inactive (6am‐6pm) periods. After three days on a normal potassium (K+) diet, the mice were switched to a nominally potassium‐free (0 K+) diet for five days with free access to water. Urine samples from the last two time points of the normal K+ and 0 K+ diets were analyzed for NH3. Under a normal K+ diet, CNTL mice displayed a significant time‐of‐day difference in urinary NH3 excretion, with values greater during the active period compared to the inactive period (66 ± 10 vs. 19 ± 5 μmol/12hr NH3, n=5, P&lt;.05). KS‐Bmal1KO exhibited a similar pattern on normal K+, excreting more NH3 during the active period (78 ± 3 vs. 16 ± 3 μmol/12hr NH3, n=5, P&lt;.05). After five days on the 0 K+ diet, the active‐inactive (A‐I) difference of NH3 excretion was exacerbated in CNTL mice, resulting in a significantly greater difference compared to normal K+diet (137 ± 15 vs. 47 ± 10 μmol/12hr NH3 P&lt;0.05). KS‐Bma11KO mice also displayed an increased A‐I difference compared to their normal K+ diet values (142 ± 33 vs. 62 ± 5 μmol/12hr NH3, P&lt;0.05). There were no differences however, in the A‐I difference between genotypes. Despite not observing any major effects of Bmal1 deletion in the distal nephron of the kidney on NH3 excretion under the conditions tested, these results suggest that the time‐of‐day differences in NH3 excretion observed in male mice are augmented in response to dietary K+ deprivation.Support or Funding InformationThis research was supported in part by the research project grant on Per1 and the Kidney Clock in Hypertension (NIH grant DK109570‐02), and the T32 training grant on Pre‐ and Postdoctoral Training in Nephrology and Hypertension (T32 DK104721).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Circadian oscillation of TNF-α gene expression regulated by clock gene, BMAL1 and CLOCK1, in the Japanese medaka (Oryzias latipes)

To date, little information is available on the effects of circadian oscillation on immune regulation in lower vertebrates, such as teleost fish. In the present study, regulation of circadian oscillation of inflammatory cytokine TNF-α gene expression by clock gene was investigated using model fish medaka (Oryzias latipes). Firstly, structural analysis of clock genes was performed, which revealed that medaka BMAL1 and CLOCK1 conserve functionally important domains, such as basic helix-loop-helix (bHLH) and period-aryl hydrocarbon receptor nuclear translocator-single-minded (PAS), seen in their counterparts in other vertebrates. Expression of medaka Bmal1, Clock1, and Per1 genes was confirmed in central and peripheral tissues. Moreover, the expression of these clock genes and TNF-α genes in medaka acclimated to a 12:12 light (L) - dark (D) cycle showed circadian oscillation. In addition, higher expression of TNF-α gene was detected in medaka embryo cells (OLHdrR-e3) overexpressing Bmal1 and Clock1 genes. It was suggested that this increase was mediated by transcriptional regulation by clock proteins, which target E-box sequence in the cis-element of TNF-α gene as was detected by luciferase reporter gene assay. Moreover, in vitro head kidney stimulation with LPS at different zeitgeber time (ZT) under LD12:12 condition affected the degree of TNF-α gene expression, which shows high and low responsiveness to LPS stimulation at ZT18 and ZT10, respectively. These results suggested that fish TNF-α exhibited circadian oscillation regulated by clock proteins and its responsiveness against immune-stimulation depends on time zone.

Intestinal clock system regulates skeletal homeostasis.

The circadian clock network is an evolutionarily conserved system involved in the regulation of metabolic homeostasis; however, its impacts on skeletal metabolism remain largely unknown. We herein demonstrated that the circadian clock network in the intestines plays pivotal roles in skeletal metabolism such that the lack of the Bmal1 gene in the intestines (Bmal1Int-/- mice) caused bone loss, with bone resorption being activated and bone formation suppressed. Mechanistically, Clock protein interaction with the vitamin D receptor (VDR) accelerated its binding to the VDR response element by enhancing histone acetylation in a circadian-dependent manner, and this was lost in Bmal1Int-/- mice because nuclear translocation of Clock required the presence of Bmal1. Accordingly, the rhythmic expression of VDR target genes involved in transcellular calcium (Ca) absorption was created, and this was not observed in Bmal1Int-/- mice. As a result, transcellular Ca absorption was impaired and bone resorption was activated in Bmal1Int-/- mice. Additionally, sympathetic tone, the activation of which suppresses bone formation, was elevated through afferent vagal nerves in Bmal1Int-/- mice, the blockade of which partially recovered bone loss by increasing bone formation and suppressing bone resorption in Bmal1Int-/- mice. These results demonstrate that the intestinal circadian system regulates skeletal bone homeostasis.

The positive circadian regulators CLOCK and BMAL1 control G2/M cell cycle transition through Cyclin B1

ABSTRACTWe previously identified a tight bidirectional phase coupling between the circadian clock and the cell cycle. To understand the role of the CLOCK/BMAL1 complex, representing the main positive regulator of the circadian oscillator, we knocked down Bmal1 or Clock in NIH3T33C mouse fibroblasts (carrying fluorescent reporters for clock and cell cycle phase) and analyzed timing of cell division in individual cells and cell populations. Inactivation of Bmal1 resulted in a loss of circadian rhythmicity and a lengthening of the cell cycle, originating from delayed G2/M transition. Subsequent molecular analysis revealed reduced levels of Cyclin B1, an important G2/M regulator, upon suppression of Bmal1 gene expression. In complete agreement with these experimental observations, simulation of Bmal1 knockdown in a computational model for coupled mammalian circadian clock and cell cycle oscillators (now incorporating Cyclin B1 induction by BMAL1) revealed a lengthening of the cell cycle. Similar data were obtained upon knockdown of Clock gene expression. In conclusion, the CLOCK/BMAL1 complex controls cell cycle progression at the level of G2/M transition through regulation of Cyclin B1 expression.

Intake of an Obesogenic Cafeteria Diet Affects Body Weight, Feeding Behavior, and Glucose and Lipid Metabolism in a Photoperiod-Dependent Manner in F344 Rats.

We previously demonstrated that chronic exposure to different photoperiods induced marked variations in several glucose and lipid metabolism-related parameters in normoweight Fischer 344 (F344) rats. Here, we examined the effects of the combination of an obesogenic cafeteria diet (CAF) and the chronic exposure to three different day lengths (L12, 12 h light/day; L18, 18 h light/day; and L6, 6 h light/day) in this rat strain. Although no changes were observed during the first 4 weeks of adaptation to the different photoperiods in which animals were fed a standard diet, the addition of the CAF for the subsequent 7 weeks triggered profound physiologic and metabolic alterations in a photoperiod-dependent manner. Compared with L12 rats, both L6 and L18 animals displayed lower body weight gain and cumulative food intake in addition to decreased energy expenditure and locomotor activity. These changes were accompanied by differences in food preferences and by a sharp upregulation of the orexigenic genes Npy and Ghsr in the hypothalamus, which could be understood as a homeostatic mechanism for increasing food consumption to restore body weight control. L18 rats also exhibited higher glycemia than the L6 group, which could be partly attributed to the decreased pAkt2 levels in the soleus muscle and the downregulation of Irs1 mRNA levels in the gastrocnemius muscle. Furthermore, L6 animals displayed lower whole-body lipid utilization than the L18 group, which could be related to the lower lipid intake and to the decreased mRNA levels of the fatty acid transporter gene Fatp1 observed in the soleus muscle. The profound differences observed between L6 and L18 rats could be related with hepatic and muscular changes in the expression of circadian rhythm-related genes Cry1, Bmal1, Per2, and Nr1d1. Although further research is needed to elucidate the pathophysiologic relevance of these findings, our study could contribute to emphasize the impact of the consumption of highly palatable and energy dense foods regularly consumed by humans on the physiological and metabolic adaptations that occur in response to seasonal variations of day length, especially in diseases associated with changes in food intake and preference such as obesity and seasonal affective disorder.