Disturbance of suprachiasmatic nucleus function improves cardiac repair after myocardial infarction by IGF2-mediated macrophage transition.

Suprachiasmatic nucleus (SCN) in mammals functions as the master circadian pacemaker that coordinates temporal organization of physiological processes with the environmental light/dark cycles. But the causative links between SCN and cardiovascular diseases, specifically the reparative responses after myocardial infarction (MI), remain largely unknown. In this study we disrupted mouse SCN function to investigate the role of SCN in cardiac dysfunction post-MI. Bilateral ablation of the SCN (SCNx) was generated in mice by electrical lesion; myocardial infarction was induced via ligation of the mid-left anterior descending artery (LAD); cardiac function was assessed using echocardiography. We showed that SCN ablation significantly alleviated MI-induced cardiac dysfunction and cardiac fibrosis, and promoted angiogenesis. RNA sequencing revealed differentially expressed genes in the heart of SCNx mice from D0 to D3 post-MI, which were functionally associated with the inflammatory response and cytokine-cytokine receptor interaction. Notably, the expression levels of insulin-like growth factor 2 (Igf2) in the heart and serum IGF2 concentration were significantly elevated in SCNx mice on D3 post-MI. Stimulation of murine peritoneal macrophages in vitro with serum isolated from SCNx mice on D3 post-MI accelerated the transition of anti-inflammatory macrophages, while antibody-mediated neutralization of IGF2 receptor blocked the macrophage transition toward the anti-inflammatory phenotype in vitro as well as the corresponding cardioprotective effects observed in SCNx mice post-MI. In addition, disruption of mouse SCN function by exposure to a desynchronizing condition (constant light) caused similar protective effects accompanied by elevated IGF2 expression on D3 post-MI. Finally, mice deficient in the circadian core clock genes (Ckm-cre; Bmal1f/f mice or Per1/2 double knockout) did not lead to increased serum IGF2 concentration and showed no protective roles in post-MI, suggesting that the cardioprotective effect observed in this study was mediated particularly by the SCN itself, but not by self-sustained molecular clock. Together, we demonstrate that inhibition of SCN function promotes Igf2 expression, which leads to macrophage transition and improves cardiac repair post-MI.

Association of Circadian Clock Gene Expression with Pediatric/Adolescent Asthma and Its Comorbidities.

The pathology of asthma is characterized by marked day-night variation, which is likely controlled by circadian clock activity. This study aimed to clarify the association of core circadian clock gene expression with clinical features of asthma. For this purpose, we accessed the National Center for Biotechnology Information database and analyzed transcriptomes of peripheral blood mononuclear cells and clinical characteristics of 134 pediatric/adolescent patients with asthma. Based on the expression patterns of seven core circadian clock genes (CLOCK, BMAL1, PER1-3, CRY1-2), we identified three circadian clusters (CCs) with distinct comorbidities and transcriptomic expressions. In the three CC subtypes, allergic rhinitis, and atopic dermatitis, both asthma comorbidities occurred in different proportions: CC1 had a high proportion of allergic rhinitis and atopic dermatitis; CC2 had a high proportion of atopic dermatitis but a low proportion of allergic rhinitis; and CC3 had a high proportion of allergic rhinitis but a low proportion of atopic dermatitis. This might be associated with the low activity of the FcεRI signaling pathway in CC2 and the cytokine-cytokine receptor interaction pathways in CC3. This is the first report to consider circadian clock gene expression in subcategories of patients with asthma and to explore their contribution to pathophysiology and comorbidity.

Persistent myopathy despite release of partial obstruction: in vivo reversal of dysfunction and transcriptional responses using rapamycin.

Current and potential medical therapy for obstruction-induced myopathic bladder dysfunction (from benign prostatic hyperplasia or posterior urethral valves) focuses on symptoms. The persistent tissue pathology and dysfunction after release of obstruction is often deemed irreversible without any systematic therapeutic approaches. As rapamycin can attenuate bladder smooth muscle hypertrophy and dysfunction during the genesis of partial obstruction in vivo, we tested whether rapamycin could improve persistent function after release of obstruction (de-obstruction or REL). Female Sprague-Dawley rat bladders were partially obstructed (PBO) by suturing around both the urethra and a para-urethral steel rod, then removing the rod. One day prior to release of obstruction (preREL), voiding parameters and residual urine volume of preREL+future rapa, preREL+future veh groups were recorded. Release of obstruction (REL) was performed by suture removal following 6 weeks of PBO. For 4 more weeks after the de-obstruction, REL animals were randomized to rapamycin (REL+rapa) or vehicle (REL+veh). PBO for 6 weeks were used as positive controls. In shams, the urethra was exposed, but no suture tied. Voiding parameters and residual urine volume were measured prior to sacrifice of sham and REL+veh or REL+rapa, and PBO. Rapamycin efficacy was tested by pair-wise comparison of changes in individual voiding data from preREL+future veh or preREL+future rapa versus REL+veh or REL+rapa, respectively, as well as by comparisons of REL+veh to REL+rapa groups. Bladders were weighed and processed for a high-throughput QPCR array, and histopathology. Bladder/body mass ratios with PBO increased significantly and remained higher in the release phase in REL+veh animals. REL+rapa versus REL+veh improved residual volumes and micturition fractions toward sham levels. Three genes encoding extracellular proteins, BMP2, SOD3, and IGFBP7, correlated with functional improvement by Pearson's correlations. The promoters of these genes showed enrichment for several motifs including circadian E-boxes. While obstruction and REL augmented CLOCK and NPAS2 expression above sham levels, rapamycin treatment during release significantly blocked their expression. This experimental design of pharmaco-intervention during the de-obstruction phase revealed a novel pathway dysregulated during the clinically relevant treatment phase of obstructive bladder myopathy.

Hydrogen sulfide regulates circadian-clock genes in C2C12 myotubes and the muscle of high-fat-diet-fed mice.

Hydrogen sulfide (H2S) is an endogenous novel gasotransmitter which is implicated in the pathophysiology of the metabolic syndrome. Core clock genes (CCG) and its controlled genes disruption is implicated in the progression of metabolic syndrome. We examined whether H2S has any effect on CCG in the skeletal muscle of mice fed a high-fat diet (HFD) and in myotubes. In the muscle of HFD-mice, the expression of H2S biosynthesis enzyme genes (CSE, CBS, and 3-Mpst) along with antioxidant genes (GCLC, GCLM, GSS, and GSR) involved in GSH biosynthesis and recycling were reduced significantly, but the oxidative stress (OS) increased. Expression of the CCG (Bmal1, Clock, RORα, Cry2, Per2) and clock-controlled genes (PPARγ, PGC-1α, RXRα) was downregulated, whereas the levels of PPARα mRNA were upregulated. Similar to that in the muscle of HFD-mice, in vitro myotubes exposed to high glucose or palmitate to mimic metabolic syndrome, showed an increased OS and decreased in CSE mRNA, H2S production and CCG mRNA levels were also downregulated. TNF and MCP-1 treatment on the myotubes was similar to that observed in HFD-muscle, with that the Rev-erbα mRNA was upregulated. Inhibition (siRNA/pharmacological inhibitors) of both CSE and GCLC (the rate-limiting enzyme in GSH biosynthesis) decreased H2S, and increased OS; Bmal1 and Clock mRNA levels were downregulated, while Rev-erbα increased significantly in these conditions. CSE KD myotubes were post-treated with an H2S donor partially restored the mRNA levels of core clock genes. These findings report that the deficiencies of H2S/GSH impair expression of CCG and treatment with H2S donor or GSH precursor exert a positive effect over CCG. Thus, suggest that H2S as a new endogenous factor for regulating circadian clock, and its donors could provide a novel chrono-pharmacological therapy to manage metabolic disorders.

The Circadian Clock as a Potential Biomarker and Therapeutic Target in Gastrointestinal Cancers.

The circadian clock consists of a hierarchical multi-oscillator network of intracellular and intercellular mechanisms throughout the body that contributes to anticipating metabolic activity and maintaining system homeostasis in response to environmental cues and intrinsic stimuli. Over the past few years, genetic variations of core clock genes have been associated with cancer risk in several epidemiological studies. A growing number of epidemiological research studies have demonstrated a direct correlation between the disturbance of circadian rhythms and the growth of tumors, indicating that shift workers are more susceptible to malignancies of the colon, prostate, ovarian, breast, lung, and liver. One of the most related cancers with circadian rhythm is Gastrointestinal (GI) cancer, which is a leading cause of cancer-related mortality nowadays. The aim of this review was to demonstrate the effect of the clock gene network on the growth of GI cancer, providing molecular targets for GI cancer treatment, possible prognostic biomarkers, and guidance for treatment choices.

Circadian Rhythm Alteration of the Core Clock Genes and the Lipid Metabolism Genes Induced by High-Fat Diet (HFD) in the Liver Tissue of the Chinese Soft-Shelled Turtle (Trionyx sinensis).

Physiology disorders of the liver, as it is an important tissue in lipid metabolism, can cause fatty liver disease. The mechanism might be regulated by 17 circadian clock genes and 18 fat metabolism genes, together with a high-fat diet (HFD). Due to their rich nutritional and medicinal value, Chinese soft-shelled turtles (Trionyx sinensis) are very popular among the Chinese people. In the study, we aimed to investigate the influence of an HFD on the daily expression of both the core clock genes and the lipid metabolism genes in the liver tissue of the turtles. The two diets were formulated with 7.98% lipid (the CON group) and 13.86% lipid (the HFD group) to feed 180 juvenile turtles, which were randomly divided into two groups with three replicates per group and 30 turtles in each replicate for six weeks, and the diet experiment was administrated with a photophase regimen of a 24 h light/dark (12L:12D) cycle. At the end of the experiment, the liver tissue samples were collected from nine turtles per group every 3 h (zeitgeber time: ZT 0, 3, 6, 9, 12, 15, 18, 21 and 24) for 24 h to investigate the daily expression and correlation analysis of these genes. The results showed that 11 core clock genes [i.e., circadian locomotor output cycles kaput (Clock), brain and muscle arnt-like protein 1 and 2 (Bmal1/2), timeless (Tim), cryptochrome 1 (Cry2), period2 (Per2), nuclear factor IL-3 gene (Nfil3), nuclear receptor subfamily 1, treatment D, member 1 and 2 (Nr1d1/2) and retinoic acid related orphan receptor α/ß/γ ß and γ (Rorß/γ)] exhibited circadian oscillation, but 6 genes did not, including neuronal PAS domain protein 2 (Npas2), Per1, Cry1, basic helix-loop-helix family, member E40 (Bhlhe40), Rorα and D-binding protein (Dbp), and 16 lipid metabolism genes including fatty acid synthase (Fas), diacylglycerol acyltransferase 1 (Dgat1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), Low-density lipoprotein receptor-related protein 1-like (Ldlr1), Lipin 1 (Lipin1), Carnitine palmitoyltransferase 1A (Cpt1a), Peroxisome proliferator activation receptor α, ß and γ (Pparα/ß/γ), Sirtuin 1 (Sirt1), Apoa (Apoa1), Apolipoprotein B (Apob), Pyruvate Dehydrogenase kinase 4 (Pdk4), Acyl-CoA synthase long-chain1 (Acsl1), Liver X receptors α (Lxrα) and Retinoid X receptor, α (Rxra) also demonstrated circadian oscillations, but 2 genes did not, Scd and Acaca, in the liver tissues of the CON group. However, in the HFD group, the circadian rhythms' expressional patterns were disrupted for the eight core clock genes, Clock, Cry2, Per2, Nfil3, Nr1d1/2 and Rorß/γ, and the peak expression of Bmal1/2 and Tim showed delayed or advanced phases. Furthermore, four genes (Cry1, Per1, Dbp and Rorα) displayed no diurnal rhythm in the CON group; instead, significant circadian rhythms appeared in the HFD group. Meanwhile, the HFD disrupted the circadian rhythm expressions of seven fat metabolism genes (Fas, Cpt1a, Sirt1, Apoa1, Apob, Pdk4 and Acsl1). Meanwhile, the other nine genes in the HFD group also showed advanced or delayed expression peaks compared to the CON group. Most importantly of all, there were remarkably positive or negative correlations between the core clock genes and the lipid metabolism genes, and their correlation relationships were altered by the HFD. To sum up, circadian rhythm alterations of the core clock genes and the lipid metabolism genes were induced by the high-fat diet (HFD) in the liver tissues of T. sinensis. This result provides experimental and theoretical data for the mass breeding and production of T. sinensis in our country.

PPARG stimulation restored lung mRNA expression of core clock, inflammation- and metabolism-related genes disrupted by reversed feeding in male mice.

The circadian rhythm system regulates lung function as well as local and systemic inflammations. The alteration of this rhythm might be induced by a change in the eating rhythm. Peroxisome proliferator-activated receptor gamma (PPARG) is a key molecule involved in circadian rhythm regulation, lung functions, and metabolic processes. We described the effect of the PPARG agonist pioglitazone (PZ) on the diurnal mRNA expression profile of core circadian clock genes (Arntl, Clock, Nr1d1, Cry1, Cry2, Per1, and Per2) and metabolism- and inflammation-related genes (Nfe2l2, Pparg, Rela, and Cxcl5) in the male murine lung disrupted by reversed feeding (RF). In mice, RF disrupted the diurnal expression pattern of core clock genes. It decreased Nfe2l2 and Pparg and increased Rela and Cxcl5 expression in lung tissue. There were elevated levels of IL-6, TNF-alpha, total cells, macrophages, and lymphocyte counts in bronchoalveolar lavage (BAL) with a significant increase in vascular congestion and cellular infiltrates in male mouse lung tissue. Administration of PZ regained the diurnal clock gene expression, increased Nfe2l2 and Pparg expression, and reduced Rela, Cxcl5 expression and IL-6, TNF-alpha, and cellularity in BAL. PZ administration at 7 p.m. was more efficient than at 7 a.m.

Diurnal Characteristics of the Orexin System Genes and Its Effects on Pathology at Early Stage in 3xTg-AD Mice.

Orexin and its receptors are closely related to the pathogenesis of Alzheimer's disease (AD). Although the expression of orexin system genes under physiological condition has circadian rhythm, the diurnal characteristics of orexin system genes, and its potential role in the pathogenesis in AD are unknown. In the present study, we hope to elucidate the diurnal characteristics of orexin system genes at the early stage of AD, and to investigate its potential role in the development of AD neuropathology. We firstly detected the mRNA levels of orexin system genes, AD risk genes and core clock genes (CCGs) in hypothalamus and hippocampus in 6-month-old male 3xTg-AD mice and C57BL/6J (wild type, WT) control mice, then analyzed diurnal expression profiles of all genes using JTK_CYCLE algorithm, and did the correlation analysis between expression of orexin system genes and AD risk genes or CCGs. In addition, the expression of ß-amyloid protein (Aß) and phosphorylated tau (p-tau) protein were measured. The results showed that the diurnal mRNA expression profiles of PPO, OX1R, OX2R, Bace2, Bmal1, Per1, Per2 and Cry1 in the hypothalamus, and gene expression of OX1R, OX2R, Bace1, Bmal1, Per1 and Cry2 in the hippocampus in 3xTg-AD mice were different from that in WT mice. Furthermore, there is positive correlation between orexin system genes and AD risk genes or CCGs in the brain in 3xTg-AD mice. In addition, the expression of Aß and p-tau in hippocampus in 3xTg-AD mice were significantly increased, and the expression of p-tau is higher in night than in day. These results indicate that the abnormal expression profiles of orexin system genes and its interaction with AD risk genes or CCGs might exert important role in the pathogenesis of AD, which will increase the expression of Aß and p-tau, and accelerate the development of AD.

Clock at the Core of Cancer Development.

To synchronize various biological processes with the day and night cycle, most organisms have developed circadian clocks. This evolutionarily conserved system is important in the temporal regulation of behavior, physiology and metabolism. Multiple pathological changes associated with circadian disruption support the importance of the clocks in mammals. Emerging links have revealed interplay between circadian clocks and signaling networks in cancer. Understanding the cross-talk between the circadian clock and tumorigenesis is imperative for its prevention, management and development of effective treatment options. In this review, we summarize the role of the circadian clock in regulation of one important metabolic pathway, insulin/IGF1/PI3K/mTOR signaling, and how dysregulation of this metabolic pathway could lead to uncontrolled cancer cell proliferation and growth. Targeting the circadian clock and rhythms either with recently discovered pharmaceutical agents or through environmental cues is a new direction in cancer chronotherapy. Combining the circadian approach with traditional methods, such as radiation, chemotherapy or the recently developed, immunotherapy, may improve tumor response, while simultaneously minimizing the adverse effects commonly associated with cancer therapies.

Moxibustion benignantly regulates circadian rhythm of REV-ERBα in RA rats.

The key clinical symptoms and previous findings of RA show a circadian variation, with more prominent joint swelling, stiffness, and pain occurring in the early morning. Moxibustion is able to relieve RA in various pass ways, however, there is no verifying study results for the pathological rhythm of RA. Therefore, we conducted this work to verify whether moxibustion could adjust RA circadian rhythm according to regulate core clock genes. Based on these previous findings that circadian timekeeping is disturbed in RA at molecular level, the aim of this study was to observe the influence of moxibustion on expression level and circadian rhythm of REV-ERBα at different tissues of RA rats. Furthermore, the expression level of core clock genes closely related to RA were evaluated by RT-PCR. 96 SD rats were randomly assigned as 1:1:1:1 ratio to 4 groups for normal control group, RA model group, 5-7 am moxibustion group, and 5-7 pm moxibustion group. RT-PCR was used to measure the relatively expression quantity of REV-ERBα, CLOCK, BMAL1, and PER2 in hypothalamus, hippocampus, and adrenal gland. In RA rats, the expression level of REV-ERBα mRNA were up-regulated in different tissues, and moxibustion potentially up-regulated them in different degrees. In untreated RA rats, the circadian rhythm of REV-ERBα mRNA in hippocampus and adrenal gland both disappeared (P>0.05) and moxibustion was able to recover them (P<0.05). The expression level of CLOCK and PER2 mRNA in hippocampus and adrenal gland were down-regulated significantly (P<0.05) in RA model rats, while moxibustion up-regulated both of them in hippocampus (P<0.05). These results suggested together that moxibustion can benign regulate circadian rhythm of REV-ERBα in different tissues of RA rats. It was revealed that moxibustion not only recovered the losing diurnal oscillation of REV-ERBα in hippocampus and adrenal gland, but also adjusted the circadian rhythm of REV-ERBα in hypothalamus, hippocampus, and adrenal gland to close the normal circadian pattern.

The effect of high fat diet on daily rhythm of the core clock genes and muscle functional genes in the skeletal muscle of Chinese soft-shelled turtle (Trionyx sinensis).

In the present study, we sought to investigate the influence of high fat diet on the core clock genes and the muscle functional genes daily expression in the skeletal muscle of Chinese soft-shelled turtle. The turtles were fed by two diets including a control fat diet (the CON treatment, 7.98% lipid) and a high fat diet (the HFD treatment, 13.86% lipid) for six weeks and administrated by the photophase regimen of 24h light/dark (12L:12D) cycle. After the feeding trial experiment, we measured the daily expression levels of 17 core clock genes (Clock, Bmal1/2, NPAS2, Tim, Cry1/2, Per1/2, DBP, AANAT, NIFL3, BHLHE40, NR1D2, RORA, RORB, RORC) and 12 muscle functional genes (FBXO32, MBNL1, MSTN, Myf5, Myf6, MyoD, MyoG, MyoM1, PPARa, PDK4, Trim63, UCP3) in the skeletal muscle of the two treatments. The results showed that except for Bmal1, NPAS2, Per2 and RORB, the expression of the other 13 core clock genes exhibited circadian oscillation in the CON treatment. Among the 12 muscle functional genes, MBNL1, PDK4 and MyoM1 did not exhibit circadian oscillation in the CON treatment. In the HFD treatment, the circadian rhythms expressional patterns of the 8 core clock genes (Clock, Bmal2, Cry2, Per1, DBP, NFIL3, BHLHE40 and RORA) and 6 muscle functional genes (MSTN, Myf5, MyoD, MyoG, PPARa and Trim63) were disrupted. In addition, compared with the CON treatment, the circadian expression of the 5 core clock genes (Tim, Cry1, AANAT, NR1D2, RORC) and the 3 muscle functional genes (FBXO32, Myf6, UCP3) showed the advanced or delayed expression peaks in the HFD treatment. In CON treatment, the circadian expression of the MyoG, MyoD, Myf6, FBXO32 and PPARa showed positive or negative correlation with the transcription pattern of Clock, Bmal2, Cry1/2, Per1/2. However, only the FBXO32 and Myf6 presented positive or negative correlation with the circadian expression of Cry1, RORB, AANAT and Tim in HFD treatment. In summary, these results demonstrate that the disruption of the circadian rhythm of the core clock genes and muscle functional genes in the skeletal muscle is closely associated with feeding high fat diet to Chinese soft-shelled turtle.

Evidence for an internal and functional circadian clock in rat pituitary cells.

In primary cultures of rat pituitary cells and in a pituitary sommatolactotroph cell line (GH4C1), endogenous core-clock- as well as hormone-genes such as prolactin displayed a rhythmic expression pattern, fitted by a sinusoidal equation in which the period value was close to the circadian one. This is consistent with the presence of a functional circadian oscillator in pituitary cells whose importance was ascertained in GH4C1 cell lines stably expressing a dominant negative mutant of BMAL1. In these cells, both endogenous core-clock- and prolactin-genes no more displayed a circadian pattern. Some genes we recently identified as mouse pituitary BMAL1-regulated genes in a DNA-microarray study, lost their circadian pattern in these cells, suggesting that BMAL1 controlled these genes locally in the pituitary. The intra-pituitary circadian oscillator could then play a role in the physiology of the gland that would not be seen anymore as a structure only driven by hypothalamic rhythmic control.