Per2 Gene Expression Research Articles

Effects of Acrolein on the Proliferation of and Per1 Gene Expression in Pulmonary Epithelial Cells

To investigate the effect of acrolein on the proliferation of pulmonary epithelial cells and its possible mechanism. Two strains of pulmonary epithelial cells, A549 cells and MLE15 cells, were used as in vitro models of pulmonary epithelial cell, and were treated with 80 μmol/L acrolein or phosphate buffer saline (PBS) as the control. The proliferation of pulmonary epithelial cells were determined with CCK-8 kit after cell culturing resumed for 12 h, 24 h, 36 h and 48 h post acrolein treatment, and the expression of period circadian regulator gene 1 ( Per1) was examined using Western blot test 24 h after acrolein treatment. In addition, after acrolein treatment, the cells were restored with transforming growth factor-β (TGF-β) added in the medium, and the cell proliferation and the expression of Per1 protein were also examined. The proliferation of A549 cells and MLE15 cells decreased significantly after being treated with 80 μmol/L acrolein for 30 min, and the expression of Per1 protein was also downregulated significantly ( P<0.05). The addition of TGF-β after acrolein treatment did not significantly change the reduction in cell proliferation caused by acrolein, but the expression of Per1 protein in pulmonary epithelial cells was significantly higher than that in cells restored without TGF-β ( P<0.05). Acrolein treatment resulted in the decreased proliferation of pulmonary epithelial cells and the Per1 expression in pulmonary epithelial cells. Although TGF-β addition did not reverse the reduction of cell proliferation after acrolein treatment, the Per1 expression levels were recovered to a certain extent compared to that in cells restored in medium without TGF-β after acrolein treatment.

Clock gene per 2 daily rhythm: Correlation with the serum level of uncoupling protein 1 (UCP1) in goat and horse.

Recent studies evidenced that the circadian rhythm of Per2 is involved in adaptive thermogenesis by the modulating transcription of uncoupling protein 1 (UCP1). For this purpose, we investigated the linkage between the daily rhythm of Per2 and UCP1 in ruminant and non-ruminant mammalian species. Five clinically healthy, not pregnant, and not lactating Maltese female goats and five clinically healthy, not pregnant, and not lactating Italian Saddle horses were enrolled in the study. All animals were housed under natural photoperiod (sunrise 05:05, sunset 20:55) and environmental temperature and humidity. Goats were kept individually in 3.0×2.0m box, horses were housed individually in 3.5×3.5m box; all boxes were equipped with an opening window. On each subject, blood samples were collected every 4h for a 48-h period. The Per2 gene expression was determined on blood samples collected in PAX gene Blood RNA Tube, whereas UCP1 concentration was assessed on serum. Per2 and UCP1 levels were statistically influenced by the species (p<0.0001) and the time of data collection (p<0.0001), but not by the day of monitoring. Per2 showed daily rhythmicity, statistically different in mesor and amplitude between the two species, diurnal in goats, nocturnal in horses; with the same robustness. UCP1 did not show daily rhythmicity. During the experimental period the two parameters showed a negative correlation in horses. According to the findings herein obtained, we can claim that the role of Per2 in the thermogenesis induced by the beige adipocytes throughout UCP1 activation did not reflect what found in other mammal species, but further studies are required to establish their correlation in equids.

Abnormal social behavior and altered gene expression in mice lacking NDRG2

N-myc downstream-regulated gene 2 (NDRG2), a member of the NDRG family, has multiple functions in cell proliferation, differentiation, and stress responses, and is predominantly expressed by astrocytes in the central nervous system. Previous studies including ours demonstrated that NDRG2 is involved in various central nervous system pathologies. However, the significance of NDRG2 in neurodevelopment is not fully understood. Here, we investigated the expression profile of NDRG2 during postnatal brain development, the role of NDRG2 in social behavior, and transcriptome changes in the brain of NDRG2-deficient mice. NDRG2 expression in the brain increased over time from postnatal day 1 to adulthood. Deletion of NDRG2 resulted in abnormal social behavior, as indicated by reduced exploratory activity toward a novel mouse in a three-chamber social interaction test. Microarray analysis identified genes differentially expressed in the NDRG2-deficient brain, and upregulated gene expression of Bmp4 and Per2 was confirmed by quantitative PCR analysis. Expression of both these genes and the encoded proteins increased over time during postnatal brain development, similar to NDRG2. Gene expression of Bmp4 and Per2 was upregulated in cultured astrocytes isolated from NDRG2-deficient mice. These results suggest that NDRG2 contributes to brain development required for proper social behavior by modulating gene expression in astrocytes.

Beneficial effects of daytime high-intensity light exposure on daily rhythms, metabolic state and affect

While the importance of the circadian system to health and well-being is extensively studied, the role of daylight exposure in these interactions is relatively poorly understood. Here we show, using a diurnal animal model naturally exposed to daylight, that daily morning exposure to 3000 lux, full spectrum electric light has beneficial health effects. Compared with controls, sand rats (Psammomys obesus) subjected to morning light treatment demonstrate daily rhythms with high peak to trough difference in activity, blood glucose levels and per2 gene expression in the suprachiasmatic nucleus, pre-frontal cortex, kidney and liver. The treated animals were also healthier, being normoglycemic, having higher glucose tolerance, lower body and heart weight and lower anxiety- and depression-like behavior. Our results suggest that exposure to high intensity light is important for the proper function of the circadian system and well-being, and are important in face of human's low exposure to daylight and extensive use of artificial light at night.

Contraction influences Per2 gene expression in skeletal muscle through a calcium-dependent pathway.

Key points Exercising at different times of day elicits different effects on exercise performance and metabolic health. However, the specific signals driving the observed time‐of‐day specific effects of exercise have not been fully identified.Exercise influences the skeletal muscle circadian clock, although the relative contribution of muscle contraction and extracellular signals is unknown.Here, we show that contraction acutely increases the expression of the core circadian clock gene Period Circadian Regulator 2 (Per2) and phase‐shifts Per2 rhythmicity in muscle cells. This contraction effect on core clock genes is mediated through a calcium‐dependant mechanism;The results obtained in the present study suggest that a proportion of the ability of exercise to entrain the skeletal muscle clock is driven directly by muscle contraction. Contraction interventions may be used to mimic some time‐of‐day specific effects of exercise on metabolism and muscle performance. Exercise entrains the central and peripheral circadian clocks, although the mechanism by which exercise modulates expression of skeletal muscle clock genes is unclear. The present study aimed to determine whether skeletal muscle contraction alone could directly influence circadian rhythmicity and uncover the underlying mechanism by which contraction modulates clock gene expression. We investigated the expression of core clock genes in human skeletal muscle after acute exercise, as well as following in vitro contraction in mouse soleus muscle and cultured C2C12 skeletal muscle myotubes. Additionally, we interrogated the molecular pathways by which skeletal muscle contraction could influence clock gene expression. Contraction acutely increased the expression of the core circadian clock gene Period Circadian Regulator 2 (Per2) and phase‐shifted Per2 rhythmicity in C2C12 myotubes in vitro. Further investigation revealed that pharmacologically increasing cytosolic calcium concentrations by ionomycin treatment mimicked the effect of contraction on Per2 expression. Similarly, treatment with a calcium channel blocker, nifedipine, blocked the effect of electric pulse stimulation‐induced contraction on Per2 expression. Increased calcium influx from contraction lead to binding of the phosphorylated form of cAMP response element‐binding protein (CREB) to the Per2 promoter, suggesting a role of CREB in contraction‐induced Per2 transcription. Thus, by dissociating the effect of muscle contraction alone from the whole effect of exercise, our investigations indicate that a proportion of the ability of exercise to entrain the skeletal muscle clock is driven directly by contraction.

MicroRNA-25 as a novel modulator of circadian Period2 gene oscillation

Circadian clock controls an organism’s biological rhythm and regulates its physiological processes in response to external time cues. Most living organisms have their own time-keeping mechanism that is maintained by transcriptional–translational autoregulatory feedback loops involving several core clock genes, such as Period. Recent studies have found the relevance between the modulation of circadian oscillation and posttranscriptional modifications by microRNAs (miRNAs). However, there are limited studies on candidate miRNAs that regulate circadian oscillation. Here, we characterize the functions of novel miRNA-25 regulating circadian Period2 (Per2) expression. Using several in silico algorithms, we identified novel miR-25-3p that, together with miR-24-3p, targets the Per2 gene. Luciferase reporter assays validated that miR-25-3p and miR-24-3p repressed Per2 expression and confirmed their predicted binding sites in the 3′-untranslated region (UTR) of Per2 mRNA. Real-time bioluminescence analyses using Per2::Luc mouse embryonic fibroblasts confirmed that PER2 protein oscillation patterns were responsive to miR-25-3p and miR-24-3. The overexpression of miR-25-3p or miR-24-3p resulted in the dampening and period shortening of the PER2::LUC oscillation, while inhibition of either miRNA increased the relative amplitude of the PER2::LUC oscillation. Notably, endogenous miR-25-3p expression in the suprachiasmatic nucleus (SCN) showed no circadian rhythmicity, but the expression levels differed in various brain regions and peripheral tissues. These results suggest that the posttranscriptional regulation of miR-25-3p and miR-24-3p may differ according to Per2 gene expression in different tissue regions. In summary, we found that novel miR-25-3p was involved in fine-tuning circadian rhythmicity by regulating Per2 oscillation at the posttranscriptional level and that it functioned synergistically with miR-24-3p to affect Per2 oscillation.

Cancer stem cell generation during epithelial-mesenchymal transition is temporally gated by intrinsic circadian clocks.

Epithelial-mesenchymal transition (EMT) is a key event preceding tumor cell metastasis that increases cell invasiveness and cancer stem cell (CSC)populations. Studies suggest that genes used in generating circadian rhythms also serve in regulating EMT. To test the role of circadian clocks in cellular EMT events two cancer cell lines were compared, one that has a well-established circadian clock, C6 from rat glioma, and one that does not, MCF-7 from human breast tumor. MCF-7 tumorsphere cultures were tested for evidence of circadian rhythms because of previously reported circadian rhythm enhancement in C6 tumorspheres shown by elevated rhythm amplitude and increased expression of circadian clock gene Per2. Bioluminescence imaging of Per2 gene expression in MCF-7 tumorspheres revealed a previously unconfirmed circadian clock in this important cancer research model. Inducing CSCgeneration through EMT in C6 and MCF-7 monolayer cultures revealed circadian oscillations in the size of the post-EMT CSCpopulation, confirming that circadian rhythms are additional processes controlling this stage of cancer progression. EMT was verified by distinct cellularmorphological changes and expression of stem cell proteins OCT4, nestin, MSI1, and CD133 along with EMT-related proteins ZEB1, vimentin, and TWIST. Quantifying single-cell events and behaviors through time-lapse imaging indicated the post-EMT population size was determined largely by circadian rhythms in epithelial-like cancer cells undergoing EMT. We then identified a specific phase of the circadian rhythm in Per2 gene activation as a potential target for therapeutic treatments that may suppress EMT, minimize CSCs, and limit metastasis.

Stability of d‐luciferin for bioluminescence to detect gene expression in freely moving mice for long durations

Circadian disturbance of clock gene expression is a risk factor for diseases such as obesity, cancer, and sleep disorders. To study these diseases, it is necessary to monitor and analyze the expression rhythm of clock genes in the whole body for a long duration. The bioluminescent reporter enzyme firefly luciferase and its substrate d-luciferin have been used to generate optical signals from tissues in vivo with high sensitivity. However, little information is known about the stability of d-luciferin to detect gene expression in living animals for a long duration. In the present study, we examined the stability of a luciferin solution over 21 days. l-Luciferin, which is synthesized using racemization of d-luciferin, was at high concentrations after 21 days. In addition, we showed that bioluminescence of Period1 (Per1) expression in the liver was significantly decreased compared with the day 1 solution, although locomotor activity rhythm was not affected. These results showed that d-luciferin should be applied to the mouse within, at most, 7 days to detect bioluminescence of Per1 gene expression rhythm in vivo.

Methyl 3-(4-hydroxyphenyl) propionate modulates plant growth and secondary metabolite accumulation by inducing metabolic changes in Perilla frutescens

As one of the components of root exudates, methyl 3-(4-hydroxyphenyl) propionate (MHPP) not only functions as a nitrification inhibitor in soil but also modulates plant growth and root system architecture (RSA); however, the physiological and molecular mechanisms underlying MHPP-mediated plant growth remain largely unclear. Here, we investigated the effects of MHPP treatment on growth and secondary metabolite accumulation by integrating physiology, transcriptome and metabolome analyses using perilla (Perilla frutescens), a type of medicinal and edible plant. MHPP reduces primary root growth but markedly induces lateral root formation in perilla seedlings. The full-length transcript sequencing combined with the comparative transcriptomic analysis revealed that the differential expression of genes involved in carbon/nitrogen metabolism and secondary metabolism contributed to the MHPP-mediated plant growth. An investigation of the differentially expressed genes showed that MHPP modulates the growth and metabolism of leaves and roots in distinct pathways. The genes involved in the phenylpropanoid metabolism pathway showed opposite expression patterns between the roots and leaves, thereby resulting in the differential accumulation of secondary metabolites in the roots and leaves. Furthermore, the differential expression of PER genes and elevated peroxidase activity in the MHPP-treated roots were responsible for modulating the equilibrium of ROS and the components of lignin and phenolic compounds, thereby affecting plant growth and secondary metabolite accumulation. MHPP modulates RSA and metabolite profiles and improves the contents of medicinal ingredients in perilla plants. These results suggested that the application of MHPP may represent a useful strategy for the medicinal plant cultivation.

Day-night rhythm of skeletal muscle metabolism is disturbed in older, metabolically compromised individuals

ObjectiveSkeletal muscle mitochondrial function and energy metabolism displays day-night rhythmicity in healthy, young individuals. Twenty-four-hour rhythmicity of metabolism has been implicated in the etiology of age-related metabolic disorders. Whether day-night rhythmicity in skeletal muscle mitochondrial function and energy metabolism is altered in older, metabolically comprised humans remains unknown. MethodsTwelve male overweight volunteers with impaired glucose tolerance and insulin sensitivity stayed in a metabolic research unit for 2 days under free living conditions with regular meals. Indirect calorimetry was performed at 5 time points (8 AM, 1 PM, 6 PM, 11 PM, 4 AM), followed by a muscle biopsy. Mitochondrial oxidative capacity was measured in permeabilized muscle fibers using high-resolution respirometry. ResultsMitochondrial oxidative capacity did not display rhythmicity. The expression of circadian core clock genes BMAL1 and REV-ERBα showed a clear day-night rhythm (p < 0.001), peaking at the end of the waking period. Remarkably, the repressor clock gene PER2 did not show rhythmicity, whereas PER1 and PER3 were strongly rhythmic (p < 0.001). On the whole-body level, resting energy expenditure was highest in the late evening (p < 0.001). Respiratory exchange ratio did not decrease during the night, indicating metabolic inflexibility. ConclusionsMitochondrial oxidative capacity does not show a day-night rhythm in older, overweight participants with impaired glucose tolerance and insulin sensitivity. In addition, gene expression of PER2 in skeletal muscle indicates that rhythmicity of the negative feedback loop of the molecular clock is disturbed. ClinicalTrials.gov IDNCT03733743.

Metabolic Effects of Light at Night are Time- and Wavelength-Dependent in Rats.

ObjectiveIntrinsically photosensitive retinal ganglion cells are most sensitive to short wavelengths and reach brain regions that modulate biological rhythms and energy metabolism. The increased exposure nowadays to artificial light at night (ALAN), especially short wavelengths, perturbs our synchronization with the 24‐hour solar cycle. Here, the time‐ and wavelength dependence of the metabolic effects of ALAN are investigated.MethodsMale Wistar rats were exposed to white, blue, or green light at different time points during the dark phase. Locomotor activity, energy expenditure, respiratory exchange ratio (RER), and food intake were recorded. Brains, livers, and blood were collected.ResultsAll wavelengths decreased locomotor activity regardless of time of exposure, but changes in energy expenditure were dependent on the time of exposure. Blue and green light reduced RER at Zeitgeber time 16‐18 without changing food intake. Blue light increased period 1 (Per1) gene expression in the liver, while green and white light increased Per2. Blue light decreased plasma glucose and phosphoenolpyruvate carboxykinase (Pepck) expression in the liver. All wavelengths increased c‐Fos activity in the suprachiasmatic nucleus, but blue and green light decreased c‐Fos activity in the paraventricular nucleus.ConclusionsALAN affects locomotor activity, energy expenditure, RER, hypothalamic c‐Fos expression, and expression of clock and metabolic genes in the liver depending on the time of day and wavelength.

Period2 gene regulates diurnal changes of nasal symptoms in an allergic rhinitis mouse model.

BackgroundAllergic rhinitis (AR) symptoms exhibit prominent 24‐hour variations associated with the biological clock. Although endogenous glucocorticoids synchronize circadian oscillator in the nasal mucosa, the precise mechanism of AR remains unclear. Therefore, using a mouse model, we investigated the association between circadian‐clock genes and AR symptoms at various time‐points.MethodsBased on the rhythmic secretion of corticosterone levels, we chose 2 time‐points, ZT4 (10:00 AM) and ZT16 (10:00 PM), to observe dynamic changes of nasal symptoms, immunologic responses, and circadian‐clock gene period (Per) expressions.ResultsIn the AR group, nasal symptom scores at ZT4 were significantly higher than at ZT16, with a greater increase in eosinophils, mast cells, and total immunoglobulin E levels at ZT4. The scores had a negative correlation with fluctuation of corticosterone levels. T‐helper 1 (Th1) cell counts and interferon‐γ levels decreased significantly at ZT4 compared with ZT16 in the AR group, whereas Th2 cells; Th17 cells; and interleukin (IL)‐4, ‐13, and ‐17A levels increased significantly at ZT4 compared with ZT16. Furthermore, Per2 gene expression levels were attenuated at ZT4 and elevated at ZT16, but correlated negatively with Th2 and Th17 responses associated with G ata3 and Rorγt expression levels that were enhanced at ZT4 and reduced at ZT16 in the AR group.ConclusionOur results suggest that the Per2 gene may influence diurnal variations of AR symptom severity, partially through its possible anti‐inflammatory effect on the circadian regulation of GATA3 and RORγt levels in immune cells. This further demonstrates the neural‐immune‐endocrinal mechanism of circadian rhythm in AR and sheds new light on chronotherapeutic approaches to AR.

Downregulation of PER2 Promotes Tumor Progression by Enhancing Glycolysis via the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway in Oral Squamous Cell Carcinoma

PER2 gene expression is downregulated in oral squamous cell carcinoma (OSCC) and may have a pivotal role in tumor suppression. However, the biological function and mechanism of action of PER2 in OSCC remain unclear. In this study, the biological functions and anticancer mechanisms of PER2 in OSCC were investigated. Both stably overexpressed and silenced PER2 OSCC cells were established as an experimental group; empty vector lentivirus and scramble short hairpin RNA lentivirus transfected-cells, as negative control groups; and untreated OSCC cells, as a blank group.Cell proliferation, apoptosis, and glycolysis potential assays were conducted. In addition, the expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), phosphorylation of protein kinase B, hexokinase 2 (HK2), pyruvate kinase M (PKM2), and lactate dehydrogenase A (LDHA) was quantified by real-time quantitative polymerase chain reaction and Western blotting.Rescue experiments were performed by the addition of AKT activators in the overexpressed cell line and by the addition of glycolysis inhibitor in the silenced cell line. These findings were verified invivo using stably transfected OSCC cells overexpressing PER2 implanted in nude mice. PER2 overexpression significantly inhibited OSCC cell proliferation and glycolysis, promoted cell apoptosis, and reduced the expression of PI3K, phosphorylation of protein kinase B, HK2, PKM2, and LDHA. The converse was observed in PER2-silenced OSCC cells. After the addition of AKT activator to cultures of PER2-overexpressed OSCC cells, reduced glucose uptake, lactic acid production, and cell proliferation, combined with increased apoptosis, were substantially reversed. In addition, after the addition of HK2 inhibitor to PER2-silenced OSCC cells to inhibit glycolysis, the reduction in apoptosis and increased proliferation were significantly countermanded. Tumorigenesis experiments invivo also confirmed that PER2 overexpression suppressed OSCC growth and decreased the expression of HK2, PKM2, and LDHA. PER2 heightened glycolysis via the PI3K/AKT pathway, heightened cell proliferation and inhibited apoptosis via glycolysis, thereby promoting the development of OSCC.

Doxorubicin modulated clock genes and cytokines in macrophages extracted from tumor-bearing mice

ABSTRACT Circadian rhythm is essential for cellular regulation of physiological, metabolic, and immune functions. Perturbations of circadian rhythms have been correlated with increased susceptibility to cancer and poor prognosis in the cancer treatment. Our aim is to investigate the role of doxorubicin (DOX) treatment on clock genes expression and inflammation in intraperitoneal macrophages and the antitumoral response. Methods: Macrophages were extracted from intraperitoneal cavity of mice without or with Lewis lung carcinoma (LLC) and treated with DOX totaling four groups (CTL, LLC, LLC+DOX and DOX) and analyzes of clock genes in six time points (ZT02, ZT06, ZT10, ZT14, ZT18 AND ZT22). Intraperitoneal macrophages cell culture was stimulated with LPS and DOX and clock genes and inflammatory profile were analyzed. In tumor were analyzed macrophages markers. Results: The expression of F4/80 (ZT22) and CD11c (ZT06) tumor tissue was significantly differed between LLC and LCC+DOX groups. In the intraperitoneal macrophages, DOX increased Clock (ZT10), Rev-Erbα (ZT18 and ZT22) and Per2 expressions (ZT18); in the LLC+DOX group was increased Bmal1 (ZT10), Per2 (ZT18) and NF-kB (ZT22) expressions; IL-6 expression increased in the LCC group (ZT02). In intraperitoneal macrophages cell culture stimulated with DOX and LPS after 24 h decreased Clock and Per1. DOX causes depression after 6 and 24 h in TNF-α content and Per2 gene expression after 24 h IL-1β expression was reduced also. Conclusion: DOX treatment in vivo disrupted cytokine and clock genes expression in intraperitoneal macrophages suppressing immune response. Moreover, macrophages cultured with DOX had decreased expression of LPS-stimulated inflammatory cytokines.

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

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

Current Status of Research on the Period Family of Clock Genes in the Occurrence and Development of Cancer.

Several physiological activities of organisms are coordinated based on periodic variations of ~24 h, which is called a circadian rhythm. Circadian rhythms, driven by circadian clock genes, play an important role in the regulation of various complex life activities of organisms, in an orderly and coordinated manner. Period (Per)1/2/3 genes are important core clock genes and part of the Per gene family. Current research has demonstrated that the abnormal expression of Per genes and disruption of circadian rhythms can lead to the occurrence and development of cancer; however, the exact mechanism has not yet been elucidated. Further study on this mechanism may lead to the discovery of new, effective therapies for the prevention and treatment of cancer. The present review summarizes the status of current research with regards to the association between the abnormal expression and rhythmic variation of the Per gene family, and carcinogenesis and progression of cancer.

Cardiomyocyte transcription is controlled by combined MR and circadian clock signalling.

We previously identified a critical pathogenic role for MR activation in cardiomyocytes that included a potential interaction between the MR and the molecular circadian clock. While glucocorticoid regulation of the circadian clock is undisputed, MR interactions with circadian clock signalling are limited. We hypothesised that the MR influences cardiac circadian clock signalling, and vice versa. 10nM aldosterone or corticosterone regulated CRY 1, PER1, PER2 and ReverbA (NR1D1) gene expression patterns in H9c2 cells over 24hr. MR-dependent regulation of circadian gene promoters containing GREs and E-box sequences was established for CLOCK, Bmal, CRY 1 and CRY2, PER1 and PER2 and transcriptional activators CLOCK and Bmal modulated MR-dependent transcription of a subset of these promoters. We also demonstrated differential regulation of MR target gene expression in hearts of mice 4hr after administration of aldosterone at 8AM versus 8PM. Our data support combined MR regulation of a subset of circadian genes and that endogenous circadian transcription factors CLOCK and Bmal modulate this response. This unsuspected relationship links MR in the heart to circadian rhythmicity at the molecular level and has important implications for the biology of MR signalling in response to aldosterone as well as cortisol. These data are consistent with MR signalling in the brain where, like the heart, it preferentially responds to cortisol. Given the undisputed requirement for diurnal cortisol release in the entrainment of peripheral clocks, the present study highlights the MR as an important mechanism for transducing the circadian actions of cortisol in addition to the GR in the heart.

Inhibition of IRS-1 Alters Retinal Circadian Clock

Background and Hypothesis: Life on Earth has adapted to a 24-hour cycle of light and darkness. Circadian physiology coordinates temporal metabolism, hormone cycling, and sleep using clock genes. It is documented that dysregulation of clock gene expression is a key factor in the pathogenesis of diabetic retinopathy. Our goal was to explore the relationship between clock genes and the retina in diabetic milieu, and we hypothesized that disrupting downstream insulin signaling in a manner similar to diabetes in the retina would also affect the circadian clock. &#x0D; Experimental Design or Project Methods: In this mouse study, we used a Per2::Luc fusion protein to perform real-time bioluminescent recording of circadian rhythms. We used SecinH3 to inhibit Insulin Receptor Signaling (IRS-1). In isolated retinas, we modulated IRS-1 to mimic the diabetic condition of impaired insulin signaling; this allowed us to directly quantify circadian rhythms in the retina. Results: Our results show that IRS-1 inhibition by SecinH3 altered the gene expression of Per2, a clock regulatory gene, over the controls. There was an increase in the period and an apparent phase shift in the presence of 100uM SecinH3. &#x0D; Conclusion and Potential Impact: Our findings can help us understand the role of insulin signaling on circadian rhythms of the retina and provide another temporal dimension to view diabetic retinopathy disease progression. Ultimately, further studies and a closer understanding of the roles of molecular clocks and insulin signaling may help to develop novel therapeutics for treating some of the harmful effects of diabetes.

The adrenal gland circadian clock exhibits a distinct phase advance in spontaneously hypertensive rats.

The circadian clock influences a multitude of cellular and biological processes, including blood pressure control. Spontaneously hypertensive rats (SHR) exhibit aberrant circadian rhythms affecting cardiovascular parameters, and they also have abnormal clock gene expression profiles in several organs. Given the important role of the adrenal gland in orchestrating circadian oscillations, we investigated the adrenal gland circadian clock in SHR and control Wistar-Kyoto rats maintained under a 12-hour light-dark cycle. Adrenal glands, livers, and serum samples were collected every 4 h and mRNA was extracted for analysis of clock gene expression. Serum levels of corticosterone and aldosterone were also analyzed. Overall, the circadian profiles of Bmal1, Per2, Per3, Cry1, RevErba, Revervb, and Dbp gene expression in SHR adrenal glands were phase-advanced relative to controls. The expression profile of StAR (a representative gene under circadian control in the adrenal gland), as well as the circadian rhythms of serum concentrations of corticosteroid and aldosterone were also phase advanced. E4bp4 gene expression was significantly higher during the dark period, yet the expression of its transcriptional activator, Rora, was significantly lower throughout the 24 h period in SHR adrenal glands than in controls. This paradoxical high E4bp4 gene expression was, however, not observed in the liver. In addition, Per1, Per2, Per3, Reverba, and Reverbb mRNA tended to be lower in SHR adrenal glands than in controls. Thus, we conclude that SHR possess an abnormal adrenal circadian clock, which may affect the transcriptional regulation of clock-controlled genes, and steroid hormone secretion by the adrenal gland.

Coordination between Prefrontal Cortex Clock Gene Expression and Corticosterone Contributes to Enhanced Conditioned Fear Extinction Recall.

Post-traumatic stress disorder (PTSD) is associated with impaired conditioned fear extinction learning, a ventromedial prefrontal cortex (vmPFC)-dependent process. PTSD is also associated with dysregulation of vmPFC, circadian, and glucocorticoid hormone function. Rats have rhythmic clock gene expression in the vmPFC that requires appropriate diurnal circulatory patterns of corticosterone (CORT), suggesting the presence of CORT-entrained intrinsic circadian clock function within the PFC. We examined the role of vmPFC clock gene expression and its interaction with CORT profiles in regulation of auditory conditioned fear extinction learning. Extinction learning and recall were examined in male rats trained and tested either in the night (active phase) or in the day (inactive phase). Using a viral vector strategy, Per1 and Per2 clock gene expression were selectively knocked down within the vmPFC. Circulating CORT profiles were manipulated via adrenalectomy (ADX) ± diurnal and acute CORT replacement. Rats trained and tested during the night exhibited superior conditioned fear extinction recall that was absent in rats that had knock-down of vmPFC clock gene expression. Similarly, the superior nighttime extinction recall was absent in ADX rats, but restored in ADX rats given a combination of a diurnal pattern of CORT and acute elevation of CORT during the postextinction training consolidation period. Thus, conditioned fear extinction learning is regulated in a diurnal fashion that requires normal vmPFC clock gene expression and a combination of circadian and training-associated CORT. Strategic manipulation of these factors may enhance the therapeutic outcome of conditioned fear extinction related treatments in the clinical setting.