Per2 Gene Expression Research Articles

Neuroglobin correlates with cryptochrome-1 in obstructive sleep apnea with primary aldosteronism.

BackgroundNeuroglobin (Ngb) is highly expressed in the suprachiasmatic nucleus, and can regulate Per1 gene expression. It is still not known whether Ngb also influences Cryptochrome (Cry). Cry is implicated in hypertension and primary aldosteronism (PA) in mice. There is a strong correlation between Obstructive Sleep Apnea (OSA) and PA. We propose to prove that Ngb and Cry play a role in OSA with PA.MethodsSubjects were recruited consecutively from residents of Jakarta, Indonesia; subjects aged 30–65 years with moderate to severe OSA and hypertension were included in the study. OSA was diagnosed using an unattended type 2 portable monitor (Alice Pdx), hypertension was diagnosed when morning blood pressure exceeded 140/90 mmHg or when taking anti-hypertensive drugs. Serum concentration of aldosterone, renin, Cry1, Cry2 and Ngb protein were determined using ELISA method. Primary aldosteronism (PA) was defined as ARR ≥20.ResultsForty subjects were recruited, 26 male and 14 female, median age 52.5 years, BMI 27.46 kg/m2, and AHI 34.8 times/hour. We found 16 subjects with PA and 24 non PA. Cry1 and Cry2 did not correlate with ARR in PA and non PA groups. Ngb correlated positively with Cry1 (Spearman’s rho = 0.455, p = 0.038) but not Cry2 in PA patients. Cry1 concentration decreased in severe hypoxia.ConclusionsNgb correlates with Cry1 in OSA with PA. There is no correlation between Cry1 or Cry2 with PA

Clock genes alterations and endocrine disorders

Various endocrine signals oscillate over the 24-hour period and so does the responsiveness of target tissues. These daily oscillations do not occur solely in response to external stimuli but are also under the control of an intrinsic circadian clock. We searched the PubMed database to identify studies describing the associations of clock genes with endocrine diseases. Various human single nucleotide polymorphisms of brain and muscle ARNT-like 1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK) genes exhibited significant associations with type 2 diabetes mellitus. ARNTL2 gene expression and upregulation of BMAL1 and PER1 were associated with the development of type 1 diabetes mellitus. Thyroid hormones modulated PER2 expression in a tissue-specific way, whereas BMAL1 regulated the expression of type 2 iodothyronine deiodinase in specific tissues. Adrenal gland and adrenal adenoma expressed PER1, PER2, CRY2, CLOCK and BMAL1 genes. Adrenal sensitivity to adrenocorticotrophin was also affected by circadian oscillations. A significant correlation between the expression of propio-melanocorticotrophin and PER 2, as well as between prolactin and CLOCK, was found in corticotroph and lactosomatotroph cells, respectively, in the pituitary. Clock genes and especially BMAL1 showed an important role in fertility, whereas oestradiol and androgens exhibited tissue-specific effects on clock gene expression. Metabolic disorders were also associated with circadian dysregulation according to studies in shift workers. Clock genes are associated with various endocrine disorders through complex mechanisms. However, data on humans are scarce. Moreover, clock genes exhibit a tissue-specific expression representing an additional level of regulation. Their specific role in endocrine disorders and their potential implications remain to be further clarified.

A Free-Choice High-Fat High-Sugar Diet Alters Day-Night Per2 Gene Expression in Reward-Related Brain Areas in Rats.

Under normal light–dark conditions, nocturnal rodents consume most of their food during the dark period. Diets high in fat and sugar, however, may affect the day–night feeding rhythm resulting in a higher light phase intake. In vitro and in vivo studies showed that nutrients affect clock-gene expression. We therefore hypothesized that overconsuming fat and sugar alters clock-gene expression in brain structures important for feeding behavior. We determined the effects of a free-choice high-fat high-sugar (fcHFHS) diet on clock-gene expression in rat brain areas related to feeding and reward and compared them with chow-fed rats. Consuming a fcHFHS diet for 6 weeks disrupted day–night differences in Per2 mRNA expression in the nucleus accumbens (NAc) and lateral hypothalamus but not in the suprachiasmatic nucleus, habenula, and ventral tegmental area. Furthermore, short-term sugar drinking, but not fat feeding, upregulates Per2 mRNA expression in the NAc. The disruptions in day–night differences in NAc Per2 gene expression were not accompanied by altered day–night differences in the mRNA expression of peptides related to food intake. We conclude that the fcHFHS diet and acute sugar drinking affect Per2 gene expression in areas involved in food reward; however, this is not sufficient to alter the day–night pattern of food intake.

Tick‐Tock, Tick‐Tock: Clock Gene Expression is Altered in Sleep Apnea

IntroductionClock genes play a fundamental role in the maintenance of human body homeostasis. Dysfunctional clock gene expression has been shown to underlie circadian rhythm sleep disorders, but its association with a far more common sleep disorder, sleep apnea (SA), is unclear. The goal of this study is to determine if there is an association between clock gene expression and the presence of SA and related nocturnal hypoxia in a cohort of Veterans with chronic kidney disease (CKD).MethodsA cross‐sectional study of Veterans enrolled in the SNORE Study, a prospective cohort study of 248 Veterans with CKD, was performed. At baseline, all participants underwent a full, unattended sleep study. A subset of 49 consecutive participants provided whole blood samples the morning after sleep study at a consistent time point for this sub‐study; samples were processed the same day to isolate RNA. We determined gene expression (normalized to β‐actin) of Bmal1, Ck1δ, Ck1ɛ, Clock, Cry1, Cry2, NPAS2, Per1, Per2, Per3, Rev‐Erb‐α, RORα, and Timeless using real‐time, quantitative RT‐PCR utilizing the ΔΔCT method. SA was defined by the apnea‐hypopnea index (AHI), measured in events per hour, as follows: none (AHI 0–4.9), mild (AHI 5–14.9), moderate (AHI 15–29.9) and severe (AHI ≥30). We also compared clock gene expression between those with and without nocturnal hypoxia, where nocturnal hypoxia was defined as ≥10% of total sleep time spent at less than 90% oxygen saturation. After excluding outliers (n=7), two‐sided t‐tests were used to compare the fold change in expression between those with no SA and those with varying degrees of SA.ResultsMean age of our cohort was 71 ±11 years and mean BMI was 30 ±5 kg/m2. Mean estimated glomerular filtration rate was 38.2 ±8.6 ml/min/1.73 m2. 51% were diabetic and 88% were hypertensive. Sleep apnea was diagnosed in 71% of our cohort with 31% having mild SA, 16% with moderate SA and 24% with severe SA. 27% met criteria for nocturnal hypoxia. Participants with SA exhibited an average of a 34.9% decrease (P=0.012) in Per3 expression compared to their counterparts without SA. Participants with nocturnal hypoxia demonstrated an average 50.4% (P=0.027), 28.7% (P=0.014), and 31% (P=0.04) decrease in NPAS2, Per1, and Rev‐Erb‐α expression, respectively, compared to those who did not present with nocturnal hypoxia.ConclusionMorning expression of Per3 was significantly down‐regulated in SA. Nocturnal hypoxia was associated with down‐regulation of Per1, NPAS2, and Rev‐Erb‐α expression. These preliminary data suggest that there are meaningful differences in the expression of key clock genes between those with and without SA and related nocturnal hypoxia. Future studies should examine the 24‐hour expression of clock genes as they relate to sleep apnea in individuals with and without CKD. Should SA lead to disruption of circadian clock gene expression, treatment of SA or targeted pharmacotherapy to affect clock gene expression may restore normal rhythms and improve health outcomes for patients with SA.Support or Funding InformationVA CSR&D Career Development Award (CX000533‐01A1) to Dr. Canales; Orlando Aerie of the Fraternal Order of Eagles to Dr. Gumz; Gatorade Trust, Division of Nephrology, Department of Medicine, University of FloridaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Light rescues circadian behavior and brain dopamine abnormalities in diurnal rodents exposed to a winter-like photoperiod.

Seasonal affective disorder (SAD), beyond mood changes, is characterized by alterations in daily rhythms of behavior and physiology. The pathophysiological conditions of SAD involve changes in day length and its first-line treatment is bright light therapy. Animal models using nocturnal rodents have been studied to elucidate the neurobiological mechanisms of depression, but might be ill suited to study the therapeutic effects of light in SAD since they exhibit light-aversive responses. Here Arvicanthis ansorgei, a diurnal rodent, was used to determine behavioral, molecular and brain dopamine changes in response to exposure to a winter-like photoperiod consisting of a light-dark cycle with 8h of light, under diminished light intensity, and 16h of darkness. Furthermore, we evaluated whether timed-daily bright light exposure has an effect on behavior and brain physiology of winter-like exposed animals. Arvicanthis under a winter-like condition showed alterations in the synchronization of the locomotor activity rhythm to the light-dark cycle. Moreover, alterations in day-night activity of dopaminergic neurotransmission were revealed in the nucleus accumbens and the dorsal striatum, and in the day-night clock gene expression in the suprachiasmatic nucleus. Interestingly, whereas dopamine disturbances were reversed in animals exposed to daily light at early or late day, altered phase of the daily rhythm of locomotion was reverted only in animals exposed to light at the late day. Moreover, Per2 gene expression in the SCN was also affected by light exposure at late day in winter-like exposed animals. These findings suggest that light induces effects on behavior by mechanisms that rely on both circadian and rhythm-independent pathways influencing the dopaminergic circuitry. This last point might be crucial for understanding the mechanisms of non-pharmacological treatment in SAD.

Entrainment of the Circadian Clock in Neural Stem Cells by Epidermal Growth Factor is Closely Associated with ERK1/2-mediated Induction of Multiple Clock-related Genes

The mitotic activity of certain tissues in the body is closely associated with circadian clock function. However, the effects of growth factors on the molecular clockwork are not fully understood. Stimulation of neural stem cells (NSCs) with epidermal growth factor (EGF), a well-known mitogen, is known to cause synchronized cell cycle progression with a period of approximately 24 h, closely associated with the Per2 gene expression rhythm. Here, we examined the effects of EGF on the molecular clockwork of NSCs. Treatment of cultured NSCs derived from embryonic mouse forebrain with EGF (20 ng/mL) caused a phase shift in the PER2::LUCIFERASE bioluminescence rhythm in a stimulation time-dependent manner. The EGF phase-response curve differed from that of forskolin (FK)—a well-known chemical resetting stimulus—both in the advance/delay ratio and stimulation time-dependency. PCR array analysis followed by quantitative PCR validation demonstrated that EGF treatment transiently induced multiple clock-related genes including Per1, Per2, Dec1, e4bp4, and Noct, whereas FK treatment induced a limited number of genes (Per1 and Dec1), suggesting that the mode of entrainment of NSC molecular clock was different for EGF and FK. EGF led to gene induction in the presence of cycloheximide, suggesting that de novo protein synthesis is unnecessary. Pretreatment with the MEK1/2 inhibitor U0126 significantly suppressed the acute induction of Per2, Dec1, and Noct by EGF and also abolished the EGF-induced phase shift of the PER2::LUCIFERASE rhythm in NSCs. These results suggest a unique effect of EGF on the molecular clockwork of NSCs.

The Interaction between Bmal1 and Per2 in Mouse BMSC Osteogenic Differentiation.

The circadian clock is a system that controls endogenous time of organisms, and it regulates the physiology and behavior of bodies. The transcription factors Brain and Muscle ARNT-like Protein 1 (BMAL1) and Period2 (Per2) are components of the circadian clock, and they play vital roles in circadian clock function. Both Bmal1−/− mice and Per2−/− mice display obvious bone volume changes. In this study, we inhibited the expression of Bmal1 in bone marrow-derived mesenchymal stem cells (BMSCs) using a lentiviral vector harboring RNAi sequences, which increased the osteogenic differentiation capability of BMSCs. We also suppressed Per2 gene expression using an adenovirus vector harboring RNAi sequences, and similarly, the osteogenic differentiation ability of BMSCs was enhanced. Furthermore, when both Bmal1 and Per2 gene expression was suppressed in BMSCs by lentiviral and adenoviral interference, the osteogenic differentiation capability was stronger than that in BMSCs following single-gene inhibition. Our data support that both Bmal1 and Per2 play negative roles in BMSC osteogenic differentiation and that Bmal1 and Per2 have a synergistic effect on the osteogenic differentiation of BMSCs.

An individual 12-h shift of the light-dark cycle alters the pancreatic and duodenal circadian rhythm and digestive function.

In mammals, behavioral and physiological rhythms are controlled by circadian clocks which are entrained by environmental light and food signals. However, how the environmental cues affect digestive tract's circadian clock remains poorly understood. Therefore, in order to elucidate the effect of light cue on the resetting of the peripheral clocks, we investigated the expressions of clock genes (Bmal1, Cry1, Rev-erbα, Per1, and Per2) and digestive function genes (Cck, Cck-1r, Sct, Sctr, and Ctrb1) in the pancreas and duodenum of rats after the light-dark (LD) cycle reversal for 7 days. We found that both the clock genes and digestive function genes exhibited a clear and similar daily rhythmicity in the pancreas and duodenum of rats. After reversal of the LD cycle for 7 days, the expressions of clock genes in pancreas, including Bmal1, Cry1, and Rev-erbα were affected; whereas the expression of Per1 gene failed to fit the cosine wave. However, in the duodenum the shifted genes were Bmal1, Rev-erbα, and Per2; in parallel, the Per1 gene expression also lost its circadian rhythm by reversal of the LD cycle. Therefore, the acrophases of the clock genes were shifted in a tissue- and gene-specific manner. Furthermore, the profiles of the digestive function genes, including Sctr and Ctrb1, were also affected by changes in LD cycle. These observations suggest that the mechanisms underlying the pancreatic and duodenal clocks are distinct, and there may be a potential linkage between the circadian clock system and the digestive system.

Day-Time Isoflurane Administration Suppresses Circadian Gene Expressions in Both the Brain and a Peripheral Organ, Liver.

The aim of this study is to investigate the effects of light and administration time of isoflurane on circadian gene expression in the brains and liver tissues of rats kept in light-dark cycle. Seventy two 15-days-old rats pups were divided into four groups. All animals were exposed to 1.5% concentration of isoflurane or to 6 L min-1 O2 for six hours between Zeitgeber Time (ZT) 0-ZT06 (day-time administration) or ZT12-ZT18 (night-time administration). Rats were sacrificed after six hours of anaesthesia with four-hour time intervals. Total RNA was isolated from brains and liver tissues. Circadian gene expression was examined using quantitative real-time Reverse transcription polymerase chain reaction (RT-PCR). BMAL1, CLOCK, PER2 and CRY2 gene expression levels were markedly suppressed after day-time anaesthesia in the both brain and liver, but night-time administration caused only temporary suppression of gene expression. The effect of isoflurane on the circadian clock is time-dependent, and administered isoflurane anaesthesia at night had minimal effect on clock gene expression. Additionally, when the treated animals were kept in a regular light-dark cycle, isoflurane-induced phase shift was not observed, possibly because of the light.

Epigenetic regulation of the circadian clock: role of 5-aza-2′-deoxycytidine

We have been investigating transcriptional regulation of the BMAL1 gene, a critical component of the mammalian clock system including DNA methylation. Here, a more detailed analysis of the regulation of DNA methylation of BMAL1 proceeded in RPMI8402 lymphoma cells. We found that CpG islands in the BMAL1 and the PER2 promoters were hyper- and hypomethylated, respectively and that 5-aza-2′-deoxycytidine (aza-dC) not only enhanced PER2 gene expression but also PER2 oscillation within 24 h in RPMI8402 cells. That is, such hypermethylation of CpG islands in the BMAL1 promoter restricted PER2 expression which was recovered by aza-dC within 1 day in these cells. These results suggest that the circadian clock system can be recovered through BMAL1 expression induced by aza-dC within a day. The RPIB9 promoter of RPMI8402 cells, which is a methylation hotspot in lymphoblastic leukemia, was also hypermethylated and aza-dC gradually recovered RPIB9 expression in 3 days. In addition, methylation-specific PCR revealed a different degree of aza-dC-induced methylation release between BMAL1 and RPIB9. These results suggest that the aza-dC-induced recovery of gene expression from DNA methylation is dependent on a gene, for example the rapid response to demethylation by the circadian system, and thus, is of importance to clinical strategies for treating cancer.

Chronic Maternal Low-Protein Diet in Mice Affects Anxiety, Night-Time Energy Expenditure and Sleep Patterns, but Not Circadian Rhythm in Male Offspring.

Offspring of murine dams chronically fed a protein-restricted diet have an increased risk for metabolic and neurobehavioral disorders. Previously we showed that adult offspring, developmentally exposed to a chronic maternal low-protein (MLP) diet, had lower body and hind-leg muscle weights and decreased liver enzyme serum levels. We conducted energy expenditure, neurobehavioral and circadian rhythm assays in male offspring to examine mechanisms for the body-weight phenotype and assess neurodevelopmental implications of MLP exposure. C57BL/6J dams were fed a protein restricted (8%protein, MLP) or a control protein (20% protein, C) diet from four weeks before mating until weaning of offspring. Male offspring were weaned to standard rodent diet (20% protein) and single-housed until 8–12 weeks of age. We examined body composition, food intake, energy expenditure, spontaneous rearing activity and sleep patterns and performed behavioral assays for anxiety (open field activity, elevated plus maze [EPM], light/dark exploration), depression (tail suspension and forced swim test), sociability (three-chamber), repetitive (marble burying), learning and memory (fear conditioning), and circadian behavior (wheel-running activity during light-dark and constant dark cycles). We also measured circadian gene expression in hypothalamus and liver at different Zeitgeber times (ZT). Male offspring from separate MLP exposed dams had significantly greater body fat (P = 0.03), less energy expenditure (P = 0.004), less rearing activity (P = 0.04) and a greater number of night-time rest/sleep bouts (P = 0.03) compared to control. MLP offspring displayed greater anxiety-like behavior in the EPM (P<0.01) but had no learning and memory deficit in fear-conditioning assay (P = 0.02). There was an effect of time on Per1, Per 2 and Clock circadian gene expression in the hypothalamus but not on circadian behavior. Thus, transplacental and early developmental exposure of dams to chronic MLP reduces food intake and energy expenditure, increases anxiety like behavior and disturbs sleep patterns but not circadian rhythm in adult male offspring.

Feeding cycle-dependent circulating insulin fluctuation is not a dominant Zeitgeber for mouse peripheral clocks except in the liver: Differences between endogenous and exogenous insulin effects

The master clock in the suprachiasmatic nucleus synchronizes peripheral clocks via humoral and neural signals in mammals. Insulin is thought to be a critical Zeitgeber (synchronizer) for peripheral clocks because it induces transient clock gene expression in cultured cells. However, the extent to which fluctuations in feeding-dependent endogenous insulin affect the temporal expression of clock genes remains unclear. We therefore investigated the temporal expression profiles of clock genes in the peripheral tissues of mice fed for 8 h during either the daytime (DF) or the nighttime (NF) for one week to determine the involvement of feeding cycle-dependent endogenous insulin rhythms in the circadian regulation of peripheral clocks. The phase of circulating insulin fluctuations was reversed in DF compared with NF mice, although those of circulating corticosterone fluctuations and nocturnal locomotor activity were identical between these mice. The reversed feeding cycle affected the circadian phases of Per1 and Per2 gene expression in the liver and not in heart, lung, white adipose and skeletal muscle tissues. On the other hand, injected exogenous insulin significantly induced Akt phosphorylation in the heart and skeletal muscle as well as the liver, and significantly induced Per1 and Per2 gene expression in all examined tissues. These findings suggest that feeding cycles and feeding cycle-dependent endogenous insulin fluctuations are not dominant entrainment signals for peripheral clocks other than the liver, although exogenous insulin might reset peripheral oscillators in mammals.

Taurine Treatment Modulates Circadian Rhythms in Mice Fed A High Fat Diet.

Close ties have been made among certain nutrients, obesity, type 2 diabetes and circadian clocks. Among nutrients, taurine has been documented as being effective against obesity and type 2 diabetes. However, the impact of taurine on circadian clocks has not been elucidated. We investigated whether taurine can modulate or correct disturbances in daily rhythms caused by a high-fat diet in mice. Male C57BL/6 mice were divided in four groups: control (C), control + taurine (C+T), high-fat diet (HFD) and HFD + taurine (HFD+T). They were administered 2% taurine in their drinking water for 10 weeks. Mice were euthanized at 6:00, 12:00, 18:00, and 24:00. HFD mice increased body weight, visceral fat and food intake, as well as higher levels of glucose, insulin and leptin, throughout the 24 h. Taurine prevented increments in food intake, body weight and visceral fat, improved glucose tolerance and insulin sensitivity and reduced disturbances in the 24 h patterns of plasma insulin and leptin. HFD downregulated the expression of clock genes Rev-erbα, Bmal1, and Per1 in pancreatic islets. Taurine normalized the gene and protein expression of PER1 in beta-cells, which suggests that it could be beneficial for the correction of daily rhythms and the amelioration of obesity and diabetes.

MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression

Serum glucocorticoids play a critical role in synchronizing circadian rhythm in peripheral tissues, and multiple mechanisms regulate tissue sensitivity to glucocorticoids. In the skeleton, circadian rhythm helps coordinate bone formation and resorption. Circadian rhythm is regulated through transcriptional and post-transcriptional feedback loops that include microRNAs. How microRNAs regulate circadian rhythm in bone is unexplored. We show that in mouse calvaria, miR-433 displays robust circadian rhythm, peaking just after dark. In C3H/10T1/2 cells synchronized with a pulse of dexamethasone, inhibition of miR-433 using a tough decoy altered the period and amplitude of Per2 gene expression, suggesting that miR-433 regulates rhythm. Although miR-433 does not directly target the Per2 3'-UTR, it does target two rhythmically expressed genes in calvaria, Igf1 and Hif1α. miR-433 can target the glucocorticoid receptor; however, glucocorticoid receptor protein abundance was unaffected in miR-433 decoy cells. Rather, miR-433 inhibition dramatically enhanced glucocorticoid signaling due to increased nuclear receptor translocation, activating glucocorticoid receptor transcriptional targets. Last, in calvaria of transgenic mice expressing a miR-433 decoy in osteoblastic cells (Col3.6 promoter), the amplitude of Per2 and Bmal1 mRNA rhythm was increased, confirming that miR-433 regulates circadian rhythm. miR-433 was previously shown to target Runx2, and mRNA for Runx2 and its downstream target, osteocalcin, were also increased in miR-433 decoy mouse calvaria. We hypothesize that miR-433 helps maintain circadian rhythm in osteoblasts by regulating sensitivity to glucocorticoid receptor signaling.

Insulin induces oscillating expression of circadian gene PER2 and steroidal acute regulatory protein gene in human granulosa cells

Insulin induces oscillating expression of circadian gene PER2 and steroidal acute regulatory protein gene in human granulosa cells

Effect of 1a, 25(OH)2D3 on circadian clock gene expression in cardiac myocytes

Objective To explore the effect of 1a, 25(OH)2D3 on circadian clock gene expressions in cardiac myocytes. Methods Cultured cardiac myocytes isolated from 7-day-old Sprague-Dawley(SD) rats were identified by immunofluorescence.The medium including 1a, 25(OH)2D3 (final concentrations were 0 nmol/L, 1 nmol/L, 10 nmol/L, 50 nmol/L and 100 nmol/L) were added to primary myocardial cells to culture for 2 h and then total RNA was extracted.Real-time polymerase chain reaction (RT-PCR) was applied to analyze myocardial cells circadian clock gene (Bmal1, Per2, Rev-erba) transcript levels to determine optimum concentration of 1a, 25(OH)2D3.Then, the primary myocardial cells cultured for 72 h were divided into 3 groups: the control group was of serum-free culture medium; serum shock group was of DMEM containing 50%volume fraction of horse serum cultured 2 h; 1a, 25(OH)2D3 treatment group receiving 1a, 25(OH)2D3 at optimal concentration cultured 2 h. The cells were collected at 7 time points (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h) and then total RNA was extracted.RT-PCR was applied to analyze circadian clock gene (Bmal1, Per2, Rev-erba) transcript levels in the myocardial cells. Results In the presence of 50 nmol/L 1 a, 25(OH)2D3, the Bmal1 mRNA expression showed the highest level, but the Per2 and Rev-erba mRNA expression levels were minimum.Compared with the control group, both 1a, 25(OH)2D3 treatment group and serum shock group caused day-cycle rhythmic oscillation in circadian clock genes(Bmal1, Per2, Rev-erba) in the cardiac myocytes.And the expressions pattern of Bmal1 and Per2 genes were in the opposite phase.While Bmal1 gene expression appeared at peak at 12 h, Per2 gene expression appeared in a trough.Expression of Rev-erba gene trend began to rise at 8 h, and the highest expression level appeared at 12-16 h. Conclusions 1a, 25(OH)2D3 can affect Bmal1, Per2 and Rev-erba mRNA expressions of circadian clock genes in the cardiac myocytes. Key words: 1a, 25(OH)2D3; Cardiac myocytes; Circadian clock; Circadian clock gene

The expression analysis of per1 gene in fugu, Takifugu rubripes administered with immunostimulants

Circadian rhythms are physical, mental and behavioral changes that follow a roughly 24-hour cycle, responding primarily to light and darkness in an organism's environment. These changes occur in most living organisms, including animals, plants and many microbes. Circadian rhythm oscillations are the daily changes in physiology and are due to involvement of numerous genes whose expression peaks and declines approximately 12 hrs apart to undergo a full cycle within 24 hrs. These circadian rhythm controlling genes are called as clock genes that include Bmal, Clock, Period (per) and Cry genes in mammals. In recent years, it has been known that clock genes regulate various physiological activities including immune response. But there is little or no information on these genes and their functions in fish. Therefore, cloning of the Japanese pufferfish, Takifugu rubripes per1 gene was conducted, and expression pattern of the gene in light and dark periods with/without immune stimulation by LPS or polyI:C was analyzed in this study. Results revealed that the per1 gene was composed of 4215 bp nucleotides that encode a 1404-amino acid residue. A rhythmic change of per1 gene expression was observed during light and dark periods. A phase change (gap) of per1 gene expression was confirmed in the pufferfish stimulated with LPS. But a gap of per1 gene expression was not confirmed in polyI:C stimulated fish. It is suggested that per1 gene participates in immune regulation at the time of the bacterial infection but not during viral infection because a phase change was seen only in LPS stimulated fish. In conclusion, fish per1 gene may have regulatory function in immune response similar to that of mammalian species.

Effect of circadian rhythm disturbance on morphine preference and addiction in male rats: Involvement of period genes and dopamine D1 receptor

It is claimed that a correlation exists between disturbance of circadian rhythms by factors such as alteration of normal light–dark cycle and the development of addiction. However, the exact mechanisms involved in this relationship are not much understood. Here we have studied the effect of constant light on morphine voluntary consumption and withdrawal symptoms and also investigated the involvement of Per1, Per2 and dopamine D1 receptor in these processes. Male wistar rats were kept under standard (LD) or constant light (LL) conditions for one month. The plasma concentration of melatonin was evaluated by enzyme-linked immunosorbent assay (ELISA). Real-time PCR was used to determine the mRNA expression of Per1, Per2 and dopamine D1 receptor in the striatum and prefrontal cortex. Morphine preference (50mg/L) was evaluated in a two-bottle-choice paradigm for 10weeks and withdrawal symptoms were recorded after administration of naloxone (3mg/kg). One month exposure to constant light resulted in a significant decrease of melatonin concentration in the LL group. In addition, mRNA levels of Per2 and dopamine D1 receptor were up-regulated in both the striatum and prefrontal cortex of the LL group. However, expression of Per1 gene was only up-regulated in the striatum of LL rats in comparison to LD animals. Furthermore, after one month exposure to constant light, morphine consumption and preference ratio and also severity of naloxone-induced withdrawal syndrome were significantly greater in LL animals. It is concluded that exposure to constant light by up-regulation of Per2 and dopamine D1 receptor in the striatum and prefrontal cortex and up-regulation of Per1 in the striatum and the possible involvement of melatonin makes animals vulnerable to morphine preference and addiction.

Circadian Rhythms in Doxorubicin Nuclear Uptake and Clock Control of C6 Glioma Cells

Alterations of drug efficacy by the circadian clock are a concern when assessing drug therapies. Circadian rhythms persist in some cancer cells and are repressed in others. A better understanding of circadian activities generated within cancer cells could indicate therapeutic approaches that selectively disrupt rhythms and deprive cells of any benefits provided by circadian timing. Another option is to induce expression of the core clock gene Per2 to suppress cancer cell proliferation. We used the C6 rat glioblastoma cell line to identify rhythmic cancer cell properties that could provide improved therapeutic targets. Nuclear uptake of the anti-cancer agent doxorubicin by C6 cells showed a circadian rhythm that was shifted six hours from the rhythm in Per2 expression. We also observed circadian expression of the Crm1 (Xpo1) gene that is responsible for a key component of molecular transport through nuclear pores. C6 cultures include glioma stem cells (GSCs) that have elevated resistance to chemotherapeutic agents. We examined C6 tumorsphere cultures formed from GSCs to determine whether Hes1 and Bmi1 genes that maintain GSCs are under circadian clock control. Unlike Per2 gene expression in tumorspheres, Hes1 and Bmi1 expression did not oscillate in a circadian rhythm. These results highlight the importance of the nuclear pore complex in cancer treatments and suggest that the nuclear export mechanism and genes maintaining the cancer stem cell state could be inhibited therapeutically at a particular phase of the circadian cycle while preserving the tumor-suppressing abilities of Per2 gene expression.

Decreased Bone Volume and Bone Mineral Density in the Tibial Trabecular Bone Is Associated with Per2 Gene by 405 nm Laser Stimulation.

Low-level laser therapy/treatment (LLLT) using a minimally invasive laser needle system (MILNS) might enhance bone formation and suppress bone resorption. In this study, the use of 405 nm LLLT led to decreases in bone volume and bone mineral density (BMD) of tibial trabecular bone in wild-type (WT) and Per2 knockout (KO) mice. Bone volume and bone mineral density of tibial trabecular bone was decreased by 405 nm LLLT in Per2 KO compared to WT mice at two and four weeks. To determine the reduction in tibial bone, mRNA expressions of alkaline phosphatase (ALP) and Per2 were investigated at four weeks after 405 nm laser stimulation using MILNS. ALP gene expression was significantly reduced in the LLLT-stimulated right tibial bone of WT and Per2 KO mice compared to the non-irradiated left tibia (p < 0.001). Per2 mRNA expression in WT mice was significantly reduced in the LLLT-stimulated right tibial bone compared to the non-irradiated left tibia (p < 0.001). To identify the decrease in tibial bone mediated by the Per2 gene, levels of runt-related transcription factor 2 (Runx2) and ALP mRNAs were determined in non-irradiated WT and Per2 KO mice. These results demonstrated significant downregulation of Runx2 and ALP mRNA levels in Per2 KO mice (p < 0.001). Therefore, the reduction in tibial trabecular bone resulting from 405 nm LLLT using MILNS might be associated with Per2 gene expression.