Glucocorticoids entrain molecular clock components in human peripheral cells.

In humans, shift work induces a desynchronization between the circadian system and the outside world, which contributes to shift work-associated medical disorders. Using a simulated night shift experiment, we previously showed that 3 d of bright light at night fully synchronize the central clock to the inverted sleep schedule, whereas the peripheral clocks located in peripheral blood mononuclear cells (PBMCs) took longer to reset. This underlines the need for testing the effects of synchronizers on both the central and peripheral clocks. Glucocorticoids display circadian rhythms controlled by the central clock and are thought to act as synchronizers of rodent peripheral clocks. In the present study, we tested whether the human central and peripheral clocks were sensitive to exogenous glucocorticoids (Cortef) administered in the late afternoon. We showed that 20 mg Cortef taken orally acutely increased PER1 expression in PBMC peripheral clocks. After 6 d of Cortef administration, the phases of central markers were not affected, whereas those of PER2-3 and BMAL1 expression in PBMCs were shifted by ∼ 9.5-11.5 h. These results demonstrate, for the first time, that human peripheral clocks are entrained by glucocorticoids. Importantly, they suggest innovative interventions for shift workers and jet-lag travelers, combining synchronizing agents for the central and peripheral clocks.

Circadian clock gene expression is impaired in gestational diabetes mellitus.

Dysfunction of the circadian clock genes is involved in the development of obesity and type 2 diabetes (T2D). Since gestational diabetes mellitus (GDM) and T2D share common genetic and phenotypic features, in the present study, we investigated the status of the circadian clock in a cohort of 40 Greek pregnant women with GDM, four with T2D and 20 normal controls. Peripheral blood mRNA transcript levels of 10 clock genes (CLOCK1, BMAL1, PER1, PER2, PER3, PPARΑ, PPARD, PPARG, CRY1 and CRY2) were determined by real-time quantitative PCR. GDM patients expressed significantly lower transcript levels of BMAL1, PER3, PPARD and CRY2 compared to control women (p < 0.05). No significant difference was documented between GDM women maintained either under insulin treatment or diet. A positive correlation was found between the expression of BMAL1 versus CRY2 (r = 0.45, p = 0.003) and BMAL1 versus PPARD (r = 0.43, p = 0.004). Further investigation on the functional relevance of these clock genes, disclosed that expression of PER3 correlated negatively with HbA1C levels (r = -0.36, p = 0.022). These data document for the first time that the expression of BMAL1, PER3, PPARD and CRY2 genes is altered in GDM compared to normal pregnant women and support the notion that deranged expression of clock genes may play a pathogenic role in GDM.

The impact of sleep deprivation and nighttime light exposure on clock gene expression in humans.

AIM: To examine the effect of acute sleep deprivation under light conditions on the expression of two key clock genes, hPer2 and hBmal1, in peripheral blood mononuclear cells (PBMC) and on plasma melatonin and cortisol levels. METHODS: Blood samples were drawn from 6 healthy individuals at 4-hour intervals for three consecutive nights, including a night of total sleep deprivation (second night). The study was conducted in April-June 2006 at the University Medical Centre Ljubljana. RESULTS: We found a significant diurnal variation in hPer2 and hBmal1 expression levels under baseline (P<0.001, F=19.7, df=30 for hPer2 and P<0.001, F=17.6, df=30 for hBmal1) and sleep-deprived conditions (P<0.001, F=9.2, df=30 for hPer2 and P<0.001, F=13.2, df=30 for hBmal1). Statistical analysis with the single cosinor method revealed circadian variation of hPer2 under baseline and of hBmal1 under baseline and sleep-deprived conditions. The peak expression of hPer2 was at 13:55 ± 1:15 hours under baseline conditions and of hBmal1 at 16:08 ± 1:18 hours under baseline and at 17:13 ± 1:35 hours under sleep-deprived conditions. Individual cosinor analysis of hPer2 revealed a loss of circadian rhythm in 3 participants and a phase shift in 2 participants under sleep-deprived conditions. The plasma melatonin and cortisol rhythms confirmed a conventional alignment of the central circadian pacemaker to the habitual sleep/wake schedule. CONCLUSION: Our results suggest that 40-hour acute sleep deprivation under light conditions may affect the expression of hPer2 in PBMCs..

Alterations in Rev-ERBα/BMAL1 ratio and glycated hemoglobin in rotating shift workers: the EuRhythDia study.

OBJECTIVE: To detect premature gluco-metabolic defects among night shift workers with disturbances in circadian rhythms. DESIGN AND METHODS: We performed a hypothesis-generating, cross-sectional analysis of anthropometric, metabolic, lipid, and inflammation parameters, comparing active (a-NSW, n = 111) and former (f-NSW, n = 98) rotating night shift workers with diurnal workers (controls, n = 69). All participants were hospital nurses. We also evaluated the Pittsburgh Sleep Quality Index (PSQI) and assessed expression of transcription factors REV-ERBα and BMAL1 in peripheral blood mononuclear cells (PBMCs), as indicators of the molecular clock. RESULTS: Both a-NSW and f-NSW participants had significantly higher glycated hemoglobin (HbA1c) and white blood cell counts (WBC) (p < 0.001 for both), PSQI global score (p = 0.001) and diastolic blood pressure levels (p = 0.024) compared with controls. Expression of REV-ERBα/BMAL1 RNA in PBMC was significantly higher in a-NSW (p = 0.05) than in f-NSW or control participants. Multivariate regression analysis showed that working status and PSQI were independent determinants of higher HbA1c levels (p < 0.001). CONCLUSIONS: We demonstrated that young, healthy night shift workers show subclinical abnormalities in HbA1c and changes in peripheral clock gene expression.

Expression of clock genes Per1 and Bmal1 in total leukocytes in health and Parkinson's disease.

BACKGROUND: There is a growing number of clinical studies that revealed a variety of behavioral and physiological desynchronies in patients with Parkinson's disease (PD). However, these desynchronies have not been defined at the molecular level. METHODS: Using real-time RT-PCR assay, we analyzed the expression profiles of two principle clock genes, PER1 and BMAL1, in total leukocytes for 12 h during the evening, overnight and morning in subjects with PD and age/sex-matched healthy controls. RESULTS: A difference in the expression pattern of BMAL1 but not PER1 was apparent during the dark span, where the relative abundance of BMAL1 was significantly lower in PD patients versus control subjects at 21:00, 00:00 and 06:00 h. Furthermore, expression levels of BMAL1 in PD patients correlated with their United Parkinson's Disease Rating Scale score at 06:00, 09:00 h, and with Pittsburgh Sleep Quality Index score at 06:00 h. CONCLUSION: These results suggest that a peripheral molecular clock, as reflected in the dampened expression of the clock genes BMAL1 in total leukocytes, is altered in PD patients. In addition, the relative BMAL1 levels correlate positively with PD severity, which could provide a molecular basis to help monitor disease progression and response to investigational drugs.

Effect of melatonin administration on the PER1 and BMAL1 clock genes in patients with Parkinson's disease.

Sleep disorders are a widespread condition in patients with Parkinson's disease (PD), which has been linked to a deregulation of the circadian cycle and therefore of the clock genes. The aim of this study was to evaluate the effect of melatonin (MEL) on the PER1 and BMAL1 clock genes in patients with PD. A double-blind, cross-over, placebo-controlled randomized clinical trial pilot study was conducted in 26 patients with stage 1-3 PD according to the Hoehn & Yahr scale, who received either 25 mg of MEL or a placebo at noon and 30 min before bedtime for three months. The relative expression of the PER1 and BMAL1 genes was measured, as well as the presence of daytime, nocturnal, and global sleepiness, and the progression of PD. The levels of the PER1 and BMAL1 genes at baseline were 0.9 (0.1-3) vs. 0.56 (0.1-2.5), respectively; while after the intervention with MEL or placebo the BMAL1 levels increased to 2.5 (0-3.70) vs. 2.2 (0.10-3.30), respectively (d = 0.387). Fifty percent (50 %) of patients had daytime sleepiness and sixty-five percent (65 %) had abnormal nighttime sleepiness, yet neither group showed changes after the intervention. Patients with PD exhibited an alteration in the levels of the clock genes: MEL increased the levels of BMAL1, but the PER1 levels remained unchanged.

Regulation of Circadian Clock Genes on Sleep Disorders in Traumatic Brain Injury Patients.

BACKGROUND: In patients with traumatic brain injury (TBI), whether sleep disorder is associated with disturbances in molecular rhythmicity is unclear. This study aimed to investigate the relationship between abnormal sleep and regulation by circadian rhythms in patients with TBI. METHODS: We sampled buccal cells and human blood samples from patients with TBI diagnosed with sleep disorders and those with normal sleep and investigated differences in the expression levels of Clock, Per2, and Bmal1 between the 2 groups. RESULTS: The expression peaks of Clock, Per2, and Bmal1 were at 12:00. There was a statistically significant difference between the sleep disorder group and the normal sleep group in the level of Clock mRNA expression (P = 0.0003 in oral mucosa and P < 0.0001 in mononuclear cells). There was no significant between-group difference in Bmal1 mRNA expression level (P = 0.1187 in oral mucosa and P = 0.2094 in mononuclear cells). There were significant between-group differences in Per2 mRNA expression levels at 12:00 (P = 0.0102 in oral mucosa and P = 0.0006 in mononuclear cells) and 18:00 (P = 0.0004 in oral mucosa and P = 0.0015 in mononuclear cells) but no significant difference at 24:00 (P = 0.7838 in oral mucosa and P = 0.2808 in mononuclear cells). CONCLUSIONS: Abnormal expression levels of Per2, Clock, and Bmal1 were detected in patients with TBI-related sleep disorders. These novel findings demonstrate disturbances in the molecular clock in TBI patients and have important implications for our understanding of the aberrant rhythms reported in this disease.

Circadian Rhythm Disruption and Sepsis in Severe Trauma Patients.

BACKGROUND: Circadian rhythms are important regulators of immune functions. Admission to an intensive care unit may impact molecular clock activity and host response. Our objective was to assess and compare the immune circadian rhythms in trauma patients who develop and in those who do not develop sepsis. METHODS: Blood samples were collected from severe trauma patients within 4 days after admission, with collections taking place every 4 h over a 24-h period. Cortisol and cytokines were measured with immunoassays. Whole-blood expression of 3 clock genes (Bmal1, Per2, and Per3) was studied by reverse transcription quantitative polymerase chain reaction. Neutrophils, monocytes, and lymphocytes were analyzed by flow cytometry. Patients with and without sepsis were compared with the cosinor mixed model to estimate mesors, amplitudes, and acrophases. RESULTS: Thirty-eight patients were enrolled in the study, and 13 developed at least 1 septic episode. The septic patients had higher levels of cortisol than the nonseptic patients (mesor at 489 nmol/L vs. 405 nmol/L, P < 0.05) and delayed acrophases (22 h vs. 15 h, P < 0.05). They also had lower lymphocyte counts (mesor at 785 vs. 1,012 cells/µL, P < 0.05), higher neutrophil counts (mesor at 7,648 vs. 7,001 cells/µL, P < 0.05), and monocyte counts (mesor at 579 vs. 473 cells/µL, P < 0.05) than the nonseptic patients. Although no amplitude difference was identified, the acrophases were significantly different between the 2 groups for lymphocytes, interleukin 10 and tumor necrosis factor. CONCLUSION: We demonstrated that all trauma patients had impaired circadian rhythms of cortisol, cytokines, leukocytes, and clock genes. Early circadian disruption was associated with the occurrence of sepsis and might be a marker of sepsis severity.

Clock-Bmal1 mediates MMP9 induction in acrolein-promoted atherosclerosis associated with gut microbiota regulation.

Circadian rhythm is believed to play important roles in atherosclerosis. The gut microbiota is found to be closely related to atherogenesis, and shows compositional and functional circadian oscillation. However, it's still unclarified whether circadian clock and intestinal microbiota are involved in the progression of atherosclerosis induced by environmental pollutant acrolein. Herein, patients with atherosclerosis showed higher MMP9, a promising biomarker for atherosclerosis, and lower Bmal1 and Clock expression in the plasma. Interestingly, acrolein exposure contributed to the increased MMP9, decreased Clock and Bmal1, and activated MAPK pathways in human umbilical vein endothelial cells (HUVECs). We found that knockdown of Clock or Bmal1 lead to upregulation of MMP9 in HUVECs, and that Clock and Bmal1 expression was elevated while MAPK pathways were blocked. Atherosclerotic apolipoproteinE-deficient mice consumed a high-fat diet were used and treated with acrolein (3 mg/kg/day) in the drinking water for 12 weeks. Upregulation of MMP9, and downregulation of Clock and Bmal1 were also observed in plasma of the mice. Besides, acrolein feeding altered gut microbiota composition at a phylum level especially for an increased Firmicutes and a decreased Bacteroidetes. Additionally, gut microbiota showed correlation with atherosclerotic plaque, MMP9 and Bmal1 levels. Therefore, our findings indicated that acrolein increased the expression of MMP9 through MAPK regulating circadian clock, which was associated with gut microbiota regulation in atherosclerosis. Circadian rhythms and gut microbiota might be promising targets in the prevention of cardiovascular disease caused by environmental pollutants.

Early sex-specific modulation of the molecular clock in trauma.

BACKGROUND: Immune system biology and most physiologic functions are tightly linked to circadian rhythms. Time of day-dependent variations in many biologic parameters also play a fundamental role in the disease process. We previously showed that the genes encoding the peripheral molecular clock were modulated in a sex-dependent manner in Q fever. METHODS: Here, we examined severe trauma patients at admission to the intensive care unit. Using quantitative real-time polymerase chain reaction, the whole-blood expression of the molecular clock components ARNTL, CLOCK, and PER2 was assessed in male and female trauma patients. Healthy volunteers of both sexes were used as controls. RESULTS: We observed a significant overexpression of both ARNTL and CLOCK in male trauma patients. CONCLUSION: We report, for the first time, the sex-related modulation of the molecular clock genes in the blood following severe trauma. These results emphasize the role of circadian rhythms in the immune response in trauma patients. LEVEL OF EVIDENCE: Epidemiologic study, level IV.

Decreased expression of Bmal2 in patients with Parkinson's disease.

Bmal1 is one of the central regulators of the clock machinery. Recently, we examined the expression profile of Bmal1 in total leukocytes for a 12h duration during the evening, overnight, and the morning, in subjects with Parkinson's disease (PD) and healthy controls. The results indicate that the expression of Bmal1 is significantly lower in PD patients versus control subjects. However, it is still unclear whether other key regulators of the clock machinery, especially Bmal2, the paralog of Bmal1, are also expressed differently in PD. To address this issue, the expression profiles of Bmal2, Clock, and Dec1 were examined in the same samples using real-time RT-PCR assay. The results show a difference in the expression pattern of Bmal2, but not Clock and Dec1. The expression of Bmal2 is significantly lower in PD at 21:00 h (p=0.005) and 00:00 h (p=0.025). These results together with our previous findings suggest that the molecular clock in total leukocytes is disturbed in PD patients.

Influence of age on clock gene expression in peripheral blood cells of healthy women.

Recent studies have demonstrated a close relationship between circadian clock function and the development of obesity and various age-related diseases. In this study, we investigated whether messenger RNA (mRNA) levels of clock genes are associated with age, body mass index, blood pressures, fasting plasma glucose, or shift work. Peripheral blood cells were obtained from 70 healthy women, including 25 shift workers, at approximately 9:00 AM. Transcript levels of clock genes (CLOCK, BMAL1, PER1, and PER3) were determined by real-time quantitative polymerase chain reaction. Stepwise multiple regression analysis demonstrated that BMAL1 mRNA levels were correlated only with age (beta = -.50, p < .001). In contrast, PER3 levels were correlated with fasting plasma glucose (beta = -.29, p < .05) and shift work (beta = .31, p < .05). These results suggest that increased age, glucose intolerance, and irregular hours independently affect the intracellular clock in humans.