The control group did not exhibit peripheral clock phase-shift.

<p>(A) Waveform of PER2::LUC expression in various tissues. (B) Correlation maps of PER2::LUC rhythms between cosinor amplitude and index of goodness of fit. The dashed line indicates 40% of cosinor amplitude and 0.1 of goodness of fit. (C) Average of PER2::LUC expression in peak phases for each organ in each group. Numbers next to plots indicate the rates of mice whose PER2::LUC expression was rhythmic (rhythmic mice numbers/total mice numbers). (D) Average cosinor amplitude for each organ in each group. All of the values are expressed as mean ± or + SEM except (B). # p<0.05 versus intact by Mann-Whitney test.</p

Gene expression, after subjecting mice to temperature pulses in a water bath for 2 h.

<p>Mice were exposed to 41°C or 37°C water bath, or a cylindrical sieve as temperature treatment control. The data of control group were set to 1. *<i>p</i><0.05 versus control; #<i>p</i><0.05 versus 37°C group, by Dunn test, respectively. n = 4 for each group except 41°C group (n = 3) of <i>Per2</i> in the liver. All values are expressed as mean + SEM.</p

Exposing mice to 41°C or 37°C water bath stimulated phase-advance.

<p>(A) Correlation maps of PER2::LUC rhythms between cosinor amplitude and index of goodness of fit. The dashed line indicates 40% of cosinor amplitude and 0.1 of goodness of fit. (B) Average PER2::LUC peak phases of each organ for each group. Numbers next to plots indicate the rates of mice whose PER2::LUC expression was rhythmic. #<i>p</i><0.05, ##<i>p</i><0.01 versus control by Tukey test. **p<0.01 versus control by Dunn test (C) Average cosinor amplitude of each organ for each group. Graphs using data from mice whose PER2::LUC expression was rhythmic. All values are expressed as mean ± or + SEM except (A).</p

Exposing mice to 41°C water bath advanced the phase in a time-dependent manner.

<p>(A) Correlation maps of PER2::LUC rhythms between cosinor amplitude and index of goodness of fit. The dashed line indicates 40% of cosinor amplitude and 0.1 of goodness of fit. (B) Average of PER2::LUC expression peak phases for each organ in each group. Numbers next to plots indicate the rates of mice whose PER2::LUC expression was rhythmic. #p<0.05, ##p<0.01, ###p<0.001 vs. control by Dunn test. Statistical analysis was not applied for the liver in figure B because of N = 1 in the 0.5 h group. (C) Average cosinor amplitude for each organ in each group. Graphs using data from mice whose PER2::LUC expression was rhythmic. (D) Average core body temperature for each group during day 1, n = 8 for 37°C and n = 16 for 41°C, control, and intact groups. All values are expressed as mean ± or + SEM except (A) and (D).</p

Dexamethasone injection did not shift phase of the peripheral clock.

<p>Dexamethasone (dex) was injected to mice at ZT 5 once a day for two continuous days. (A) Average PER2::LUC peak phases of each organ for each group. Numbers next to plots indicate the rates of mice whose PER2::LUC expression was rhythmic. (B) Averaged cosinor amplitude of each organ for each group. Graphs using data from mice whose PER2::LUC expression was rhythmic. All values are expressed as mean ± or + SEM.</p

Feeding and adrenal entrainment stimuli are both necessary for normal circadian oscillation of peripheral clocks in mice housed under different photoperiods

<div><p>The mammalian circadian rhythm is entrained by multiple factors, including the light–dark cycle, the organism’s feeding pattern and endocrine hormones such as glucocorticoids. Both a central clock (the suprachiasmatic nucleus, or SCN) and peripheral clocks (i.e. in the liver and lungs) in mice are entrained by photoperiod. However, the factors underlying entrainment signals from the SCN to peripheral clocks are not well known. To elucidate the role of entrainment factors such as corticosterone and feeding, we examined whether peripheral clock rhythms were impaired by adrenalectomy (ADX) and/or feeding of 6 meals per day at equal intervals under short-day, medium-day and long-day photoperiods (SP, MP and LP, respectively). We evaluated the waveform and phase of circadian rhythms in the liver, kidney and salivary gland by <i>in vivo</i> imaging of PER2::LUCIFERASE knock-in mice. In intact mice, the waveforms of the peripheral clocks were similar among all photoperiods. The phases of peripheral clocks were well adjusted by the timing of the “lights-off”-operated evening (E) oscillator but not the “lights-on”-operated morning (M) oscillator. ADX had almost no effect on the rhythmicity and phase of peripheral clocks, regardless of photoperiod. To reduce the feeding-induced signal, we placed mice on a restricted feeding regimen with 6 meals per day (6 meals RF). This caused advances of the peripheral clock phase in LP-housed mice (2–5 h) and MP-housed mice (1–2 h) but not SP-housed mice. Thus, feeding pattern may affect the phase of peripheral clocks, depending on photoperiod. More specifically, ADX + 6 meals RF mice showed impairment of circadian rhythms in the kidney and liver but not in the salivary gland, regardless of photoperiod. However, the impairment of peripheral clocks observed in ADX + 6 meals RF mice was reversed by administration of dexamethasone for 3 days. The phase differences in the salivary gland clock among SP-, MP- and LP-housed mice became very small following treatment with ADX + 6 meals RF, suggesting that the effect of photoperiod was reduced by ADX and 6 meals RF. Because the SCN rhythm (as evaluated by PER2 immunohistochemistry) was not disrupted by ADX + 6 meals RF, impairment of peripheral clocks in these mice was not because of impaired SCN clock function. In addition, locomotor activity rhythm and modifications of the feeding pattern may not be completely responsible for determining the phase of peripheral clocks. Thus, this study demonstrates that the phase of peripheral clocks responds to a photoperiodic lights-off signal, and suggests that signals from normal feeding patterns and the adrenal gland are necessary to maintain the oscillation and phase of peripheral clocks under various photoperiods.</p></div

Dose-dependent effects of chronic treatment with various flavonoids on circadian rhythm period and amplitude.

<p>Various flavonoids: <b>(A)</b> flavone, <b>(B)</b> flavonol, <b>(C)</b> isoflavone, <b>(D)</b> catechin, and <b>(E)</b> PMF were chronically applied to the culture medium of MEFs. The circadian rhythm period and amplitude in the presence of these flavonoids were compared with that in the presence of vehicle (VEH; 0.25% DMSO). The amplitudes (left) and the periods (right) of the PER2::LUC waveform. VEH average amplitude value is normalized to indicate 100 (circle), and all normalized amplitude points are indicated (rhombus). Period value is analyzed by sin-fitting, and each value (rhombus) and average (circle) are indicated. Values are mean ± SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01 vs. VEH (Tukey or Dunn’s test).</p

Involvement of ERK in the nobiletin-induced phase delay of the circadian rhythm in PER2::LUC MEFs.

<p><b>(A)</b> Western blotting. MEFs from PER2::LUC knock-in mice were cultured in a 35-mm dish to a density of 1 × 10⁶ cells and then incubated with nobiletin (50 μM) or DMSO (0.25%; vehicle) for 15 or 60 min. Blotted proteins were detected with antibodies against ERK1/2, phosphor-ERK1/2, orβ-actin. <b>(B and C)</b> The amount of protein was measured as the chemiluminescent signal. The ratio of phosphorylated ERK1/2 toβ-actin is shown. Values are mean ± SEM (n = 3 per group). *<i>p</i> < 0.05 vs. VEH (independent <i>t</i>-test). <b>(D and E)</b> Transient application of nobiletin (50 μM) at CT14–14.5 caused a phase delay in peak 2 (red). When 25 μM U0126 (an ERK inhibitor) was added 5 min before nobiletin application (blue), the phase delay induced by nobiletin was blocked. Values are mean ± SEM (n = 8 per group). **<i>p</i> < 0.01 vs. VEH (two-way ANOVA, <i>post-hock</i> Tukey’s test).</p

The <i>r</i> values and <i>p</i> values of amplitude-period correlations.

<p>The <i>r</i> values and <i>p</i> values of amplitude-period correlations.</p

Chemical structures of flavonoids.

<p><b>(A)</b> flavone, <b>(B)</b> flavonol, <b>(C)</b> isoflavone, <b>(D)</b> catechin, and <b>(E)</b> PMF.</p