Fish oil accelerates diet-induced entrainment of the mouse peripheral clock via GPR120

The circadian peripheral clock is entrained by restricted feeding (RF) at a fixed time of day, and insulin secretion regulates RF-induced entrainment of the peripheral clock in mice. Thus, carbohydrate-rich food may be ideal for facilitating RF-induced entrainment, although the role of dietary oils in insulin secretion and RF-induced entrainment has not been described. The soybean oil component of standard mouse chow was substituted with fish or soybean oil containing docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). Tuna oil (high DHA/EPA), menhaden oil (standard), and DHA/EPA dissolved in soybean oil increased insulin secretion and facilitated RF-induced phase shifts of the liver clock as represented by the bioluminescence rhythms of PER2::LUCIFERASE knock-in mice. In this model, insulin depletion blocked the effect of tuna oil and fish oil had no effect on mice deficient for GPR120, a polyunsaturated fatty acid receptor. These results suggest food containing fish oil or DHA/EPA is ideal for adjusting the peripheral clock

Potent Effects of Flavonoid Nobiletin on Amplitude, Period, and Phase of the Circadian Clock Rhythm in PER2::LUCIFERASE Mouse Embryonic Fibroblasts.

Flavonoids are natural polyphenols that are widely found in plants. The effects of flavonoids on obesity and numerous diseases such as cancer, diabetes, and Alzheimer's have been well studied. However, little is known about the relationships between flavonoids and the circadian clock. In this study, we show that continuous or transient application of flavonoids to the culture medium of embryonic fibroblasts from PER2::LUCIFERASE (PER2::LUC) mice induced various modifications in the circadian clock amplitude, period, and phase. Transient application of some of the tested flavonoids to cultured cells induced a phase delay of the PER2::LUC rhythm at the down slope phase. In addition, continuous application of the polymethoxy flavonoids nobiletin and tangeretin increased the amplitude and lengthened the period of the PER2::LUC rhythm. The nobiletin-induced phase delay was blocked by co-treatment with U0126, an ERK inhibitor. In summary, among the tested flavonoids, polymethoxy flavones increased the amplitude, lengthened the period, and delayed the phase of the PER2::LUC circadian rhythm. Therefore, foods that contain polymethoxy flavones may have beneficial effects on circadian rhythm disorders and jet lag

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

Effects of continuous application of flavone, 7-hydroxyflavone, and 5-hydroxyflavone on the amplitude and period of the circadian clock rhythm.

<p><b>(A)</b> Experimental schedule for continuous application of flavonoids. <b>(B)</b> Wave forms of the bioluminescence rhythm in MEFs derived from PER2::LUC mice. Flavone, 5-hydroxyflavone, 7-hydroxyflavone (10 μM each), or 0.25% DMSO as vehicle (VEH), was applied to assess the effect of slight structural differences in flavone structure on the circadian clock rhythm. <b>(C)</b> The phase shift of the first peak. 7-hydroxyflavone induces phase delay compared with VEH. VEH average value was normalized to indicate 0. <b>(D)</b> The period length in the presence of flavone, 5-hydroxyflavone, or 7-hydroxyflavone. Among these three flavones, the period length differed slightly. <b>(E)</b> Amplitudes were not affected by any of the three tested flavones when compared with VEH. In figure C and D, values indicate each point and the average. Values are mean ± SEM (n = 4 per group). **<i>p</i> < 0.01 vs. VEH (Tukey’s test).</p

Effect of chronic or transient application of nobiletin on the circadian rhythm of PER2::LUC liver slices.

<p>Nobiletin was applied chronically <b>(A)</b> or transiently <b>(B)</b> in <i>ex vivo</i> culture medium with PER2::LUC liver slices. <b>(A)</b> The figures shown are the deviated waveforms generated by the PER2::LUC imaging during exposure to nobiletin 50 μM, 100 μM, or vehicle (0.25% DMSO) (left). Bar graph shows the average values analyzed by sin-fitting (right). Values are mean ± SEM (n = 8 per group). *<i>p</i> < 0.05 vs. VEH (Tukey’s test). <b>(B)</b> The figures shown are the deviated waveforms generated by the PER2::LUC imaging during exposure to nobiletin 100 μM, 200 μM, or vehicle (0.25% DMSO) (left). The purple triangle indicates the application time point, and the purple arrow indicates the imaging-restart time point. The phase shift of peak 2 is shown in the right panel. VEH average phase changed value was normalized to indicate 0. Values are mean ± SEM (n = 5 per group). *<i>p</i> < 0.05, **<i>p</i> < 0.01 vs. VEH (Tukey’s test).</p

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

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

Effect of transient application of flavonoids (100 μM or 200 μM) at CT14–14.5 on the phase of the circadian rhythm.

<p><b>(A)</b> Experimental schedule for transient application of flavonoids. Flavonoids [<b>(B)</b> flavone, <b>(C)</b> flavonol, <b>(D)</b> isoflavone, <b>(E)</b> catechin (at 100 μM or 200 μM), or <b>(F)</b> PMF (at 50 μM, 100 μM, or 200 μM)] or vehicle (0.25% DMSO) were transiently applied at CT14–14.5 for 30 min to compare their effects on the phase. The figures shown are the deviated waveforms generated by the PER2::LUC imaging during exposure to 100 μM (left) or 200 μM (middle) flavonoid <b>(B-E)</b>. <b>(F)</b> PMF was added at 50 μM concentration. The phase shift of peak 2 is shown in the right panel. VEH average phase changed value was normalized to indicate 0. The purple triangle indicates the application time point, and the purple arrow indicates the imaging-restart time point. Values are mean ± SEM (n = 4 per group). *<i>p</i> < 0.05, **<i>p</i> < 0.01 vs. VEH (Tukey’s test).</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

Glucagon and/or IGF-1 Production Regulates Resetting of the Liver Circadian Clock in Response to a Protein or Amino Acid-only Diet

The circadian system controls the behavior and multiple physiological functions. In mammals, the suprachiasmatic nucleus (SCN) acts as the master pacemaker and regulates the circadian clocks of peripheral tissues. The SCN receives information regarding the light-dark cycle and is thus synchronized to the external 24-hour environment. In contrast, peripheral clocks, such as the liver clock, receive information from the SCN and other factors; in particular, food intake which leads to insulin secretion induces strong entrainment of the liver clock. On the other hand, the liver clock of insulin-depleted mice treated with streptozotocin (STZ) has been shown to be entrained by scheduled feeding, suggesting that insulin is not necessary for entrainment of the liver clock by feeding. In this study, we aimed to elucidate additional mechanism on entraining liver clock by feeding a protein-only diet and/or amino-acid administration which does not increase insulin levels. We demonstrated that protein-only diet and cysteine administration elicit entrainment of the liver clock via glucagon secretion and/or insulin-like growth factors (IGF-1) production. Our findings suggest that glucagon and/or IGF-1 production are additional key factors in food-induced entrainment