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. 2018 Jan 23;8(1):1395.
doi: 10.1038/s41598-018-19836-7.

Gut Microbiota-Derived Short Chain Fatty Acids Induce Circadian Clock Entrainment in Mouse Peripheral Tissue

Yu Tahara  1   2 , Mayu Yamazaki  1 , Haruna Sukigara  1 , Hiroaki Motohashi  1 , Hiroyuki Sasaki  1 , Hiroki Miyakawa  1 , Atsushi Haraguchi  1 , Yuko Ikeda  1 , Shinji Fukuda  3   4 , Shigenobu Shibata  5
Affiliations

Gut Microbiota-Derived Short Chain Fatty Acids Induce Circadian Clock Entrainment in Mouse Peripheral Tissue

Yu Tahara et al. Sci Rep. .

Abstract

Microbiota-derived short-chain fatty acids (SCFAs) and organic acids produced by the fermentation of non-digestible fibre can communicate from the microbiome to host tissues and modulate homeostasis in mammals. The microbiome has circadian rhythmicity and helps the host circadian clock function. We investigated the effect of SCFA or fibre-containing diets on circadian clock phase adjustment in mouse peripheral tissues (liver, kidney, and submandibular gland). Initially, caecal SCFA concentrations, particularly acetate and butyrate, induced significant day-night differences at high concentrations during the active period, which were correlated with lower caecal pH. By monitoring luciferase activity correlated with the clock gene Period2 in vivo, we found that oral administration of mixed SCFA (acetate, butyrate, and propionate) and an organic acid (lactate), or single administration of each SCFA or lactate for three days, caused phase changes in the peripheral clocks with stimulation timing dependency. However, this effect was not detected in cultured fibroblasts or cultured liver slices with SCFA applied to the culture medium, suggesting SCFA-induced indirect modulation of circadian clocks in vivo. Finally, cellobiose-containing diets facilitated SCFA production and refeeding-induced peripheral clock entrainment. SCFA oral gavage and prebiotic supplementation can facilitate peripheral clock adjustment, suggesting prebiotics as novel therapeutic candidates for misalignment.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Daily fluctuations of caecal short chain fatty acid (SCFA), lactate content, and caecal pH in different conditions. (A,B) Caecal total SCFA content (A, acetate, butyrate, and propionate), each SCFA and lactate content (B, n = 3 tubes for SCFA measurement, 3 caecal samples were pooled in 1 tube). (C) Caecal pH was measured every 4 hours over 24 hours in ICR mice in ad libitum with high or low fibre diets (n = 10 for high fibre diets, n = 4 for low fibre diets in each time point). (D,E) Caecal picture (D) and pH (E) from vehicle or antibiotic-treated (at least a month by drinking) C57BL mice (n = 4 for each). All values are expressed as mean ± SEM. The P value of the one- or two-way ANOVA is indicated in the lower right side of each graph if significant. *p < 0.05, **p < 0.01, ***p < 0.001 (Tukey’s multiple comparisons test).
Figure 2
Figure 2
Effect of antibiotic treatment on peripheral PER2::LUC rhythms. Vehicle (water) or mixed antibiotics was administered to mice in drinking water over a month. (A) Average waveform of in vivo peripheral PER2::LUC imaging in the kidney, liver, and submandibular gland (sub gla). (B,C) Peak phase and amplitude of PER2::LUC rhythms. All the values are expressed as mean ± SEM. The number of mice used in this study is indicated in Table S1. More details on the statistics used is presented in Table S2. *p < 0.05, **p < 0.01, vs. vehicle gavaged group (t-test). P value of the two-way ANOVA is indicated in the lower right side of each graph if significant.
Figure 3
Figure 3
Administration of short chain fatty acids (SCFA) and organic acid (lactate) changed the phase of peripheral PER2::LUC rhythms with time-of-day dependency of treatment timing in antibiotic-induced microbiota depleted mice. (A) Experimental schedule. After drinking antibiotic-containing water for at least a month, mice were orally gavaged with vehicle (water) or SCFA + lactate mix (400 mM in each) (0.1 ml/10 g mouse weight) for 3 consecutive days at ZT0, 5, 12, or 17. Then, peripheral PER2::LUC bioluminescence rhythms were monitored every 4 hours for 24 hours starting at ZT7 after final injection. (B,C) Representative image or averaged waveform of in vivo peripheral PER2::LUC imaging in mice orally gavaged with vehicle or SCFA + lactate at ZT5. (D) Peak phase changes of PER2::LUC rhythm compared with the peak in the vehicle treatment group. The value of peak phase of the vehicle treatment was set as 0. All the values are expressed as mean ± SEM. The number of mice used in this study is indicated in Table S1. More details on the statistics is presented in Table S2. *p < 0.05, **p < 0.01, ***p < 0.001, vs. vehicle gavaged group (Tukey’s multiple comparisons or Mann Whitney test). P value of the two-way ANOVA is indicated in the lower right side of each graph if significant.
Figure 4
Figure 4
Administration of short chain fatty acids (SCFA) changed the phase of clock gene expression rhythms in the kidney of antibiotic-induced microbiota depleted mice. (A) Similar to Fig. 1A, antibiotic-induced microbiota depleted mice were orally gavaged SCFA + lactate mix (400 mM in each) at ZT5 for three consecutive days; then, kidneys were collected every 4 hours for 24 hours starting at ZT7 after the last injection. (B) Clock gene expression measured by RT-PCR. Peak phase analysis is shown in Table S3. Gene expression levels were normalized to Gapdh. All the values are expressed as mean ± SEM. P value of the two-way ANOVA is indicated in the lower right side of each graph if significant. **p < 0.01, ***p < 0.001, vs. vehicle gavaged group (Sidak’s multiple comparisons test).
Figure 5
Figure 5
Administration of each short chain fatty acid (SCFA) changed the phase of peripheral PER2::LUC rhythms in antibiotic-induced microbiota depleted mice. (A) Similar to Fig. 1A, antibiotic-induced microbiota depleted mice were orally gavaged with each SCFA (400 or 1600 mM) at ZT5 for three consecutive days, and then we measured peripheral PER2::LUC every 4 hours for 24 hours starting at ZT7 after the last injection. (B) Peak phase difference compared to the peak of the vehicle only treatment is shown. The value of peak change of the vehicle-gavaged group was set as 0. All values are expressed as mean ± SEM. The number of mice used in this study is indicated in Table S1. *p < 0.05 vs. vehicle only treatment (t-test or Mann Whitney test).
Figure 6
Figure 6
Application of short chain fatty acids (SCFA) failed to change PER2::LUC rhythms of mouse embryonic fibroblasts or cultured PER2::LUC liver slices, with dose and time-of-day dependency. (A) Representative PER2::LUC rhythms of each treatment timing. Circadian time (CT) 0 and CT12 were defined as the bottom and peak of the bioluminescence rhythm, respectively. Vehicle (water) or SCFA + lactate (100 μM) was applied to the media for 30 min (see methods section). Arrows indicate the treatment time and arrow heads indicate the first and second peaks we analysed. (B,C) Dose- or time-dependent phase changes of the first or second peak and amplitude of the first peak after SCFA treatment at CT1, 8, 15, or 22. Value of peak change of the vehicle treatment was set as 0. Value of amplitude of the vehicle treatment was set as 100. All the values are expressed as mean ± SEM (n = 4 in each group, except vehicle at CT22 n = 3). *p < 0.05 vs. vehicle only treatment (Mann Whitney test). (D,E) Representative PER2::LUC rhythms of each treatment timing in cultured liver slices. No difference was seen at the first peak after treatment between the vehicle and SCFA + lactate.
Figure 7
Figure 7
High fibre diets facilitated refeeding-induced phase resetting of peripheral PER2::LUC rhythms. (A) Experimental schedule. Control: Ad libitum feeding of low fibre diets. Fasting: Mice were fasted overnight. 1 day or 2 days refeeding: Mice were fasted from ZT12 and refed low or high fibre diets (1 g) at ZT4 for 1 day or 2 days. Peripheral PER2::LUC was monitored every 4 hours for 24 hours beginning at ZT7 after refeeding. (B,C) Caecal pH and SCFA content at 4 hours after refeeding of low or high fibre diets in overnight fasted mice (n = 6 for each, except n = 10 for pH of fasting group). (D,E) Averaged PER2::LUC rhythms in each condition. (F) Peak phase changes of PER2::LUC rhythm compared with the peak of the control group. The value of the peak phase of the control was set as 0. All values are expressed as mean ± SEM. The number of mice used in this study is indicated in Table S1. More detail on the statistics used is included in Table S2. *p < 0.05, **p < 0.01, ***p < 0.001, vs. vehicle or low fibre treatments (t-test or Tukey’s multiple comparisons test). P value of the two-way ANOVA is indicated in the lower right side of each graph if significant.
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