Fig 1.
Microbially modified bile acids (DCA & CDCA) oscillate clock controlled genes in synchronised Caco-2 cells.
Caco-2 cells were synchronized via serum starvation followed by a serum shock and treated with bile acids at 100 μM or with their corresponding bile salts. The cells were harvested for every 6h for a total of 48 hours. The relative expression levels of clock-regulated genes were measured using qRT-PCR and plotted in the graph versus time. The red colour graph represents the vehicle, pink represents the TDCA, brown represents TCDCA, blue represents DCA and green represents CDCA. Data represents pooled results from three independent biological replicates with two technical replicates.
Fig 2.
The periodic gene expression of CLOCK and CLOCK-controlled genes in synchronised Caco2 cells in the absence of bile acids.
Caco2 cells were synchronized via serum starvation followed by a serum shock as outlined for Fig 1. The cells were harvested every 6h for a total of 48 hours. The relative expression levels of clock genes were measured using qRT-PCR and plotted in the graph versus time. Data represents pooled results from three independent biological replicates with two technical replicates.
Fig 3.
Unconjugated bile acids (DCA & CDCA) influence expression of clock related genes in murine peripheral organs.
C57BL/6 mice were given vehicle (Corn oil) or DCA or CDCA each at 9 μmol/kg bodyweight dissolved in corn oil via oral gavage. The mice were gavaged three times (t = 0, t = 24h and t = 48h) with corn oil or bile acids and were fasted for 3h prior to harvesting of tissues from (A, B) Ileum, (C, D) Colon and (E, F) Liver. All tissues were harvested within the same 45 minute window during the light phase to minimize variation between subjects (see Materials & Methods). Total RNA was isolated from tissues and mRNA expression was measured using qRT-PCR. The expression of clock-regulated genes was analysed. Data are plotted relative to β-actin expression. The white bar represents the results from vehicle, grey bar represents the DCA treatment group and the black bar represents the CDCA group. Error bars denote SEM. Statistical significance determined by one way ANOVA (*P<0.05, **P<0.01, ***P<0.001), n = 4.
Fig 4.
Unconjugated bile acids (DCA and CDCA) influence expression of genes encoding input regulators of clock-regulated genes in murine peripheral organs.
C57BL/6 mice were given vehicle (Corn oil) or DCA or CDCA each at 9 μmol/ kg bodyweight dissolved in corn oil via oral gavage. The mice were gavaged three times (t = 0, t = 24h and t = 48h) with corn oil or bile acids and were fasted for 3h prior to harvesting of tissues from (A, B) Ileum, (C, D) Colon and (E, F) Liver. All tissues were harvested within the same 45 minute window during the light phase to minimize variation between subjects (see Materials & Methods). Total RNA was isolated from tissues and mRNA expression was measured using qRT-PCR. The expression of clock-controlled genes was analysed. Data are plotted relative to β-actin expression. The white bar represents the results from vehicle, grey bar represents the DCA treatment group and the black bar represents the CDCA group. Error bars denote SEM. Statistical significance was determined by one way ANOVA (*P<0.05, **P<0.01, ***P<0.001), n = 4.
Fig 5.
Model by which microbially-modified bile acids may influence expression of circadian genes.
Bile acids are synthesised from cholesterol and conjugated with taurine or glycine in the liver and stored in the gall bladder. Upon food intake, the bile salts are released into the duodenum and aid in fat digestion and adsorption. Gut microbes in the intestinal lumen deconjugate bile salts to yield unconjugated bile acids. Recent work from other groups has demonstrated that diet, antibiotics and probiotics may influence this microbial activity (see discussion). Within the ileal enterocytes, unconjugated bile acids influence the amplitude and periodicity of circadian gene expression. Nearly 95% of bile salts and bile acids are reabsorbed in the terminal ileum and transported back to the liver via the hepatic portal circulation. Upon reaching the liver, the bile acids further influence circadian gene expression profiles.