Investigating the Influence of Gut Cell Circadian Rhythm on Mitochondrial Metabolism and Toxicological Responses

Circadian rhythms regulate numerous cellular and physiological processes, aligning cellular metabolic activities with day and night cycles. This time-keeping mechanism, driven by transcriptional/translational feedback loops of core clock genes, is present in nearly all mammalian cells, including the cells in intestinal epithelium. Mitochondrial metabolism in gut cells is linked with circadian regulation and both processes are influenced by the metabolites derived from gut microbiome. These interactions help coordinate essential physiological processes including nutrient absorption, energy metabolism, and intestinal barrier maintenance. In this project I used human intestinal epithelial (Caco-2) cells to investigate how microbiome-associated metabolites influence gut cell circadian rhythmicity and mitochondria metabolism. Caco-2 cells entrained with dexamethasone were exposed to Melatonin, Butyric acid, Indole-3-carboxaldehyde and L-ornithine and the oxygen consumption rates of the cells were monitored alongside the expression of BMAL1 and PER2 clock genes. We observed that Caco-2 cells can be successfully entrained with 1 µM dexamethasone to display circadian rhythms in clock gene expression during 24 hour period. However, exposure to microbiome-derived metabolites disrupted or completely removed these rhythmic patterns and led to alterations in mitochondrial respiration compared to the control, including changes in OCR amplitude and rhythmicity, indicating interference with the Caco-2 cells’ synchronized metabolic state. The interconnection between clock gene expression and mitochondrial metabolism appeared relatively weak, however it may require more optimized experimental conditions. Despite these challenges this work has laid the foundation to consider Caco-2 cell culture as a valuable model for interactions between circadian regulation, metabolism and toxicological responses highlighting the importance of optimized and developed experimental strategies for future research.