Project description:MS/MS fragmentation data on bile acid standards were acquired on the QE - with a gradient developed to separate between isomeric pairs on a Polar C18 column and a fragmentation energy of NCE 45.
Project description:Specific bile acids are potent signaling molecules that modulate metabolic pathways affecting lipid, glucose and bile acid homeostasis, and the microbiota. Bile acids are synthesized from cholesterol in the liver, and the key enzymes involved in bile acid synthesis (Cyp7a1, Cyp8b1) are regulated transcriptionally by the nuclear receptor FXR. We have identified an FXR-regulated pathway upstream of a transcriptional repressor that controls multiple bile acid metabolism genes. We identify MafG as an FXR target gene and show that hepatic MAFG overexpression represses genes of the bile acid synthetic pathway and modifies the biliary bile acid composition. In contrast, loss-of-function studies using MafG(+/-) mice causes de-repression of the same genes with concordant changes in biliary bile acid levels. Finally, we identify functional MafG response elements in bile acid metabolism genes using ChIP-seq analysis. Our studies identify a molecular mechanism for the complex feedback regulation of bile acid synthesis controlled by FXR.
Project description:Bile acids are multifunctional signaling molecules that play significant roles in maintaining microbial homeostasis. N6-methyladenine (m6A), the most abundant epitranscriptomic modification, mediates various biological processes by modulating RNA metabolism. However, the precise regulatory mechanisms of m6A methylation in bile acid metabolism, and its downstream effects on microbiota remain unclear. In this study, liver-specific Mettl14 knockout (Mettl14-LKO) reshaped bile acid profile and expression levels of protein related to bile acid metabolism, namely CYP7A1, FXR, and BSEP. M6A-seq data revealed m6A methylated peaks on CYP7A1. Mettl14-LKO significantly elevated expression of m6A “reader” IGF2BP3. Knockdown of IGF2BP3 inhibited CYP7A1 expression by decreasing mRNA stability. Mechanistically, Mettl14-LKO promoted bile acid synthesis by upregulating CYP7A1 expression in an m6A-IGF2BP3-dependent manner. Interestingly, Mettl14-LKO reduced bile acid content in ileum due to decreased BSEP level in liver. Noteworthy, we discovered for the first time that Mettl14 knockout in the liver altered fecal microbiota composition. Specifically, it changed the abundance of Cyanobacteria and Patescibacteria at phylum level, and Lachnochostridium, Candidatus-Saccharimonas, and Roseburia at genera level. Remarkably, Roseburia was negatively correlated with the bile acid levels and CYP7A1 expression. Our findings provide new insights into the role of METTL14 in regulating bile acid homeostasis and its impact on fecal microbiota. Roseburia emerges as a potential target for addressing metabolic diseases linked to disrupted METTL14 signaling.
Project description:Liver sinusoidal endothelial cells (LSEC) are unique endothelial cell typelining the sinusoids of the liver and we have shown that these cells respond in a unique matter when exposed to saturated and unsaturated free fatty acids (FFA) and bile acids. We used microarray to analyze the transcriptional differences between the LSEC exposed to free fatty acids and bile acid receptor agonists to further shed light on their role in non-alcoholic fatty liver disease. The Murine Liver Sinusoidal Endothelial Cell Line (TSEC) was treated with palmitic and oleic acid or the bile acid receptor agonist INT-767 for 8 hours. Total RNA was then harvested to determine transcriptional differences.
Project description:Bile acid return from the intestine and attendant signaling is necessary for liver regeneration after partial hepatectomy or CCl4 injury
Project description:Bile acids act as ligands for several nuclear receptors expressed in the small intestine, especially the terminal ileum. Bile acid pool composition can be influenced by health and disease states. Here, we used RNA-seq to investigate the effects of altering bile acid pool composition on gene expression in ileal organoids.