Reduction in hepatic secondary bile acids caused by short-term antibiotic-induced dysbiosis decreases mouse serum glucose and triglyceride levels.
ABSTRACT: Antibiotic-caused changes in intestinal flora (dysbiosis) can have various effects on the host. Secondary bile acids produced by intestinal bacteria are ligands for specific nuclear receptors, which regulate glucose, lipid, and drug metabolism in the liver. The present study aimed to clarify the effect of changes in secondary bile acids caused by antibiotic-induced dysbiosis on the host physiology, especially glucose, lipid, and drug metabolism. After oral administration of non-absorbable antibiotics for 5 days, decreased amounts of secondary bile acid-producing bacteria in faeces and a reduction in secondary bile acid [lithocholic acid (LCA) and deoxycholic acid (DCA)] levels in the liver were observed. Serum glucose and triglyceride levels were also decreased, and these decreases were reversed by LCA and DCA supplementation. Quantitative proteomics demonstrated that the expression levels of proteins involved in glycogen metabolism, cholesterol, bile acid biosynthesis, and drug metabolism (Cyp2b10, Cyp3a25, and Cyp51a1) were altered in the liver in dysbiosis, and these changes were reversed by LCA and DCA supplementation. These results suggested that secondary bile acid-producing bacteria contribute to the homeostasis of glucose and triglyceride levels and drug metabolism in the host, and have potential as therapeutic targets for treating metabolic disease.
Project description:The gut microbiome can impact brain health and is altered in Parkinson’s disease (PD) patients. The vermiform appendix is a lymphoid tissue implicated in the storage and regulation of the gut microbiome. Here, we investigate changes in the functional microbiome in the appendix of PD patients relative to controls by metatranscriptomic analysis. In the PD appendix, we find microbial dysbiosis affecting lipid metabolism, particularly an upregulation of bacteria responsible for secondary bile acid synthesis. Likewise, proteomic and transcript analysis in the PD gut corroborates a disruption in cholesterol homeostasis and lipid catabolism. Bile acid analysis in the PD appendix reveals an increase in the microbially-derived, toxic secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA). Synucleinopathy in mice induces similar microbiome alterations to those of PD patients and heightens microbial changes to gut inflammation. As observed in PD, the mouse model of synucleinopathy has elevated DCA and LCA. Raised levels of DCA and LCA can lead to liver injury, and an analysis of blood markers of liver dysfunction shows evidence of biliary abnormalities in PD patients, including elevated alkaline phosphatase and bilirubin. Increased bilirubin levels are also evident before PD diagnosis, in individuals at-risk of developing PD. In sum, microbially-derived toxic bile acids are heightened in PD and biliary changes may even precede the onset of overt motor symptoms.
Project description:The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 μM and are known to block the growth of Clostridium difficile<sup>1</sup>, promote hepatocellular carcinoma<sup>2</sup> and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. <sup>3</sup>). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids<sup>4</sup>; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.
Project description:<i>Extibacter muris</i> is a newly described mouse gut bacterium which metabolizes cholic acid (CA) to deoxycholic acid (DCA) via 7α-dehydroxylation. Although bile acids influence metabolic and inflammatory responses, few <i>in vivo</i> models exist for studying their metabolism and impact on the host. Mice were colonized from birth with the simplified community Oligo-MM<sup>12</sup> with or without <i>E. muris</i>. As the metabolism of bile acids is known to affect lipid homeostasis, mice were fed either a low- or high-fat diet for eight weeks before sampling and analyses targeting the gut and liver. Multiple Oligo-MM<sup>12</sup> strains were capable of deconjugating primary bile acids <i>in vitro. E. muris</i> produced DCA from CA either as pure compound or in mouse bile. This production was inducible by CA <i>in vitro</i>. Ursodeoxycholic, chenodeoxycholic, and β-muricholic acid were not metabolized under the conditions tested. All gnotobiotic mice were stably colonized with <i>E. muris</i>, which showed higher relative abundances after HF diet feeding. The presence of <i>E. muris</i> had minor, diet-dependent effects on Oligo-MM<sup>12</sup> communities. The secondary bile acids DCA and surprisingly LCA and their taurine conjugates were detected exclusively in <i>E. muris</i>-colonized mice. <i>E. muris</i> colonization did not influence body weight, white adipose tissue mass, liver histopathology, hepatic aspartate aminotransferase, or blood levels of cholesterol, insulin, and paralytic peptide (PP). However, proteomics revealed shifts in hepatic pathways involved in amino acid, glucose, lipid, energy, and drug metabolism in <i>E. muris</i>-colonized mice. Liver fatty acid composition was substantially altered by dietary fat but not by <i>E. muris.</i>In summary, <i>E. muris</i> stably colonized the gut of mice harboring a simplified community and produced secondary bile acids, which affected proteomes in the liver. This new gnotobiotic mouse model can now be used to study the pathophysiological role of secondary bile acids <i>in vivo</i>.
Project description:Bile acids are known to play important roles as detergents in the absorption of hydrophobic nutrients and as signaling molecules in the regulation of metabolism. We tested the novel hypothesis that naturally occurring bile acids interfere with protein-mediated hepatic long chain free fatty acid (LCFA) uptake. To this end, stable cell lines expressing fatty acid transporters as well as primary hepatocytes from mouse and human livers were incubated with primary and secondary bile acids to determine their effects on LCFA uptake rates. We identified ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA) as the two most potent inhibitors of the liver-specific fatty acid transport protein 5 (FATP5). Both UDCA and DCA were able to inhibit LCFA uptake by primary hepatocytes in a FATP5-dependent manner. Subsequently, mice were treated with these secondary bile acids in vivo to assess their ability to inhibit diet-induced hepatic triglyceride accumulation. Administration of DCA in vivo via injection or as part of a high-fat diet significantly inhibited hepatic fatty acid uptake and reduced liver triglycerides by more than 50%.The data demonstrate a novel role for specific bile acids, and the secondary bile acid DCA in particular, in the regulation of hepatic LCFA uptake. The results illuminate a previously unappreciated means by which specific bile acids, such as UDCA and DCA, can impact hepatic triglyceride metabolism and may lead to novel approaches to combat obesity-associated fatty liver disease.
Project description:The gut microbiome can impact brain health and is altered in Parkinson's disease (PD). The vermiform appendix is a lymphoid tissue in the cecum implicated in the storage and regulation of the gut microbiota. We sought to determine whether the appendix microbiome is altered in PD and to analyze the biological consequences of the microbial alterations. We investigated the changes in the functional microbiota in the appendix of PD patients relative to controls (<i>n</i> = 12 PD, 16 C) by metatranscriptomic analysis. We found microbial dysbiosis affecting lipid metabolism, including an upregulation of bacteria responsible for secondary bile acid synthesis. We then quantitatively measure changes in bile acid abundance in PD relative to the controls in the appendix (<i>n</i> = 15 PD, 12 C) and ileum (<i>n</i> = 20 PD, 20 C). Bile acid analysis in the PD appendix reveals an increase in hydrophobic and secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA). Further proteomic and transcriptomic analysis in the appendix and ileum corroborated these findings, highlighting changes in the PD gut that are consistent with a disruption in bile acid control, including alterations in mediators of cholesterol homeostasis and lipid metabolism. Microbially derived toxic bile acids are heightened in PD, which suggests biliary abnormalities may play a role in PD pathogenesis.
Project description:Although the unconjugated secondary bile acids, specifically deoxycholic acid (DCA) and lithocholic acid (LCA), are considered to be risk factors for colorectal cancer, the precise mechanism(s) by which they regulate carcinogenesis is poorly understood. We hypothesize that the cytotoxic bile acids may promote stemness in colonic epithelial cells leading to generation of cancer stem cells (CSCs) that play a role in the development and progression of colon cancer.Normal human colonic epithelial cells (HCoEpiC) were used to study bile acid DCA/LCA-mediated induction of CSCs. The expression of CSC markers was measured by real-time qPCR. Flow cytometry was used to isolate CSCs. T-cell factor/lymphoid-enhancing factor (TCF/LEF) luciferase assay was employed to examine the transcriptional activity of ?-catenin. Downregulation of muscarinic 3 receptor (M3R) was achieved through transfection of corresponding siRNA.We found DCA/LCA to induce CSCs in normal human colonic epithelial cells, as evidenced by the increased proportion of CSCs, elevated levels of several CSC markers, as well as a number of epithelial-mesenchymal transition markers together with increased colonosphere formation, drug exclusion, ABCB1 and ABCG2 expression, and induction of M3R, p-EGFR, matrix metallopeptidases, and c-Myc. Inhibition of M3R signaling greatly suppressed DCA/LCA induction of the CSC marker ALDHA1 and also c-Myc mRNA expression as well as transcriptional activation of TCF/LEF.Our results suggest that bile acids, specifically DCA and LCA, induce cancer stemness in colonic epithelial cells by modulating M3R and Wnt/?-catenin signaling and thus could be considered promoters of colon cancer.
Project description:Necrotic enteritis (NE), mainly induced by the pathogens of <i>Clostridium perfringens</i> and coccidia, causes huge economic losses with limited intervention options in the poultry industry. This study investigated the role of specific bile acids on NE development. Day-old broiler chicks were assigned to six groups: noninfected, NE, and NE with four bile diets of 0.32% chicken bile, 0.15% commercial ox bile, 0.15% lithocholic acid (LCA), or 0.15% deoxycholic acid (DCA). The birds were infected with <i>Eimeria maxima</i> at day 18 and <i>C. perfringens</i> at day 23 and 24. The infected birds developed clinical NE signs. The NE birds suffered severe ileitis with villus blunting, crypt hyperplasia, epithelial line disintegration, and massive immune cell infiltration, while DCA and LCA prevented the ileitis histopathology. NE induced severe body weight gain (BWG) loss, while only DCA prevented NE-induced BWG loss. Notably, DCA reduced the NE-induced inflammatory response and the colonization and invasion of <i>C. perfringens</i> compared to NE birds. Consistently, NE reduced the total bile acids in the ileal digesta, while dietary DCA and commercial bile restored it. Together, this study showed that DCA and LCA reduced NE histopathology, suggesting that secondary bile acids, but not total bile acid levels, play an essential role in controlling the enteritis.
Project description:The secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), formed by gut microbiota from primary bile acids via a multi-step 7?-dehydroxylation reaction, have wide-ranging effects on host metabolism and play an important role in health and disease. A few 7?-dehydroxylating strains have been isolated, where bile acid-inducible (bai) genes were organized in a gene cluster and encoded major enzymes involved. However, only little is known on diversity and abundance of intestinal bacteria catalysing DCA/LCA formation in the human gut in situ. In this study, we took the opportunity to screen metagenome-assembled genomes (MAGs) from sequence data of stool samples provided by two recent studies along with newly available gut-derived isolates for the presence of the bai gene cluster. We revealed in total 765 and 620 MAGs encoding the potential to form DCA/LCA that grouped into 21 and 26 metagenomic species, respectively. The majority of MAGs (92.4 and 90.3%) were associated with a Ruminococcaceae clade that still lacks an isolate, whereas less MAGs belonged to Lachnospiraceae along with eight new isolates (n total?=?11) that contained the bai genes. Only a few MAGs were linked to Peptostreptococcaceae. Signatures for horizontal transfer of bai genes were observed. This study gives a comprehensive overview of the diversity of bai-exhibiting bacteria in the human gut highlighting the application of metagenomics to unravel potential functions hidden from current isolates. Eventually, isolates of the identified main MAG clade are required in order to prove their capability of 7?-dehydroxylating primary bile acids.
Project description:A perinatal high-salt (HS) diet was reported to elevate plasma triglycerides. This study aimed to investigate the hypothesis that a perinatal HS diet predisposed offspring to non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of abnormal lipid metabolism, and the possible mechanism. Female C57BL/6 mice were fed a control diet (0.5% NaCl) or HS diet (4% NaCl) during pregnancy and lactation and their offspring were sacrificed at weaning. The perinatal HS diet induced greater variation in fecal microbial beta-diversity (β-diversity) and increased bacteria abundance of <i>Proteobacteria</i> and <i>Bacteroides</i>. The gut microbiota dysbiosis promoted bile acid homeostasis disbalance, characterized by the accumulation of lithocholic acid (LCA) and deoxycholic acid (DCA) in feces. These alterations disturbed gut barrier by increasing the expression of tight junction protein (<i>Tjp</i>) and occludin (<i>Ocln</i>), and increased systemic lipopolysaccharide (LPS) levels and hepatic inflammatory cytokine secretion (TNF-α and IL-6) in the liver. The perinatal HS diet also inhibited hepatic expression of hepatic FXR signaling (<i>CYP7A1</i> and <i>FXR</i>), thus triggering increased hepatic expression of pro-inflammatory cytokines (<i>TNF-α</i> and <i>IL-6</i>) and hepatic lipid metabolism-associated genes (<i>SREBP-1c</i>, <i>FAS</i>, <i>ACC</i>), leading to unique characteristics of NAFLD. In conclusion, a perinatal HS diet induced NAFLD in weanling mice offspring; the possible mechanism was related to increased bacteria abundance of <i>Proteobacteria</i> and <i>Bacteroides</i>, increased levels of LCA and DCA in feces, and increased expressions of hepatic FXR signaling.
Project description:An improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method was established for the simultaneous analysis of various bile acids (BA) and applied to investigate liver BA content in C57BL/6 mice fed 1% cholic acid (CA), 0.3% deoxycholic acid (DCA), 0.3% chenodeoxycholic acid (CDCA), 0.3% lithocholic acid (LCA), 3% ursodeoxycholic acid (UDCA), or 2% cholestyramine (resin). Results indicate that mice have a remarkable ability to maintain liver BA concentrations. The BA profiles in mouse livers were similar between CA and DCA feedings, as well as between CDCA and LCA feedings. The mRNA expression of Cytochrome P450 7a1 (Cyp7a1) was suppressed by all BA feedings, whereas Cyp7b1 was suppressed only by CA and UDCA feedings. Gender differences in liver BA composition were observed after feeding CA, DCA, CDCA, and LCA, but they were not prominent after feeding UDCA. Sulfation of CA and CDCA was found at the 7-OH position, and it was increased by feeding CA or CDCA more in male than female mice. In contrast, sulfation of LCA and taurolithocholic acid (TLCA) was female-predominant, and it was increased by feeding UDCA and LCA. In summary, the present systematic study on BA metabolism in mice will aid in interpreting BA-mediated gene regulation and hepatotoxicity.