Unconjugated Bile Acids Research Articles

The influence of probiotics on individual fecal secondary bile acid levels: a two-case study of schizophrenic patients receiving an atypical antipsychotic drug

Background: Probiotics is used as a promising approach in the prevention and treatment of hypercholesterolemia. Modification of bile acid metabolism through the deconjugation of bile salts by microbial bile salt hydrolase (BSH) is considered to be the core mechanism of the hypocholesterolemic effects of probiotics. Nevertheless, BSH activity is reported to be detrimental to the human host due to the generation of toxic secondary bile acids. Thus, the influence of probiotic intake on bile acid metabolism needs to be elucidated. We analyzed the bile acid levels and microbiota in human fecal samples after probiotic supplementation to assess the influence of probiotic intake on fecal bile acid levels. Two patients hospitalized for schizophrenia and dyslipidemia, receiving an atypical antipsychotic drug, were enrolled in this study (Subjects A and B). Both subjects received Lactobacillus rhamnosus GG (LGG) for 4 weeks, and no probiotics for the following 4 weeks. Fecal samples were collected at baseline and after 4 and 8 weeks.Results: Conjugated bile acids may be modified by indigenous intestinal bacteria into unconjugated bile acids and secondary bile acids through deconjugation reactions by BSH activity and the subsequent 7a-dehydroxylation pathway, respectively. In the fecal microbiota from Subject A, the relative abundance of Bifidobacterium increased after LGG supplementation (30%–49%). Most Bifidobacterium and Lactobacillus strains that colonize mammalian intestines may report BSH activity, and in general bifidobacteria reveals a higher BSH activity than lactobacilli. The fecal unconjugated bile acid and secondary bile acid levels in Subject A increased after the LGG supplementation (0.36–1.79 and 1.82–16.19 mmol/g respectively). Although the LGG supplementation appears to promote bile acid deconjugation, most of the unconjugated bile acids in Subject A appear to have been modified into secondary bile acids. Alternatively, in Subject B there were no significant changes throughout the study.Conclusion: We observed a significant increase in the fecal secondary bile acid levels after probiotic administration in one of our cases. Further studies are needed to elucidate the factors affecting 7a-dehydroxylation of bile acids to confirm the safety of using probiotics.Keywords: bile salt hydrolase; BSH; dihydroxylation; Bifidobacterium

Impaired vagus function in rats suppresses bile acid synthesis in the liver by disrupting tight junctions and activating Fxr-Fgf15 signaling in the intestine

Bile acids (BAs) circulate between the liver and intestine, and regulate the homeostasis of glucose, lipid, and energy. Recent studies demonstrated an essential role of BAs in neurological diseases, suggesting an interaction between BAs and the nervous system. In the present study, we showed that impaired vagus function in rats induced by vagotomy resulted in an increase in bile flow without causing liver injury. The concentrations of unconjugated and glycine-conjugated BAs were increased in both serum and bile of rats after vagotomy, which was due to impaired tight junctions and thus increased passive absorption of BAs in the intestine. Vagotomy markedly suppressed the expression of the rate-limiting BA synthetic enzyme Cyp7a1, which was not due to activation of Fxr-Shp signaling in the liver, but due to activation of Fxr-Fgf15 signaling in the intestine. Furthermore, vagotomy produced a BA profile in the bile favorable for Fxr activation by decreasing tauro-β-muricholic acid, a natural Fxr antagonist, and increasing glyco-chenodeoxycholic acid, a natural Fxr agonist. In summary, the present study provides the first comprehensive analysis of the critical role of the vagus nerve in regulating BA metabolism and signaling pathway.

Analyses of gut microbiota and plasma bile acids enable stratification of patients for antidiabetic treatment

Antidiabetic medication may modulate the gut microbiota and thereby alter plasma and faecal bile acid (BA) composition, which may improve metabolic health. Here we show that treatment with Acarbose, but not Glipizide, increases the ratio between primary BAs and secondary BAs and plasma levels of unconjugated BAs in treatment-naive type 2 diabetes (T2D) patients, which may beneficially affect metabolism. Acarbose increases the relative abundances of Lactobacillus and Bifidobacterium in the gut microbiota and depletes Bacteroides, thereby changing the relative abundance of microbial genes involved in BA metabolism. Treatment outcomes of Acarbose are dependent on gut microbiota compositions prior to treatment. Compared to patients with a gut microbiota dominated by Prevotella, those with a high abundance of Bacteroides exhibit more changes in plasma BAs and greater improvement in metabolic parameters after Acarbose treatment. Our work highlights the potential for stratification of T2D patients based on their gut microbiota prior to treatment.

Comparison of the composition of bile acids in bile of patients with adenocarcinoma of the pancreas and benign disease

Bile acids have been implicated in the development of gastrointestinal malignancies. Both the specific nature of individual bile acids and their concentration appear key factors in the carcinogenic potency of bile. Using liquid chromatography mass spectrometry (LC–MS) we performed quantitative profiling of bile extracted directly from the common bile duct in 30 patients (15 patients with pancreatic cancer and 15 patients with benign disease). Separation and detection of bile acids was performed using a 1.7μm particle size reversed-phase C18 LC column at a flow rate of 200μL/min with negative electrospray ionization MS. A significant difference (p=0.018) was seen in the concentration of unconjugated cholic acid in the malignant group (0.643mmol/L) compared to the benign group (0.022mmol/L), with an overall significant difference (p=0.04) seen in the level of total unconjugated bile acids in the malignant group (1.816mmol/L) compared to the benign group (0.069mmol/L). This finding may offer the possibility of both understanding the biology of cancer development in the pancreas, as well as offering a potential diagnostic avenue to explore. However, a larger study is necessary to confirm the alterations in bile acid profiles reported here and explore factors such as diet and microbial populations on the bile acid profiles of these patient groups.

Serum bile acid level and fatty acid composition in Chinese children with non-alcoholic fatty liver disease.

To determine serum bile acid (BA) and fatty acid (FA) profiles in Chinese children with non-alcoholic fatty liver disease (NAFLD). A total 76 children aged 4-17 years were categorized into three groups according to the presence and absence of as well as the severity of NAFLD, that is, non-NAFLD (control), mild and moderate to severe NAFLD groups, respectively, based on their liver ultrasonography findings. Serum BA and FA profiles were quantified separately by mass spectrometry and gas chromatography. General linear models were performed to assess the differences among the groups. After adjusted for potential confounders, children with NAFLD had higher levels of chenodeoxycholic acid (CDCA), unconjugated primary BAs (CDCA + cholic acid) but lower levels of deoxycholic acid (DCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), total DCA (DCA + TDCA + GDCA), glycolithocholic acid (GLCA) and total lithocholic acid (GLCA + taurolithocholic acid) than children without NAFLD. As for FAs, children with mild and moderate to severe NAFLD had higher levels of n-7 monounsaturated FA. Circulating BA and FA profiles may change in children with NAFLD. Further studies are needed to determine their associations and to understand the underlying mechanism of action.

Diet-induced obesity and weight loss alter bile acid concentrations and bile acid–sensitive gene expression in insulin target tissues of C57BL/6J mice

Bile acids (BAs) influence the metabolism of glucose, lipids, and energy expenditure. We hypothesized that BA concentrations and related gene expression would be altered in lean (low-fat diet fed; LFD) vs diet-induced obese (high-fat diet fed; HFD) groups of mice and that some detected changes would remain after weight loss in an HFD group switched to the LFD (SW). Taurine conjugates dominated the bile acid composition of the liver, epididymal white adipose tissue (eWAT), and hypothalamus, with the latter having lower levels (~95%, ~95%, and ~80%, respectively; P<.05). Plasma conjugated bile acids were elevated in the HFD relative to the LFD and SW animals. Total hepatic BA concentrations decreased in obese mice fed HFD, and levels returned to preobese levels in the SW group. Subtle changes in unconjugated bile acids were detected in the eWAT, hypothalamus, and muscle. Liver expression of a variety of enzymes involved in BA synthesis (eg, Cyp27a1, Acox2), BA transport (eg, Slc22a8), and BA-sensitive receptors (Fxr, Tgr5) were unchanged by HFD feeding but decreased with SW. Other hepatic enzymes were induced in the SW group (eg, Amacr and Bal). In eWAT, Cyp27a1 and Acox2 also declined in the SW group, whereas the HFD group showed reduced expression of BA transporters (eg, Abcc3), and changes in Fxr and Tgr5 were unclear. Therefore, although most detectable changes in BA metabolism associated with diet-induced obesity are reversed by diet-induced weight loss, some effects on BA composition, concentrations, and gene expression can persist after weight loss.

Unconjugated bile acids in rat brain: Analytical method based on LC/ESI-MS/MS with chemical derivatization and estimation of their origin by comparison to serum levels

Although some studies have revealed the implication of bile acids (BAs) and neurological diseases, the levels and origin of the BAs in the brain are not fully understood. In this study, we first developed and validated a sensitive and specific method for the determination of three unconjugated BAs [cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)] in the rat brain by liquid chromatography/electrospray ionization-tandem mass spectrometry combined with chemical derivatization. The measured brain concentrations (mean±standard deviation, n=10) of normal rats were 58.7±48.8, 14.2±11.7 and 13.2±8.7ng/g tissue for CA, CDCA and DCA, respectively. For their origin, we developed the hypothesis that they might be mostly derived from the periphery. To test this hypothesis, the brain BA levels were compared with the serum levels. The brain levels had high correlations with the serum levels, and were always lower than the serum levels for the three unconjugated BAs. Furthermore, the higher brain-to-serum concentration ratios were found for the BAs with higher logD values (higher lipophilicity). Moreover, the brains of the rats intraperitoneally administered with deuterium-labeled CA and CDCA were also analyzed; the deuterium-labeled BAs were detected in the brain of the rats administered with these compounds. Based on all the results, we concluded that the BAs found in the brain are mostly derived from the periphery and the major mechanism for the transportation of the unconjugated BAs to the brain is by passive diffusion.

Factors affecting separation and detection of bile acids by liquid chromatography coupled with mass spectrometry in negative mode.

Bile acids (BAs) are cholesterol metabolites with important biological functions. They undergo extensive host-gut microbial co-metabolisms during the enterohepatic circulation, creating a vast structural diversity and resulting in great challenges to separate and detect them. Based on the bioanalytical reports in the past decade, this work developed three chromatographic gradient methods to separate a total of 48 BA standards on an ethylene-bridged hybrid (BEH) C18 column and high-strength silica (HSS) T3 column and accordingly unraveled the factors affecting the separation and detection of them by liquid chromatography coupled with mass spectrometry (LC-MS). It was shown that both the acidity and ammonium levels in mobile phases reduced the electrospray ionization (ESI) of BAs as anions of [M-H]-, especially for those unconjugated ones without 12-hydroxylation. It was also found that the retention of taurine conjugates on the BEH C18 column was sensitive to the strength of formic acid and ammonium in mobile phases. By using the volatile buffers with an equivalent ammonium level as mobile phases, we comprehensively demonstrated the effects of the elution pH value on the retention behaviors of BAs on both the BEH C18 column and HSS T3 column. Based on the retention data acquired on a C18 column, we presented the ionization constants (pK a) of various BAs with the widest coverage beyond those of previous reports. When we made attempts to establish the structure-retention relationships (SRRs) of BAs, the lack of discriminative structural descriptors for BA stereoisomers emerged as the bottleneck problem. The methods and results presented in this work are especially useful for the development of reliable, sensitive, high-throughput, and robust LC-MS bioanalytical protocols for the quantitative metabolomic studies. Graphical Abstract Nonlinear curve fitting of capacity factors and elution pH value for the separation of common unconjugated bile acids.

The anti-cholesterolaemic effect of a consortium of probiotics: An acute study in C57BL/6J mice

Hypercholesterolaemia is a major risk factor for cardiovascular disease and it has been found that some probiotic bacteria possess cholesterol-lowering capabilities. In this study, the ability of the Lab4 probiotic consortium to hydrolyse bile salts, assimilate cholesterol and regulate cholesterol transport by polarised Caco-2 enterocytes was demonstrated. Furthermore, in wild-type C57BL/6J mice fed a high fat diet, 2-weeks supplementation with Lab4 probiotic consortium plus Lactobacillusplantarum CUL66 resulted in significant reductions in plasma total cholesterol levels and suppression of diet-induced weight gain. No changes in plasma levels of very low-density lipoprotein/low-density lipoprotein, high-density lipoprotein, triglycerides, cytokines or bile acids were observed. Increased amounts of total and unconjugated bile acids in the faeces of the probiotic-fed mice, together with modulation of hepatic small heterodimer partner and cholesterol-7α-hydroxylase mRNA expression, implicates bile salt hydrolase activity as a potential mechanism of action. In summary, this study demonstrates the cholesterol-lowering efficacy of short-term feeding of the Lab4 probiotic consortium plus L. plantarum CUL66 in wild-type mice and supports further assessment in human trials.

Quantitative profiling of 19 bile acids in rat plasma, liver, bile and different intestinal section contents to investigate bile acid homeostasis and the application of temporal variation of endogenous bile acids

Bile acid homeostasis is maintained by liver synthesis, bile duct secretion, microbial metabolism and intestinal reabsorption into the blood. When drug insults result in liver damage, the variances of bile acids (BAs) are related to the physiological status of the liver. Here, we established a method to simultaneously quantify 19 BAs in rat plasma, liver, bile and different intestinal section contents (duodenum, jejunum, ileum, cecum and colon) using high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) to reveal the pattern of bile acid homeostasis in the enterohepatic circulation of bile acids in physiological situations. Dynamic changes in bile acid composition appeared throughout the enterohepatic circulation of the BAs; taurine- and glycine-conjugated BAs and free BAs had different dynamic homeostasis levels in the circulatory system. cholic acid (CA), beta-muricholic acid (beta-MCA), lithocholic acid (LCA), glycocholic acid (GCA) and taurocholic acid (TCA) greatly fluctuated in the bile acid pool under physiological conditions. Taurine- and glycine-conjugated bile acids constituted more than 90% in the bile and liver, whereas GCA and TCA accounted for more than half of the total bile acids and the secretion of bile mainly via conjugating with taurine. While over 80% of BAs in plasma were unconjugated bile acids, CA and HDCA were the most abundant elements. Unconjugated bile acids constituted more than 90% in the intestine, and CA, beta-MCA and HDCA were the top three bile acids in the duodenum, jejunum and ileum content, but LCA and HDCA were highest in the cecum and colon content. As the main secondary bile acid converted by microflora in the intestine, LCA was enriched in the cecum and DCA mostly in the colon. As endogenous substances, the concentrations of plasma BAs were closely related to time rhythm and diet. In conclusion, analyzing detailed BA profiles in the enterohepatic circulation of bile acids in a single run is possible using LC–MS/MS. Based on the physiological characteristics of the metabolic profiling of 19 BAs in the total bile acid pool and the time rhythm variation of the endogenous bile acids, this study provided a new valuable method and theoretical basis for the clinical research of bile acid homeostasis.

Age‐ and species‐specific activation of CAR on bile acid homeostasis in mice

Pharmacological activation of the constitutive androstane receptor (CAR) has been shown to protect against bile acid (BA)‐induced liver injury in adults (Beilke et al., 2009). During development, targeting CAR by agonists has been suggested to be one of the ultimate goals in the management and treatment of total parenteral nutrition related cholestasis in newborns (Hendaus, 2013). However, very little is known regarding the age‐ and species‐specific effects of CAR activation on BA homeostasis during development. The goal of this study was to use RNA sequencing and BA metabolomics to investigate the effects of CAR activation on the expression of genes involved in BA homeostasis and the BA profiles in 5‐ and 60‐day‐old wild‐type (WT) and humanized CAR transgenic (hCAR‐TG) mice. Male mice were i.p. administered corn oil or a species‐appropriate CAR ligand (TCPOBOP at 3mg/kg for mCAR, and CITCO at 30mg/kg for hCAR) once daily for 4 days. In control mice of both genotypes, from newborns to adulthood, there was a marked decrease in total serum BAs likely due to the maturation of the enterohepatic system. Specifically, serum total primary BAs and conjugated BAs were down‐regulated with age in both WT and hCAR‐TG mice. Conversely, there was a marked increase in serum total secondary BAs and unconjugated BAs, likely due to an ontogenic increase in intestinal bacteria for BA‐dehydroxylation and deconjugation. Following mCAR‐activation, there was an increase in serum total BAs, primary BAs, secondary BAs, and conjugated BAs at both Day 5 and 60. However, the increase in serum BAs was not observed following hCAR activation. In fact, serum cholic acid was decreased by CITCO in 60‐day‐old hCAR‐TG mice. Regarding hepatic BA synthesis, Cyp7a1 mRNA was minimally influenced by CAR activation regardless of the age or species. Conversely, the regulation of Cyp8b1 mRNA seems age‐specific, because it was up‐regulated by CAR of both species at Day 5, but was down‐regulated at Day 60. The regulation of many other BA‐synthetic enzymes at mRNA level was mCAR‐specific, evidenced by an induction of Akr1d1, but a reduction of Amacr, Hsd17b4, Hsd3b7, and Scp2 at both ages only by activation of mCAR but not hCAR. Age‐ and/or species‐specific regulatory effects of CAR activation were also observed for genes involved in BA conjugation and transport. However, several BA‐processing genes appeared to be “universal targets” of CAR of both species and at both ages, including a consistent up‐regulation of Cyp39a1, Akr1c14, Mrp3, and Mrp4. However, in general, mCAR activation produced greater degree of induction as compared to hCAR activation at the same age. In conclusion, the present study suggests that hCAR activation has less effects on serum BA metabolites as compared to mCAR, whereas mCAR and hCAR activation produce both age‐ and species‐specific effects on the hepatic expression of genes involved in BA homeostasis. Understanding the age‐ and species‐differences in the pharmacodynamics of CAR activation is essential in precision medicine for pediatric patients.Support or Funding InformationGrace Liejun Guo glg48@eohsi.rutgers.edu

Tu1864 - Longitudinal Characterization of Dysbiosis and Unconjugated Bile acid Profiles in the Feces of Dogs with Inflammatory Bowel Disease

Tu1864 - Longitudinal Characterization of Dysbiosis and Unconjugated Bile acid Profiles in the Feces of Dogs with Inflammatory Bowel Disease

Su1092 - Unconjugated Bile Acid Reduces Desmosomal Gene Expression in Esophageal Cells by Inhibiting BMP Signaling Through Nuclear Receptor Mediated FGF-19 Transcription

Su1092 - Unconjugated Bile Acid Reduces Desmosomal Gene Expression in Esophageal Cells by Inhibiting BMP Signaling Through Nuclear Receptor Mediated FGF-19 Transcription

Influence of ad Libitum Feeding of Piglets With Bacillus Subtilis Fermented Liquid Feed on Gut Flora, Luminal Contents and Health

Some scholars caution that long-term ad libitum feeding with probiotic fermented food poses potential health risks to baby animals. We conducted a feeding experiment to investigate the influence of ad libitum feeding of pre-and post-weaned piglets with a Bacillus subtilis fermented diet on the gut microbiome, gut metabolomic profiles, bile acid metabolism, proinflammatory cytokines and faecal consistency. Compared with piglets fed a Bacillus subtilis-supplemented pellet diet, piglets fed the Bacillus subtilis fermented liquid diet had lower intestinal bacterial diversity (P > 0.05), higher intestinal fungal diversity (P > 0.05), more Firmicutes (P > 0.05), fewer Bacteroidetes, Actinobacteria and Proteobacteria (P > 0.05), higher concentrations of 3-hydroxypropionic acid (P < 0.05), orotic acid (P < 0.05), interleukin-6 (P < 0.01), lactic acid (P < 0.01), deoxycholic acid (P > 0.05) and lithocholic acid (P < 0.01) and a higher incidence of diarrhoea (P > 0.05). The data show that ad libitum feeding of piglets with a Bacillus subtilis fermented liquid diet during the suckling and early post-weaning periods promotes the growth of lactic acid bacteria, bile salt hydrolase-active bacteria and 7a-dehydroxylase-active bacteria in the intestinal lumen; disturbs the normal production of lactic acid, orotic acid and unconjugated bile acids; and increases circulating interleukin-6 levels and diarrhoea incidence.

Antibiotic-Induced Elevations of Plasma Bile Acids in Rats Independent of Bsep Inhibition.

Drug-induced liver injury (DILI) is a common toxicity observed in drug development and can lead to withdrawal of approved drugs from the market. To better understand the numerous mechanisms of DILI, recent efforts have focused on transporter inhibition, specifically liver canalicular bile salt export pump (Bsep) as one mechanism of DILI, and on the potential use of plasma bile acids as monitorable mechanism-based biomarkers of Bsep inhibition. To explore alternative mechanisms of bile acid increases in plasma, 6 antibiotic and 2 nonantibiotic drugs unlikely to be Bsep inhibitors were evaluated in rat studies. Surprisingly, all 6 antibiotics demonstrated 2- to 14-fold increases of plasma taurocholic acid (TCA). Also, unconjugated primary bile acids and secondary bile acids (both taurine-conjugated and unconjugated) were decreased in rat plasma after antibiotic treatments, but not with the nonantibiotic drugs. These results suggest alternative mechanisms of bile acids regulation such as attenuation of bacterial deconjugation of bile acids following reduction of gut microflora by antibiotics. Measurements of TCA transport in rat hepatocytes and Bsep-containing membrane vesicles suggest that inhibition of uptake into hepatocytes could also contribute to increases in plasma bile acid concentrations, while excluding inhibition of Bsep as a mechanism. These studies further demonstrate that there are several mechanisms that can lead to conjugated bile acid increases in plasma. By carefully considering the time course and magnitude of changes of individual bile acids relative to any changes seen in transaminases and bilirubin, interpretations and conclusions of the involvement of Bsep inhibition are enabled.

Preference of Conjugated Bile Acids over Unconjugated Bile Acids as Substrates for OATP1B1 and OATP1B3.

Bile acids, the metabolites of cholesterol, are signaling molecules that play critical role in many physiological functions. They undergo enterohepatic circulation through various transporters expressed in intestine and liver. Human organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3 contribute to hepatic uptake of bile acids such as taurocholic acid. However, the transport properties of individual bile acids are not well understood. Therefore, we selected HEK293 cells overexpressing OATP1B1 and OATP1B3 to evaluate the transport of five major human bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid) together withtheir glycine and taurine conjugates via OATP1B1 and OATP1B3. The bile acids were quantified by liquid chromatography-tandem mass spectrometry. The present study revealed that cholic acid, chenodeoxyxcholic acid, and deoxycholic acid were transported by OATP1B1 and OATP1B3, while ursodeoxycholic acid and lithocholic acid were not significantly transported by OATPs. However, all the conjugated bile acids were taken up rapidly by OATP1B1 and OATP1B3. Kinetic analyses revealed the involvement of saturable OATP1B1- and OATP1B3-mediated transport of bile acids. The apparent Km values for OATP1B1 and OATP1B3 of the conjugated bile acids were similar (0.74–14.7 μM for OATP1B1 and 0.47–15.3 μM for OATP1B3). They exhibited higher affinity than cholic acid (47.1 μM for OATP1B1 and 42.2 μM for OATP1B3). Our results suggest that conjugated bile acids (glycine and taurine) are preferred to unconjugated bile acids as substrates for OATP1B1 and OATP1B3.

Identification of Taurine-Responsive Genes in Murine Liver Using the Cdo1-Null Mouse Model.

The cysteine dioxygenase (Cdo1)-null mouse is unable to synthesize hypotaurine and taurine by the cysteine/cysteine sulfinate pathway and has very low taurine levels in all tissues. The lack of taurine is associated with a lack of taurine conjugation of bile acids, a dramatic increase in the total and unconjugated hepatic bile acid pools, and an increase in betaine and other molecules that serve as organic osmolytes. We used the Cdo1-mouse model to determine the effects of taurine deficiency on expression of proteins involved in sulfur amino acid and bile acid metabolism. We identified cysteine sulfinic acid decarboxylase (Csad), betaine:homocysteine methytransferase (Bhmt), cholesterol 7α-hydroxylase (Cyp7a1), and cytochrome P450 3A11 (Cyp3a11) as genes whose hepatic expression is strongly regulated in response to taurine depletion in the Cdo1-null mouse. Dietary taurine supplementation of Cdo1-null mice restored hepatic levels of these four proteins and their respective mRNAs to wild-type levels, whereas dietary taurine supplementation had no effect on abundance of these proteins or mRNAs in wild-type mice.

Unconjugated Bile Acids Influence Expression of Circadian Genes: A Potential Mechanism for Microbe-Host Crosstalk

Disruptions to circadian rhythm in mice and humans have been associated with an increased risk of obesity and metabolic syndrome. The gut microbiota is known to be essential for the maintenance of circadian rhythm in the host suggesting a role for microbe-host interactions in the regulation of the peripheral circadian clock. Previous work suggested a role for gut bacterial bile salt hydrolase (BSH) activity in the regulation of host circadian gene expression. Here we demonstrate that unconjugated bile acids, known to be generated through the BSH activity of the gut microbiota, are potentially chronobiological regulators of host circadian gene expression. We utilised a synchronised Caco-2 epithelial colorectal cell model and demonstrated that unconjugated bile acids, but not the equivalent tauro-conjugated bile salts, enhance the expression levels of genes involved in circadian rhythm. In addition oral administration of mice with unconjugated bile acids significantly altered expression levels of circadian clock genes in the ileum and colon as well as the liver with significant changes to expression of hepatic regulators of circadian rhythm (including Dbp) and associated genes (Per2, Per3 and Cry2). The data demonstrate a potential mechanism for microbe-host crosstalk that significantly impacts upon host circadian gene expression.

Bile acid: a potential inducer of colon cancer stem cells.

BackgroundAlthough 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.MethodsNormal 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 β-catenin. Downregulation of muscarinic 3 receptor (M3R) was achieved through transfection of corresponding siRNA.ResultsWe 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.ConclusionsOur results suggest that bile acids, specifically DCA and LCA, induce cancer stemness in colonic epithelial cells by modulating M3R and Wnt/β-catenin signaling and thus could be considered promoters of colon cancer.

Functional human induced hepatocytes (hiHeps) with bile acid synthesis and transport capacities: A novel in vitro cholestatic model.

Drug-induced cholestasis is a leading cause of drug withdrawal. However, the use of primary human hepatocytes (PHHs), the gold standard for predicting cholestasis in vitro, is limited by their high cost and batch-to-batch variability. Mature hepatocyte characteristics have been observed in human induced hepatocytes (hiHeps) derived from human fibroblast transdifferentiation. Here, we evaluated whether hiHeps could biosynthesize and excrete bile acids (BAs) and their potential as PHH alternatives for cholestasis investigations. Quantitative real-time PCR (qRT-PCR) and western blotting indicated that hiHeps highly expressed BA synthases and functional transporters. Liquid chromatography tandem mass spectrometry (LC-MS/MS) showed that hiHeps produced normal intercellular unconjugated BAs but fewer conjugated BAs than human hepatocytes. When incubated with representative cholestatic agents, hiHeps exhibited sensitive drug-induced bile salt export pump (BSEP) dysfunction, and their response to cholestatic agent-mediated cytotoxicity correlated well with that of PHHs (r2 = 0.8032). Deoxycholic acid (DCA)-induced hepatotoxicity in hiHeps was verified by elevated aspartate aminotransferase (AST) and γ-glutamyl-transferase (γ-GT) levels. Mitochondrial damage and cell death suggested DCA-induced toxicity in hiHeps, which were attenuated by hepatoprotective drugs, as in PHHs. For the first time, hiHeps were reported to biosynthesize and excrete BAs, which could facilitate predicting cholestatic hepatotoxicity and screening potential therapeutic drugs against cholestasis.