Comparative analysis of proteomic adaptations in Enterococcus faecalis and Enterococcus faecium after long term bile acid exposure.

BACKGROUND: All gastrointestinal pathogens, including Enterococcus faecalis and Enterococcus faecium, undergo adaptation processes during colonization and infection. In this study, we investigated by data-independent acquisition mass spectrometry (DIA-MS) two crucial adaptations of these two Enterococcus species at the proteome level. Firstly, we examined the adjustments to cope with bile acid concentrations at 0.05% that the pathogens encounter during a potential gallbladder infection. Therefore, we chose the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) as well as the secondary bile acid deoxycholic acid (DCA), as these are the most prominent bile acids. Secondly, we investigated the adaptations from an aerobic to a microaerophilic environment, as encountered after oral-fecal infection, in the absence and presence of deoxycholic acid (DCA). RESULTS: Our findings showed similarities, but also species-specific variations in the response to the different bile acids. Both Enterococcus species showed an IC50 in the range of 0.01- 0.023% for DCA and CDCA in growth experiments and both species were resistant towards 0.05% CA. DCA and CDCA had a strong effect on down-expression of proteins involved in translation, transcription and replication in E. faecalis (424 down-expressed proteins with DCA, 376 down-expressed proteins with CDCA) and in E. faecium (362 down-expressed proteins with DCA, 391 down-expressed proteins with CDCA). Proteins commonly significantly altered in their expression in all bile acid treated samples were identified for both species and represent a "general bile acid response". Among these, various subunits of a V-type ATPase, different ABC-transporters, multi-drug transporters and proteins related to cell wall biogenesis were up-expressed in both species and thus seem to play an essential role in bile acid resistance. Most of the differentially expressed proteins were also identified when E. faecalis was incubated with low levels of DCA at microaerophilic conditions instead of aerobic conditions, indicating that adaptations to bile acids and to a microaerophilic atmosphere can occur simultaneously. CONCLUSIONS: Overall, these findings provide a detailed insight into the proteomic stress response of two Enterococcus species and help to understand the resistance potential and the stress-coping mechanisms of these important gastrointestinal bacteria.

BaiJ and BaiB are key enzymes in the chenodeoxycholic acid 7α-dehydroxylation pathway in the gut microbe Clostridium scindens ATCC 35704.

Bile acid transformation is a common gut microbiome activity that produces secondary bile acids, some of which are important for human health. One such process, 7α-dehydroxylation, converts the primary bile acids, cholic acid and chenodeoxycholic acid, to deoxycholic acid and lithocholic acid, respectively. This transformation requires a number of enzymes, generally encoded in a bile acid-inducible (bai) operon and consists of multiple steps. Some 7α-dehydroxylating bacteria also harbor additional genes that encode enzymes with potential roles in this pathway, but little is known about their functions. Here, we purified 11 enzymes originating either from the bai operon or encoded at other locations in the genome of Clostridium scindens strain ATCC 35704. Enzyme activity was probed in vitro under anoxic conditions to characterize the biochemical pathway of chenodeoxycholic acid 7α-dehydroxylation. We found that more than one combination of enzymes can support the process and that a set of five enzymes, including BaiJ that is encoded outside the bai operon, is sufficient to achieve the transformation. We found that BaiJ, an oxidoreductase, exhibits an activity that is not harbored by the homologous enzyme from another C. scindens strain. Furthermore, ligation of bile acids to coenzyme A (CoA) was shown to impact the product of the transformation. These results point to differences in the 7α-dehydroxylation pathway among microorganisms and the crucial role of CoA ligation in the process.

Dietary chenodeoxycholic acid attenuates high-fat diet-induced growth retardation, lipid accumulation and bile acid metabolism disorder in the liver of yellow catfish Pelteobagrus fulvidraco.

This experiment was conducted to investigate whether dietary chenodeoxycholic acid (CDCA) could attenuate high-fat (HF) diet-induced growth retardation, lipid accumulation and bile acid (BA) metabolism disorder in the liver of yellow catfish Pelteobagrus fulvidraco. Yellow catfish (initial weight: 4·40 (sem 0·08) g) were fed four diets: the control (105·8 g/kg lipid), HF diet (HF group, 159·6 g/kg lipid), the control supplemented with 0·9 g/kg CDCA (CDCA group) and HF diet supplemented with 0·9 g/kg CDCA (HF + CDCA group). CDCA supplemented in the HF diet significantly improved growth performance and feed utilisation of yellow catfish (P < 0·05). CDCA alleviated HF-induced increment of hepatic lipid and cholesterol contents by down-regulating the expressions of lipogenesis-related genes and proteins and up-regulating the expressions of lipololysis-related genes and proteins. Compared with the control group, CDCA group significantly reduced cholesterol level (P < 0·05). CDCA significantly inhibited BA biosynthesis and changed BA profile by activating farnesoid X receptor (P < 0·05). The contents of CDCA, taurochenodeoxycholic acid and glycochenodeoxycholic acid were significantly increased with the supplementation of CDCA (P < 0·05). HF-induced elevation of cholic acid content was significantly attenuated by the supplementation of CDCA (P < 0·05). Supplementation of CDCA in the control and HF groups could improve the liver antioxidant capacity. This study proved that CDCA could improve growth retardation, lipid accumulation and BA metabolism disorder induced by HF diet, which provided new insight into understanding the physiological functions of BA in fish.

Loss of Farnesoid X receptor (FXR) accelerates dysregulated glucose and renal injury in db/db mice.

Background: End-stage renal disease is primarily caused by diabetic kidney disease (DKD). The Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has anti-inflammatory, lipid-lowering and hypoglycemic properties. It also inhibits renal fibrosis. Although its physiological role is not fully understood, it also plays a role in the control of diabetic nephropathy (DN). Methods: In the present study, we examined male FXR & leptin receptor double knockout mice, in which weight, blood glucose, body fat, and other indicators were monitored. After 6 months of rearing, blood and urine samples were collected and biochemical parameters were measured. Fibrosis was assessed by Masson's stain, while the assessment of the resuscitation case's condition was performed using succinate dehydrogenase (SDHA) stain immunohistochemistry, which measures aerobic respiration. Expression of molecules such as connective tissue growth factor (CTGF), SMAD family members 3 (Smad3) and 7 (Smad7), and small heterodimer partner were detected by RT-PCR and Western blotting as part of the application. Results: FXR knockout decreased body weight and body fat in db/db mice, but increased blood glucose, urine output, and renal fibrosis. Primary mesangial cells (P-MCs) from FXR+/ + mice stimulated with transforming growth factor ß1 (TGFß1) showed significantly higher levels of related fibrosis factors, TGFß1 and Smad3 mRNA and protein, and significantly reduced levels of Smad7. These effects were reversed by the action of FXR agonist chenodeoxycholic acid (CDCA). P-MCs from FXR-/ - mice stimulated with TGFß1 resulted in an increase in the expression and protein levels of collagen I and TGFß1, and the addition of CDCA had no significant effect on TGFß1 stimulation. However, compared with FXR+/ +db/db mice, the rate of oxygen consumption, the rate of carbon dioxide production, and the rate of energy conversion were increased in FXR-/ -db/db mice, whereas the SDHA succinate dehydrogenase, a marker enzyme for aerobic respiration, was significantly decreased. Conclusions: These results provide evidence that FXR plays a critical role in the regulation of mesangial cells in DN. The likely mechanism is that aberrant FXR expression activates TGFß1, which induces extracellular matrix accumulation through the classical Smad signaling pathway, leading to mitochondrial dysfunction.

The implication of LPS/TLR4 and FXR receptors in hepatoprotective efficacy of indole-3-acetic acid and chenodeoxycholic acid.

AIM: Valproic acid (VPA) belongs to the first-generation antiepileptic drugs, yet its prolonged use can cause life-threatening liver damage. The importance of our study is to investigate the protective effect of indole-3-acetic acid (IAA), chenodeoxycholic acid (CDCA) and their combination on VPA-induced liver injury focusing on lipopolysaccharides (LPS)/toll-like receptor 4 (TLR4) pathway and farnesoid X receptor (FXR). METHODS: Thirty rats were randomly assigned into five groups, normal control group, VPA group received 500 mg/kg of VPA intraperitoneally. The remaining groups were orally treated with either 40 mg/kg of IAA, 90 mg/kg of CDCA, or a combination of both, along with VPA. All treatments were administered one hour after the administration of VPA for three weeks. KEY FINDINGS: VPA group showed significant elevations in the liver weight/body weight ratio, serum aminotransferases, triglyceride, and total cholesterol levels. Hepatic glutathione (GSH) level and superoxide dismutase (SOD) activity were significantly decreased, while malondialdehyde (MDA) level, tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1ß), lipopolysaccharide (LPS) and caspase 3 were significantly increased. Likewise, immunohistochemical analysis revealed that TLR4 expression was elevated, whereas FXR expression was downregulated in hepatocytes. IAA substantially ameliorated all previously altered parameters, whereas CDCA treatment showed a partial improvement compared to IAA. Surprisingly, combination therapy of IAA with CDCA showed an additive effect only in the hepatic expression of TLR4 and FXR proteins. SIGNIFICANCE: IAA could be a promising protective agent against VPA-induced liver injury.

Bile acids promote the development of HCC by activating inflammasome.

BACKGROUND: Hepatocellular carcinoma (HCC) is associated with chronic inflammation caused by different factors; especially, the interaction of inflammatory pathways and bile acids (BAs) can affect hepatocyte proliferation, death, and regeneration, but whether BAs promote HCC progression through inflammatory pathways and the mechanisms is still unclear. METHODS AND RESULTS: By examining cancer and tumor-adjacent tissue BA levels and genes associated with BA homeostasis in 37 HCC patients, we found that total bile acids (TBAs) were decreased by 36% and varying degrees of changes in factors regulating BA homeostasis (p < 0.05). In addition, we found that BA homeostasis was disturbed in diethylnitrosamine-induced HCC mouse models, and TBA was correlated with inflammasome activation during HCC progression (6-24 W) (p < 0.05). Similarly, the inflammasome and chenodeoxycholic acid (CDCA) content were suppressed in cholestasis model mice (Mrp2-deficient mice) (p < 0.05). In vitro, CDCA significantly promoted the malignant transformation of hepatocytes (p < 0.001), activated the inflammasome by triggering the release of mitochondrial reactive oxygen species and mitochondrial DNA, and ultimately induced pyroptosis. Furthermore, we found that CDCA has a targeted binding effect with HO-1 through molecular docking and Cellular Thermal Shift Assay experiments. CONCLUSIONS: In conclusion, we found that CDCA can trigger the excessive accumulation of mitochondrial reactive oxygen species by targeting HO-1 to promote the activation of the inflammasome and ultimately promote the progression of HCC. Our study provides a novel mechanism by which BAs promote HCC by activating the inflammasome and establishes the important role of BA homeostasis imbalance in the progression of HCC from the aspect of inflammation.

Farnesoid X receptor represses human sulfotransferase 1A3 expression through direct binding to the promoter.

Human sulfotransferases 1A3 (SULT1A3) has received particular interest, due to their functions of catalyzing the sulfonation of numerous phenolic substrates, including bioactive endogenous molecules and therapeutic agents. However, the regulation of SULT1A3 expression and the underlying mechanism remain unclear. Here, we aimed to investigate the regulation effects of bile acid-activated farnesoid X receptor (FXR) on SULT1A3 expression, and to shed light on the mechanism thereof. Our results demonstrated that FXR agonists (CDCA and GW4064) significantly inhibit the expression of SULT1A3 at mRNA and protein levels. In addition, overexpression of FXR led to decrease in SULT1A3 expression and knockdown of FXR significantly induced the expression of SULT1A3 in protein and mRNA levels, confirming that FXR expression manifestly showed negative regulatory effect on basal SULT1A3 expression. Furthermore, a combination of luciferase reporter gene and CHIP assays showed that FXR repressed SULT1A3 transcription through direct binding to the region at base pair positions -664 to -654. In conclusion, this study for the first time confirmed FXR was a negative transcriptional regulator of human SULT1A3 enzyme.

Chenodeoxycholic acid rescues axonal degeneration in induced pluripotent stem cell-derived neurons from spastic paraplegia type 5 and cerebrotendinous xanthomatosis patients.

BACKGROUND: Biallelic mutations in CYP27A1 and CYP7B1, two critical genes regulating cholesterol and bile acid metabolism, cause cerebrotendinous xanthomatosis (CTX) and hereditary spastic paraplegia type 5 (SPG5), respectively. These rare diseases are characterized by progressive degeneration of corticospinal motor neuron axons, yet the underlying pathogenic mechanisms and strategies to mitigate axonal degeneration remain elusive. METHODS: To generate induced pluripotent stem cell (iPSC)-based models for CTX and SPG5, we reprogrammed patient skin fibroblasts into iPSCs by transducing fibroblast cells with episomal vectors containing pluripotency factors. These patient-specific iPSCs, as well as control iPSCs, were differentiated into cortical projection neurons (PNs) and examined for biochemical alterations and disease-related phenotypes. RESULTS: CTX and SPG5 patient iPSC-derived cortical PNs recapitulated several disease-specific biochemical changes and axonal defects of both diseases. Notably, the bile acid chenodeoxycholic acid (CDCA) effectively mitigated the biochemical alterations and rescued axonal degeneration in patient iPSC-derived neurons. To further examine underlying disease mechanisms, we developed CYP7B1 knockout human embryonic stem cell (hESC) lines using CRISPR-cas9-mediated gene editing and, following differentiation, examined hESC-derived cortical PNs. Knockout of CYP7B1 resulted in similar axonal vesiculation and degeneration in human cortical PN axons, confirming a cause-effect relationship between gene deficiency and axonal degeneration. Interestingly, CYP7B1 deficiency led to impaired neurofilament expression and organization as well as axonal degeneration, which could be rescued with CDCA, establishing a new disease mechanism and therapeutic target to mitigate axonal degeneration. CONCLUSIONS: Our data demonstrate disease-specific lipid disturbances and axonopathy mechanisms in human pluripotent stem cell-based neuronal models of CTX and SPG5 and identify CDCA, an established treatment of CTX, as a potential pharmacotherapy for SPG5. We propose this novel treatment strategy to rescue axonal degeneration in SPG5, a currently incurable condition.

The FXR mediated anti-depression effect of CDCA underpinned its therapeutic potentiation for MDD.

Emerging evidence from animal and human studies has suggested that small microbial metabolites generated in the gut influence host mood and behavior. Our previous study reported that patients with major depressive disorder (MDD) reduced the abundance of genera Blautia and Eubacterium, the microbials critically regulating cholesterol and bile acid metabolism in the gut. In this study, we further demonstrated that the levels of plasma bile acid chenodeoxycholic acid (CDCA) were significantly lower in Chinese MDD patients (142) than in healthy subjects (148). Such low levels of plasma CDCA in MDD patients were rescued in remitters but not in nonremitters following antidepressant treatment. In a parallel animal study, Chronic Social Defeat Stress (CSDS) depressed mice reduced the plasma CDCA and expression level in prefrontal cortex (PFC) of bile acid receptor (FXR) protein, which is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. We found that CDCA treatment restored the level of FXR in the CSDS mice, suggesting the involvement of bile acid receptors in MDD. We observed that CDCA decreased the activity of the NLRP3 inflammasome and caspase-1 and subsequently increased the levels of phosphorylation and expression of PFC glutamate receptors (GluA1) in the PFC. In addition, CDCA showed antidepressant effects in the tests of sucrose preference, tail suspension, and forced swimming in CSDS mouse model of depression. Finally, in agreement with this idea, blocking these receptors by a FXR antagonist GS abolished CDCA-induced antidepressant effect. Moreover, CDCA treatment rescued the increase of IL-1ß, IL-6, TNF α and IL-17, which also were blocked by GS. These results suggest that CDCA is a biomarker and target potentially important for the diagnosis and treatment of MDD.

Microbiota-induced lipid peroxidation impairs obeticholic acid-mediated antifibrotic effect towards nonalcoholic steatohepatitis in mice.

Obeticholic acid (OCA) has been examined to treat non-alcoholic steatohepatitis (NASH), but has unsatisfactory antifibrotic effect and deficient responsive rate in recent phase III clinical trial. Using a prolonged western diet-feeding murine NASH model, we show that OCA-shaped gut microbiota induces lipid peroxidation and impairs its anti-fibrotic effect. Mechanically, Bacteroides enriched by OCA deconjugates tauro-conjugated bile acids to generate excessive chenodeoxycholic acid (CDCA), resulting in liver ROS accumulation. We further elucidate that OCA reduces triglycerides containing polyunsaturated fatty acid (PUFA-TGs) levels, whereas elevates free PUFAs and phosphatidylethanolamines containing PUFA (PUFA-PEs), which are susceptible to be oxidized to lipid peroxides (notably arachidonic acid (ARA)-derived 12-HHTrE), inducing hepatocyte ferroptosis and activating hepatic stellate cells (HSCs). Inhibiting lipid peroxidation with pentoxifylline (PTX) rescues anti-fibrotic effect of OCA, suggesting combination of OCA and lipid peroxidation inhibitor could be a potential antifibrotic pharmacological approach in clinical NASH-fibrosis.

Bile acid distributions, sex-specificity, and prognosis in colorectal cancer.

BACKGROUND: Bile acids are known to be genotoxic and contribute to colorectal cancer (CRC). However, the link between CRC tumor bile acids to tumor location, patient sex, microbiome, immune-regulatory cells, and prognosis is not clear. METHODS: We conducted bile acid analysis using targeted liquid chromatography-mass spectrometry (LC-MS) on tumor tissues from CRC patients (n = 228) with survival analysis. We performed quantitative immunofluorescence (QIF) on tumors to examine immune cells. RESULTS: Twelve of the bile acids were significantly higher in right-sided colon tumors compared to left-sided colon tumors. Furthermore, in male patients, right-sided colon tumors had elevated secondary bile acids (deoxycholic acid, lithocholic acid, ursodeoxycholic acid) compared to left-sided colon tumors, but this difference between tumors by location was not observed in females. A high ratio of glycoursodeoxycholic to ursodeoxycholic was associated with 5-year overall survival (HR = 3.76, 95% CI = 1.17 to 12.1, P = 0.026), and a high ratio of glycochenodeoxycholic acid to chenodeoxycholic acid was associated with 5-year recurrence-free survival (HR = 3.61, 95% CI = 1.10 to 11.84, P = 0.034). We also show correlation between these bile acids and FoxP3 + T regulatory cells. CONCLUSIONS: This study revealed that the distribution of bile acid abundances in colon cancer patients is tumor location-, age- and sex-specific, and are linked to patient prognosis. This study provides new implications for targeting bile acid metabolism, microbiome, and immune responses for colon cancer patients by taking into account primary tumor location and sex.

Chenodeoxycholic acid suppresses AML progression through promoting lipid peroxidation via ROS/p38 MAPK/DGAT1 pathway and inhibiting M2 macrophage polarization.

PURPOSE: Bile acids are steroid synthesized in liver, which are essential for fat emulsification, cholesterol excretion and gut microbial homeostasis. However, the role of bile acids in leukemia progression remains unclear. We aim at exploring the effects and mechanisms of chenodeoxycholic acid (CDCA), a type of bile acids, on acute myeloid leukemia (AML) progression. RESULTS: Here, we found that CDCA was decreased in feces and plasma of AML patients, positively correlated with the diversity of gut microbiota, and negatively associated with AML prognosis. We further demonstrated that CDCA suppressed AML progression both in vivo and in vitro. Mechanistically, CDCA bound to mitochondria to cause mitochondrial morphology damage containing swelling and reduction of cristae, decreased mitochondrial membrane potential and elevated mitochondrial calcium level, which resulted in the production of excessive reactive oxygen species (ROS). Elevated ROS further activated p38 MAPK signaling pathway, which collaboratively promoted the accumulation of lipid droplets (LDs) through upregulating the expression of the diacylglycerol O-acyltransferase 1 (DGAT1). As the consequence of the abundance of ROS and LDs, lipid peroxidation was enhanced in AML cells. Moreover, we uncovered that CDCA inhibited M2 macrophage polarization and suppressed the proliferation-promoting effects of M2 macrophages on AML cells in co-cultured experiments. CONCLUSION: Our findings demonstrate that CDCA suppresses AML progression through synergistically promoting LDs accumulation and lipid peroxidation via ROS/p38 MAPK/DGAT1 pathway caused by mitochondrial dysfunction in leukemia cells and inhibiting M2 macrophage polarization.

Ileal bile acid transporter inhibition in Cyp2c70 KO mice ameliorates cholestatic liver injury.

Cyp2c70 is the liver enzyme in rodents responsible for synthesis of the primary 6-hydroxylated muricholate bile acid (BA) species. Cyp2c70 KO mice are devoid of protective, hydrophilic muricholic acids, leading to a more human-like BA composition and subsequent cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be therapeutic in cholestatic models. Here, we aimed to determine if IBAT inhibition with SC-435 is protective in Cyp2c70 KO mice. As compared to WT mice, we found male and female Cyp2c70 KO mice exhibited increased levels of serum liver injury markers, and our evaluation of liver histology revealed increased hepatic inflammation, macrophage infiltration, and biliary cell proliferation. We demonstrate serum and histologic markers of liver damage were markedly reduced with SC-435 treatment. Additionally, we show hepatic gene expression in pathways related to immune cell activation and inflammation were significantly upregulated in Cyp2c70 KO mice and reduced to levels indistinguishable from WT with IBAT inhibition. In Cyp2c70 KO mice, the liver BA content was significantly increased, enriched in chenodeoxycholic acid, and more hydrophobic, exhibiting a hydrophobicity index value and red blood cell lysis properties similar to human liver BAs. Furthermore, we determined IBAT inhibition reduced the total hepatic BA levels but did not affect overall hydrophobicity of the liver BAs. These findings suggest that there may be a threshold in the liver for pathological accretion of hydrophobic BAs and reducing hepatic BA accumulation can be sufficient to alleviate liver injury, independent of BA pool hydrophobicity.

Effects of dietary chenodeoxycholic acid supplementation in a low fishmeal diet on growth performance, lipid metabolism, autophagy and intestinal health of Pacific white shrimp, Litopenaeus vannamei.

An 8-week feeding trial was conducted to evaluate the effects of chenodeoxycholic acid (CDCA) on growth performance, body composition, lipid metabolism, and intestinal health of juvenile white shrimp, Litopenaeus vannamei fed a low fishmeal diet. Four practical diets were formulated: HFM (25% fishmeal), LFM (15% fishmeal), LB1 (LFM + 0.04% CDCA), LB2 (LFM + 0.08% CDCA). Each diet was assigned to four tanks with forty shrimp (initial weight 0.33 ± 0.03 g) per tank. The results indicated that the growth performance of shrimp were similar between the four groups; the crude lipid content of shrimp fed the LB2 diet was significantly lower than those fed the HFM diet (P < 0.05). The lipase activity content in hepatopancreatic were significantly higher in the two CDCA supplemented groups than that in LFM group; the contents of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol in hemolymph were significantly lower in LFM group, LB1 group and LB2 group than that in HFM group (P < 0.05). The shrimp fed LB1 diet was significantly decreased the intestinal expression levels of tube than those fed in HFM diet; the intestinal gene expression of imd and toll were significantly lower in LB2 group than those in HFM group (P < 0.05). The results of hepatopancreas gene expression suggest that shrimp fed the LFM diet showed significantly upregulated expression levels of sterol regulatory element-binding protein (srebp), acetyl-CoA carboxylase (acc), and carnitine palmitoyltransferase 1 (cpt-1) than those fed the HFM diet; shrimp fed the LB1 diet showed significantly upregulated expression levels of srebp, acc, and AMP-activated protein kinase (ampk) than those fed the HFM diet; shrimp fed the LB2 diet had higher expression levels of srebp, acc, and cpt-1 than those fed the HFM diet (P < 0.05). In the hepatopancreas, the shrimp fed the LFM diet shown significantly up-regulated the expression levels of beclin1 compared to those fed HFM diet; the expression levels of autophagy-related protein13 (atg3), autophagy-related protein 12 (atg12) of in shrimp fed the LB1 diet were significantly higher than those fed the HFM diet; and the expression levels of autophagy-related protein13 (atg13), beclin1, atg3, atg12, autophagy-related protein 9 (atg9) of shrimp fed LB2 diet were significantly higher than those fed the HFM diet (P < 0.05). The atg3 in intestine of shrimp fed the LB2 diet were significantly higher than those fed the HFM diet (P < 0.05). Intestinal mucous fold were damaged, hepatic tubules were disorganized and B cells appeared to be swollen in LFM group. The fold height and width of shrimp fed the diets supplemented with CDCA increased significantly than those fed the LFM diet (P < 0.05), the hepatic tubules were neatly arranged, and R cells increased. In conclusion, supplementary CDCA in a low fishmeal diet promoted lipid metabolism, enhanced autophagy of shrimp, also improved the health of the intestine and hepatopancreas.

Impaired Bile Acid Synthesis in a Taurine-Deficient Cat Model.

Deficiency of the functional amino acid-like compound taurine induced in cats by taurine-depleted food was previously shown to significantly decrease the levels of taurine-conjugated bile acids (BAs) and significantly increase the levels of unconjugated BAs, with a significant decrease in total BA concentration in the bile. Because the ratios of primary BAs (cholic acid [CA] and chenodeoxycholic acids [CDCA]) have also been shown to be altered in the bile, taurine has been suggested to play an important role in BA synthesis in the liver. The present study showed that in the liver of taurine-deficient cats, CYP7A1 protein expression and its metabolites (7α-hydroxycholesterol and α-hydroxy-4-cholesten-3-one) were significantly increased and, therefore, the ratio of the CA product in this pathway was decreased. On the other hand, the expression of the mitochondrial CYP27A1 protein and its metabolite 27-hydroxycholesterol (27HC) were significantly decreased in the taurine-deficient liver. Thus, a significantly decreased ratio of CDCA, which is the main product of 27HC, was found. The decreased activity of the CDCA-producing pathway might be related to mitochondrial dysfunction induced by taurine deficiency. In addition, a significant decrease in cholesterol levels in the liver was induced by a decrease in intestinal cholesterol absorption because of decreased hepatic-intestinal circulation of taurine-conjugated BAs. The results of this study showed that taurine deficiency alters both the quality and quantity of BAs through inactivity of the mitochondrial CDCA production pathway caused by impaired mitochondrial function and inhibited the absorption of cholesterol in the intestine.

Repression of Organic Anion Transporting Polypeptide (OATP) 1B Expression and Increase of Plasma Coproporphyrin Level as Evidence for OATP1B Downregulation in Cynomolgus Monkeys Treated with Chenodeoxycholic Acid.

Farnesoid X receptor (FXR) is a nuclear receptor known to markedly alter expression of major transporters and enzymes in the liver. However, its effects toward organic anion transporting polypeptides (OATP) 1B1 and 1B3 remain poorly characterized. Therefore, the present study was aimed at determining the effects of chenodeoxycholic acid (CDCA), a naturally occurring FXR agonist, on OATP1B expression in cynomolgus monkeys. Multiple administrations of 50 and 100 mg/kg of CDCA were first shown to significantly repress mRNA expression of SLCO1B1/3 approximately 60% to 80% in monkey livers. It also suppressed cytochrome P450 (CYP)7A1-mRNA and induced OSTα/ß-mRNA, which are well known targets of FXR and determinants of bile acid homeostasis. CDCA concomitantly decreased OATP1B protein abundance by approximately 60% in monkey liver. In contrast, multiple doses of 15 mg/kg rifampin (RIF), a pregnane X receptor agonist, had no effect on hepatic OATP1B protein, although it induced the intestinal P-glycoprotein and MR2 proteins by ∼2-fold. Moreover, multiple doses of CDCA resulted in a steady ∼2- to 10-fold increase of the OATP1B biomarkers coproporphyrins (CPs) in the plasma samples collected prior to each CDCA dose. Additionally, 3.4- to 11.2-fold increases of CPI and CPIII areas under the curve were observed after multiple administrations compared with the single dose and vehicle administration dosing groups. Taken together, these data suggest that CDCA represses the expression of OATP1B1 and OATP1B3 in monkeys. Further investigation of OATP1B downregulation by FXR in humans is warranted, as such downregulation effects may be involved in bile acid homeostasis and potential drug interactions in man. SIGNIFICANCE STATEMENT: Using gene expression and proteomics tools, as well as endogenous biomarker data, for the first time, we have demonstrated that OATP1B expression was suppressed and its activity was reduced in the cynomolgus monkeys following oral administration of 50 and 100 mg/kg/day of chenodeoxycholic acid (CDCA), a Farnesoid X receptor agonist, for 8 days. These results lead to a better understanding of OATP1B downregulation by CDCA and its role on bile acid and drug disposition.

Synthesis of the Alkylsulfonate Metabolites Cysteinolic Acid, 3-Amino-2-hydroxypropanesulfonate, and 2,3-Dihydroxypropanesulfonate.

Chiral hydroxy- and aminohydroxysulfonic acids are widespread in the marine and terrestrial environment. Here we report simple methods for the synthesis of d- and l-cysteinolic acid (from (Boc-d-Cys-OH)2 and (Boc-l-Cys-OH)2, respectively), R- and S-3-amino-2-hydroxypropanesulfonate (from S- and R-epichlorohydrin, respectively), and R- and S-2,3-dihydroxypropanesulfonate (from S- and R-epichlorohydrin, respectively). d-Cysteinolate bile salts were generated by coupling with cholic and chenodeoxycholic acids. A series of single-crystal 3D X-ray structures confirmed the absolute configurations of the aminosulfonates. By comparison of optical rotation, we assign naturally occurring 3-amino-2-hydroxypropanesulfonate from Gateloupia livida as possessing the R-configuration. This simple synthetic approach will support future studies of the occurrence, chemotaxonomic distribution, and metabolism of these alkylsulfonates.

Intestinal Flora is a Key Factor in Insulin Resistance and Contributes to the Development of Polycystic Ovary Syndrome.

CONTEXT: The key gut microbial biomarkers for polycystic ovarian syndrome (PCOS) and how dysbiosis causes insulin resistance and PCOS remain unclear. OBJECTIVE: To assess the characteristics of intestinal flora in PCOS and explore whether abnormal intestinal flora can affect insulin resistance and promote PCOS and whether chenodeoxycholic acid (CDCA) can activate intestinal farnesoid X receptor (FXR), improving glucose metabolism in PCOS. SETTING AND DESIGN: The intestinal flora of treatment-naïve PCOS patients and hormonally healthy controls was analyzed. Phenotype analysis, intestinal flora analysis, and global metabolomic profiling of caecal contents were performed on a letrozole-induced PCOS mouse model; similar analyses were conducted after 35 days of antibiotic treatment on the PCOS mouse model, and glucose tolerance testing was performed on the PCOS mouse model after a 35-day CDCA treatment. Mice receiving fecal microbiota transplants from PCOS patients or healthy controls were evaluated after 10 weeks. RESULTS: Bacteroides was significantly enriched in treatment-naïve PCOS patients. The enrichment in Bacteroides was reproduced in the PCOS mouse model. Gut microbiota removal ameliorated the PCOS phenotype and insulin resistance and increased relative FXR mRNA levels in the ileum and serum fibroblast growth factor 15 levels. PCOS stool-transplanted mice exhibited insulin resistance at 10 weeks but not PCOS. Treating the PCOS mouse model with CDCA improved glucose metabolism. CONCLUSIONS: Bacteroides is a key microbial biomarker in PCOS and shows diagnostic value. Gut dysbiosis can cause insulin resistance. FXR activation might play a beneficial rather than detrimental role in glucose metabolism in PCOS.

Bile Acids Elevated in Chronic Periaortitis Could Activate Farnesoid-X-Receptor to Suppress IL-6 Production by Macrophages.

Chronic periaortitis (CP) is a rare autoimmune disease without effective treatment. By analyzing the serum bile acid spectrum in 28 CP patients with the ultra-performance liquid chromatography-tandem mass spectrometry, we found that the bile acids were significantly altered in CP patients, with significant increases in chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA) and decrease in deoxycholic acid (DCA). Signaling pathway enrichment analysis from the RNA sequencing results suggested that the altered gene sets in PBMC of CP patients were associated with bile acid metabolism. Furthermore, we found that pathological concentration of CDCA could significantly inhibited IL-6 expression in RAW 264.7 cells after LPS stimulation. Since CDCA is a well-known natural high-affinity ligand for the bile acid receptor farnesoid-x-receptor (FXR) while GW4064 is the synthetic specific agonist of this receptor, we then revealed that GW4064 significantly decreased IL-6 expression in RAW 264.7 cells and bone marrow-derived macrophages but not in FXR-/- macrophages upon LPS stimulation. The western blot results with the anti-FXR antibody showed significantly increased expression in the nuclear proportion, suggesting that FXR agonist promoted the transportation of FXR into the nucleus but did not increase the FXR expression in macrophages. Dual-luciferase report assay and ChIP assay demonstrated that upon activation, FXR could directly bind to the promoter site of IL-6, leading to the decreased expression of IL-6. Thus, bile acids, especially CDCA, may operate to damp inflammation via FXR-mediated downregulation of IL-6 in mononuclear cells and provide a protective mechanism for CP patients.

Assessment of Statin Interactions With the Human NTCP Transporter Using a Novel Fluorescence Assay.

Sodium taurocholate cotransporting polypeptide (NTCP), which is highly expressed in the sinusoidal membrane of hepatocytes, maintains bile acid homeostasis and participates in the hepatic disposition of a variety of endogenous substances as well as xenobiotics. Manifested by the involvement of organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) in the hepatic uptake of statin drugs, sinusoidal membrane transporters play an important role in the pharmacokinetics and pharmacodynamics of these agents. It has been speculated that NTCP may function as an alternative pathway for statin hepatic uptake, complementary to OATP1B1 and OATP1B3. In the current study, we produced stable NTCP-expressing human embryonic kidney 293 (HEK293) cells and developed a fluorescence-based assay using flow cytometry for measuring NTCP transport with chenodeoxycholyl-(Nε-7-nitrobenz-2-oxa-1,3-diazole)-lysine (CDCA-NBD) as the substrate. NTCP-mediated CDCA-NBD transport was time-dependent and exhibited typical Michaelis-Menten kinetics, with a Km of 6.12 µM. Compounds known to interact with NTCP, including chenodeoxycholic acid and taurocholic acid, displayed concentration-dependent inhibition of NTCP-mediated CDCA-NBD transport. We report here a systematic evaluation of the interaction between statins and the NTCP transporter. Utilizing this system, several statins were either found to inhibit NTCP-dependent transport or act as substrates. We find a good correlation between the reported lipophilicity of statins and their ability to inhibit NTCP. The objective was to develop a higher-throughput system to evaluate potential inhibitors such as the statins. The in vitro assays using CDCA-NBD as fluorescent substrate are convenient, rapid, and have utility in screening drug candidates for potential drug-NTCP interactions.