Diet-dependent induction of life-history changes in Daphnia: 5α-cyprinol sulfate is not the kairomone

Elevated levels of conjugated bile acids (BA) such as taurochenodeoxycholic acid (TCDCA) and taurocholic acid (TCA) are found in serum of patients with chronic inflammatory liver diseases and liver cancers. Increased lymphangiogenesis has been documented in several chronic liver diseases, as lymphatics are integral to the hepatic microcirculation and also plays an important role in early metastasis of liver cancers like hepatocellular and cholangiocarcinoma. High levels of BAs have been shown in metastatic tumor-bearing LNs compared to naive LNs, however, the effects of BAs on lymphatic endothelial cells (LECs) remain completely undefined. The present study focuses on investigating the effects of TCDCA and TCA on inducing molecular and metabolic changes in LECs. LECs were treated with TCDCA (100 uM) and TCA (100 uM) for 24hrs in presence or absence of specific BA receptor inhibitor (FXR or TGR5: 50uM) and the changes in proliferation, migration, tube formation was determined. Effects of BAs on LEC cellular metabolism was assessed by Seahorse analysis and lactate levels, ATP production and ROS was measured by fluorometric assays. mRNA levels of bile acid receptors- FXR, TGR5, VDR as well as receptors for VEGF and FGF, chemokines and chemokine receptors were analyzed by real time quantitative PCR. Inhibitor for TGR5 was used to test the role of TGR5. Our results showed that LECs express the BA receptors FXR, TGR5 and VDR with TGR5 showing highest basal level expression. Both TCDCA and TCA induced significant induction in migration rates as well as increased LEC tube formation. Among the two BA used, TCDCA induced more metabolic changes in LECs than TCA with significant increase in maximal glycolytic capacity as well as basal rate of ATP production. These alterations were abrogated in presence of the TGR5 inhibitor. BAs, also increased levels of Yes-associated protein (YAP) (associated with increased lymphangiogenesis and tumor metastasis) and several downstream mediators in the LECs. Further, TCDCA elevated several lymphangiogenic molecules such as VEGFR1, VEGFR3, FGFR2 and FGFR4, and other inflammatory chemokines and chemokine receptors that play an important role in LEC immune cell crosstalk such as CCL19, CXCL5, CCR4, CCR5. TCA on the other hand, significantly increased mRNA expression of VEGFR3, MCP1 and CCR4 indicating specific roles for the different BAs on LECs. From these data we conclude that TCDCA and TCA alters morphological and phenotypic characteristics of LECs, induces lymphangiogenesis and promotes inflammatory signaling. We also predict that these activities of BA are initiated through the FXR and TGR5 receptors and targeting these pathways could suppress lymphangiogenesis and progression of inflammation.

Hepatocyte nuclear factor 4alpha (HNF4alpha) has an important role in regulating the expression of liver-specific genes. Because bile acids are produced from cholesterol in liver and many enzymes involved in their biosynthesis are preferentially expressed in liver, the role of HNF4alpha in the regulation of bile acid production was examined. In mice, unconjugated bile acids are conjugated with taurine by the liver-specific enzymes, bile acid-CoA ligase and bile acid-CoA:amino acid N-acyltransferase (BAT). Mice lacking hepatic HNF4alpha expression exhibited markedly decreased expression of the very long chain acyl-CoA synthase-related gene (VLACSR), a mouse candidate for bile acid-CoA ligase, and BAT. This was associated with markedly elevated levels of unconjugated and glycine-conjugated bile acids in gallbladder. HNF4alpha was found to bind directly to the mouse VLACSR and BAT gene promoters, and the promoter activities were dependent on HNF4alpha-binding sites and HNF4alpha expression. In conclusion, HNF4alpha plays a central role in bile acid conjugation by direct regulation of VLACSR and BAT in vivo.

Among bile acids whose levels increase greatly during hepatic failure, glycocholate (GC), taurocholate (TC), glycochenodeoxycholate (GCDC), and taurochenodeoxycholate (TCDC) are potentially hepatotoxic. Most bioartificial liver systems utilize isolated or cultured hepatocytes, and therefore the injuriousness of these bile acids might constitute a clinical drawback. To assess the possible hepatocellular injuriousness of these bile acids, changes in gluconeogenesis, ureagenesis, alanine aminotransferase (ALT) in medium, DNA contents, and morphological changes in hepatocytes were analyzed. The measurements were made 1, 3, and 5 days after cultured hepatocytes were exposed to the bile acids. The concentration of each bile acid was 2 mM, which is lower than their reported critical micelle concentrations (CMCs). TCDC and GCDC showed significantly high elevation of ALT, significantly low gluconeogenesis and ureagenesis activities, and low DNA contents. They also caused serious injury to hepatocytes on morphological study. TCDC showed heavier hepatocyte toxicity than did GCDC. Although the DNA contents in the GCDC group were relatively preserved throughout the experimental period, marked necrosis and deformity of hepatocytes were observed, whereas GC and TC were not seen to cause any injury to hepatocytes. The CMCs of TCDC and GCDC were reported to be 2.5 mM. However, our experiment showed that 2 mM of TCDC or GCDC has strong toxicity to hepatocytes. This raises the possibility that the CMCs of TCDC and GCDC could be 2 mM or even lower. In the clinical use of a bioartificial liver, removal of elevated TCDC and GCDC must be considered. Even if the levels of individual bile acids are lower than these CMCs, their cumulative hepatotoxic effect must be considered and requires further investigation.

The sodium taurocholate cotransporting polypeptide (Ntcp) is the major bile salt uptake transporter at the sinusoidal membrane of hepatocytes. Short-term feedback regulation of Ntcp by primary bile salts has not yet been investigated in vivo. Subcellular localization of Ntcp was analyzed in Ntcp-transfected HepG2-cells by flow cytometry and in immunofluorescence images from tissue sections by a new automated image analysis method. Net bile salt uptake was investigated in perfused rat liver by a pulse chase technique. In Flag-Ntcp-EGFP (enhanced green fluorescent protein) expressing HepG2-cells, taurochenodeoxycholate (TCDC), but not taurocholate (TC), induced endocytosis of Ntcp. TCDC, but not TC, caused significant internalization of Ntcp in perfused rat livers, as shown by an increase in intracellular Ntcp immunoreactivity, whereas Bsep distribution remained unchanged. These results correlate with functional studies. Rat livers were continuously perfused with 100 μmol/L of TC. 25 μmol/L of TCDC, taurodeoxycholate (TDC), tauroursodeoxycholate (TUDC), or TC were added for 30 minutes, washed out, followed by a pulse of (3) [H]-TC. TCDC, but not TDC, TUDC, or TC significantly increased the amount of (3) [H]-TC in the effluent, indicating a reduced sinusoidal net TC uptake. This effect was sensitive to chelerythrine (protein kinase C inhibitor) and cypermethrin (protein phosphatase 2B inhibitor). Phosphoinositide 3-kinase (PI3K) inhibitors had an additive effect, whereas Erk1/2 (extracellular signal activated kinase 1/2), p38MAPK, protein phosphatase 1/2A (PP1/2A), and reactive oxygen species (ROS) were not involved. TCDC regulates bile salt transport at the sinusoidal membrane by protein kinase C- and protein phosphatase 2B-mediated retrieval of Ntcp from the plasma membrane. During increased portal bile salt load this mechanism may adjust bile salt uptake along the acinus and protect periportal hepatocytes from harmful bile salt concentrations.

Objective: To analyze serum bile acid profiles in pregnant women with normal pregnancy, intrahepatic cholestasis of pregnancy (ICP) and asymptomatic hypercholanemia of pregnancy (AHP), and to evaluate the application value of serum bile acid profiles in the diagnosis of ICP and AHP. Methods: The clinical data of 122 pregnant women who underwent prenatal examination in Xuzhou Maternal and Child Health Care Hospital from June 2022 to May 2023 were collected, including 54 cases of normal pregnancy group, 28 cases of ICP group and 40 cases of AHP group. Ultraperformance liquid chromatography-tandem mass spectrometry was used to measure the levels of 15 serum bile acids in each group, including cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), glycolcholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), glycoursodeoxycholic acid (GUDCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA) and tauroursodeoxycholic acid (TUDCA). Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to screen differential bile acids. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of differential bile acids and combined indicators between groups. Results: (1) Compared with normal pregnancy group, the serum levels of LCA, GCA, GCDCA, GDCA, GLCA, UDCA, TCA, TCDCA, TDCA, TLCA, GUDCA and TUDCA in ICP group were significantly different (all P<0.05), while the levels of LCA, DCA, GCA, GCDCA, GDCA, GLCA, TCA, TCDCA, TDCA, TLCA, GUDCA and TUDCA in AHP group were significantly different (all P<0.05). Compared with ICP group, the serum levels of CDCA, DCA, UDCA, TDCA, GUDCA and TUDCA in AHP group were significantly different (all P<0.05). (2) In the OPLS-DA model, the differential bile acids between ICP group and AHP group were TUDCA, TCA, UDCA, GUDCA and GCA, and their variable importance in projection (VIP) were 1.489, 1.345, 1.344, 1.184 and 1.111, respectively. TCA, GCDCA, GCA, TDCA, GDCA and TCDCA were the differentially expressed bile acids between AHP group and normal pregnancy group, and their VIP values were 1.236, 1.229, 1.197, 1.145, 1.139 and 1.138, respectively. (3) ROC analysis showed that the area under the curve (AUC) of TUDCA, TCA, UDCA, GUDCA and GCA in the differential diagnosis of ICP and AHP was 0.860, and the sensitivity and specificity were 67.9% and 95.0%, respectively. The AUC of TCA, GCDCA, GCA, TDCA, GDCA and TCDCA in the diagnosis of AHP was 0.964, and the sensitivity and specificity were 95.0% and 93.1%, respectively. Conclusions: There are differences in serum bile acid profiles among normal pregnant women, ICP and AHP. The serum bile acid profiles of pregnant women have potential application value in the differential diagnosis of ICP and AHP and the diagnosis of AHP.

We present a method using a combination of enzymatic deconjugation and targeted LC-multiple reaction monitoring (MRM)-MS analysis for analyzing all common bile acids (BAs) in piglet urine, and in particular, for detecting conjugated BAs either in the absence of their standards, or when present in low concentrations. Initially, before enzymatic deconjugation, 19 unconjugated BAs (FBAs) were detected where the total concentration of the detected FBAs was 9.90 μmol/l. Sixty-seven conjugated BAs were identified by LC-MRM-MS analysis before and after enzymatic deconjugation. Four enzymatic assays were used to deconjugate the BA conjugates. FBAs in urine after cholylglycine hydrolase/sulfatase treatment were 33.40 μmol/l, indicating the urinary BAs were comprised of 29.75% FBAs and 70.25% conjugated BAs in single and multiple conjugated forms. For the conjugates in single form, released FBAs from cholylglycine hydrolase deconjugation indicated that the conjugates with amino acids were 14.54% of urinary BAs, 16.27% glycosidic conjugates were found by β-glucuronidase treatment, and sulfatase with glucuronidase inhibitor treatment liberated FBAs that constituted 16.67% of urinary BAs. Notably, chenodeoxycholic acid (CDCA) was initially detected only in trace amounts in urine, but was found at significant levels after the enzymatic assays above. These results support that CDCA is a precursor of γ-muricholic acid in BA biosynthesis in piglets.

Traumatic brain injury is one of the major causes of morbidity and mortality worldwide. With the development of bile acids as a potential treatment, to identify the influence of traumatic brain injury on bile acid metabolism shows growing importance. This present study did a preliminary exploration of the bile acid profile alteration among traumatic brain injury patients. In total, 14 patients and 7 healthy volunteers were enrolled. The bile acid profile of the blood samples were detected by an Ultra-performance Liquid Chromatography Mass Spectrometer/Mass Spectrometer system. It was found that 6 bile acids were statistically decreased in traumatic brain injury patients comparing with healthy volunteers: glycocholic acid (median level 44.4 ng/ml vs 98.7 ng/ml, p = 0.003), taurocholic acid (median level 10.9 ng/ml vs 19.5 ng/ml, p = 0.006), glycoursodeoxycholic acid (median level 17.4 ng/ml vs 71.4 ng/ml, p = 0.001), ursodeoxycholic acid (median level <1 ng/ml vs 32.4 ng/ml, p = 0.002), taurochenodeoxycholic acid (median level <1 ng/ml vs 53.6 ng/ml, p = 0.003) and glycochenodeoxycholic acid (GCDCA, median level 160 ng/ml vs 364 ng/ml, p<0.001). In conclusion, traumatic brain injury events are able to induce bile acid metabolism alteration in plasma and might cause reduction in glycocholic, taurocholic, glycoursodeoxycholic, ursodeoxycholic, taurochenodeoxycholic and glycochenodeoxycholic acid levels.

Dietary Protein Source (Soybean vs. Casein) and Taurine Status Affect Kinetics of the Enterohepatic Circulation of Taurocholic Acid in Cats

– Migratory banded kokopu juveniles exhibit a species‐specific attraction to adult conspecifics. The potential for bile acids to function as a component of a migratory pheromone in banded kokopu was investigated by examining which bile acids are present in the gallbladder of adult fish, are released into the water and are detected by adult fish. High‐performance liquid chromatography (HPLC) and mass spectrometry (MS) of gallbladder extracts showed that banded kokopu contained high quantities of taurocholic acid (TCA) and taurochenodeoxycholic acid (TCD), and a very small quantity of cyprinol sulphate (5α‐CS). The holding water of adult banded kokopu was found to contain only TCA and TCD. In addition, three unknown peaks were evident. Electro‐olfactogram recordings revealed that banded kokopu showed strong responses to four bile acids [5α‐CS, petromyzonol (P), petromyzonol sulphate (PS), and allocholic acid (ACA)] and low responses to TCA and TCD (&lt;30% of the l‐serine standard). The bile acids TCA and TCD, produced and released by adult banded kokopu, are common among teleosts and are not species specific. Therefore, it is unlikely that these bile acids will function as a migratory pheromone in banded kokopu.

To investigate the effects of bile salts on the proliferation and differentiation of human normal esophageal mucosal epithelial cells on cultured. Normal human esophageal mucosa was obtained during operation. The esophageal epithelial cells were isolated, cultured, and treated with 6 different conjugated bile salts [glycocholate (GC), glycochenodeoxycholate (GCDC), glycodeoxycholate (GDC), taurochenodeoxycholate (TCDC), and taurodeoxycholate (TDC), all of the concentration of 50 micromol/L, and taurocholate (TC) of the concentration of 20 micromol/L], and their mixtures the concentration of 50 micromol/L respectively. One, three, and five days later MTT assay was applied to detect the cell proliferation. The cell cycle was assayed by flow cytometry with propidium iodide staining 1 and 3 d after treating with the bile salts. The cytokeratin 13 (CK13) in the differentiated cells and cytokeratin 14 (CK14) in the proliferating cells were detected by immunocytochemical assay. The concentration of intercellular calcium ([Ca(2+)]i) was analyzed by Laser Scanning Confocal Microscope (LSCM) in cells with TC, mixed bile salts and GC. The cultured esophageal epithelial cells treated by the bile salts, except those treated by GC for 1 - 3 days, became larger and shuttle-like, with the cell proliferation inhibited, the percentages of cells in G(0)-G(1) phase increased and percentages of cells in S phase decreased. The percentages of CK14 positive cells were increased, but the percentages of CK13 positive cells were decreased time-dependently in cells treated for 1 - 5 days. The most obvious effects were seen in those cells treated with TC. The percentage of CK13 positive cells reached 74% +/- 8% in those cells treated with TC for 5 days, higher significantly than the percentage in the control cell (22% +/- 7%), (P < 0.01). The [Ca(2+)]i increased significantly only several minutes after treatment of TC and mixed bile salts, however, GC failed to cause the increase of [Ca(2+)]i. GCDC, GDC, TC, TCDC, TDC and their mixture all induce differentiation of cultured human normal esophageal mucosal epithelial cells, but nor does GC. Increased [Ca(2+)]i is related to the TC-induced differentiation in those esophageal mucosal epithelial cells.

Hepatocellular Uptake of Bile Acids in the Dog: Evidence for a Common Carrier-Mediated Transport System: An indicator dilution study

Bile acid profiles and mRNA abundance of bile acid-related genes in adipose tissue of dairy cows with high versus normal body condition

Influence of hydroxylation and conjugation in cross-inhibition of bile acid transport across the human trophoblast basal membrane

TLC-analysis for bile acid composition was carried out on the bile samples obtained by bile duct-cannulation from the rats fasted for 48 hr and from those kept continuously on a diet supplemented with 1% cholesterol or 2% thyroid powder. The secretion of total taurine-conjugated bile acids decreased after fasting, only except the secretion of taurodeoxycholic acid which was maintained at normal level. The fasted group, thus, demonstrated an increase in the ratio of trihydroxy to dihydroxy bile acids and a decrease in another ratio of bile acid components, (taurocholic acid+taurodeoxycholic acid)/(taurochenodeoxycholic acid+tauromuricholic acid+taurohyodeoxycholic acid), as compared with the normal group. Cholesterol-feeding increased slightly the biliary output of total bile acids. However, both of the above ratios for this group were nearly the same to those for the normal group. On the other hand, thyroid powder-feeding caused an increase of taurocholic acid accompanied by a concomitant decrease of taurodihydroxy bile acids within the normal range of total bile acid secretion, resulting in increases of both the ratios over the normal levels. In all the experimental groups including the normal, the incorporation of intravenously injected taurine-35S into biliary taurocholic, tauromuricholic and taurodihydroxy bile acids was approximately proportional to the secreted amounts of these fractions.

5alpha-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5alpha-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was approximately 4 mM. At concentrations >1 mM, 5alpha-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5alpha-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax congruent with 0.5 micromol/min/kg) as compared with taurocholate. 5alpha-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5alpha-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5alpha-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5alpha-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit. These studies indicate that 5alpha-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish.