Taurine-conjugated bile acids are the predominant form in hens and have potential impact on lipid metabolism in the liver

Abstract In mammals, bile acids composition varies with species, age, physiological conditions. However, the role of bile acids in the laying cycle of hens and their impact on lipid metabolism in chicken liver is not well understood. This study aimed to elucidate the role of bile acids in hen laying cycles, investigating their distribution across ages and assessing the impact of taurine-conjugated lithocholic acid (TLCA) on oleic acid-induced fatty liver cells in chickens. Results revealed an age-related increase in total bile acid concentration in hens blood, with taurine-conjugated bile acids prevailing. Among specific bile acids analysed, taurodeoxycholic acid (TDCA) decreased with age, while taurochenodeoxycholic acid (TCDCA) increased (n = 8). TLCA concentration also decreased gradually, while lycocholic acid (GCA) was elevated in 16-week-old hens (n = 8). Primary bile acids, cholic acid (CA), and chenodeoxycholic acid (CDCA) were higher at 16 weeks compared to egg production commencement (22 weeks) and 34 weeks (n = 8). Moreover, 5ß-Cholenic acid-7a-ol-3-one concentration was elevated in 74-week-old hens post-laying (n = 8). Investigating TLCA's impact on chicken liver lipid metabolism, TLCA significantly reduced lipid droplet accumulation, especially at 20 μM, while upregulating TLCA concentrations correlated with decreased SREBP1 protein expression responsible for triglyceride synthesis. In conclusion, taurine-conjugated bile acids potentially regulate lipid metabolism in hens, offering avenues for enhancing hens health and production through further mechanistic exploration and their potential integration into hens feed. HIGHLIGHTS Age-related changes in bile acid composition in hens were observed, with taurine-conjugated bile acids being predominant. Taurine-conjugated lithocholic acid showed promising effects in reducing lipid droplet accumulation and suppressing SREBP1 protein expression in fatty liver cells.


Introduction
Bile acids, derived from hepatic cholesterol conversion, hold diverse physiological roles.Triggered by dietary intake, blood bile acid levels, and cholecystokinin secretion, they traverse bile ducts to reach the small intestine, where they are concentrated in the gallbladder (Argenzio 2004).Bile acids serve as critical emulsifiers, facilitating lipid digestion by reducing oil-water interfacial tension.In the small intestine, they emulsify cholesterol, fat-soluble vitamins, and lipids, aiding their absorption by encapsulating them in micelles.Enterohepatic circulation, involving transporters like the Na þ -dependent bile-salt transporter and multidrug resistance-associated protein 3, reabsorbs 95% of bile acids into the intestinal lumen, with the remaining 5% excreted (Argenzio 2004).Additionally, bile acids impact glucose and lipid metabolism, energy homeostasis, and regulate their own biosynthesis.
Bile acids, amphipathic molecules structurally resembling cholesterol, stem from cholesterol conversion via hepatic enzymes, primarily through the primary synthesis pathway (Schwarz et al. 1996).Liver-synthesized primary bile acids can conjugate with amino acids, enhancing hydrophilicity.In the intestine, anaerobic bacteria modify primary bile acids into secondary forms, including deoxycholic acid, lithocholic acid (LCA), and ursodeoxycholic acid, via bile salt hydrolase (BSH)-mediated cleavage.Bile acids orchestrate biological responses through nuclear receptors such as the Farnesoid X receptor (FXR), Pregnane X receptor (PXR), Vitamin D receptor (VDR), and a G protein-coupled receptor, Takeda G proteincoupled receptor 5 (TGR5).FXR activation initiates SHPmediated negative feedback, reducing triglyceride (TG) and very low-density lipoprotein concentrations, and affecting lipogenesis and cholesterol metabolism.TGR5 impacts various cell types beyond the liver (Maruyama et al. 2002;Sato et al. 2007;Pols et al. 2011;Keitel and H€ aussinger 2012).
This study aims to explore bile acid dynamics in laying hens, an area with limited research.We seek to elucidate their roles in hen laying cycles, considering bile acid distribution across different age groups and evaluating the effects of taurine-conjugated lithocholic acid (TLCA) on oleic acid-induced fatty liver cells in hens.

Animals and sample collection
The aim of this study was to investigate the profile of bile acids in laying hens during their egg-laying period.Eight Commercial Lohmann LSL-Ultra Lite Layers were purchased and housed individually in cages (33 cm � 48 cm � 52 cm) inspected and approved by the Experimental Animal Management Team of National Taiwan University.The temperature of the hen house was controlled between 20 � C and 28 � C, and excessive heat was reduced by water mist and fan ventilation.Humidity was maintained between 50% and 70%.The lighting period, feed formulation, and vaccination program for different growth stages were based on the Lohmann LSL-Ultra Lite Layers management guide.The detailed feed formulations are presented in Tables 1-4.The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of National Taiwan University (NTU110-EL-00024).
During the experiment, the hens were fed with feed formulations tailored to their growth needs based on the Lohmann management guide.Blood samples were collected from the wing vein of each hen during the pre-laying period, early period, peak period, and later period.The egg-laying rate was used as the criterion for determining the different periods: pre-laying period, before egg-laying began (egg-laying rate ¼ 0%); early period, before reaching the peak egg-laying rate, which was approximately 1%-89%; peak period, when the egg-laying rate reached 95%; and later period, when the egg-laying rate fell below 85%.The temperature and humidity were recorded daily, and egg production was recorded.
supernatant were transferred to bile acid analysis.The analysis was performed on Waters ultra-highperformance liquid chromatography coupled with Waters Xevo TQS MS (Waters Corp.).For Mass analysis, chromatographic separation was performed on a Waters ACQUITY BEH C8 column (2.1 mm � 100 mm � 1.7 mm).Column temperature was maintained at 60 � C. For optimised parameters, the mobile phase A was 10% acetonitrile with 0.01% formic acid and the mobile phase B was isopropanol/acetonitrile (50:50, v/v) with 0.01% formic acid.Mass analysis was performed using the Waters Xevo TQ-S system in positive-ion ESI mode.The capillary voltage was set at 1.5 KV.The desolvation gas flow rate was set at 1000 L/h, and cone gas flow was maintained at 150 L/h.The desolvation and source temperatures were set at 600 � C and 150 � C, respectively.QC sample (laboratory quality control) and mix QC sample (a mixture of all samples) were prepared for analysed during the analytical runs after every 10th sample.For total bile acid (TBA) analysis, a Colorimetric Assay kit (MyBioSource, CA, USA) was used.The assay involved adding specific reagents and sample volumes according to product instruction, followed by placing the 96well plate in a spectrophotometer to measure the absorbance at 405 nm at 0 and 3 min.The total concentration was then calculated using the following formula.
DA ¼ absorbance at 405 nm at 3 min − absorbance at 405 nm at 0 min

Cell culture
The hepatocellular gallus cell line (LMH; ATCC-CRL2117; Japan) was used as the primary material for cell experiments.The culture dishes were first coated with 0.1% gelatine and left to stand at 4 � C for 10 min.
After removing the gelatine solution, the dishes were air-dried and then filled with Waymouth's medium (Gibco, Thermo Fisher Scientific, MA, USA) containing 10% FBS (Thermo Fisher Scientific, MA, USA) and 1% penicillin (Thermo Fisher Scientific, MA, USA).The cells were cultured at 37 � C with 5% CO 2 .The cells were passaged when they reached 80-90% confluency, and all cell experiments were conducted between passages 10-20.After the LMH cells were passaged, they were cultured in a medium containing 10% FBS and 1% penicillin for 48 h before being induced with 500 lM oleic acid to become fatty liver cells.Different concentrations of tauroursodeoxycholic acid (Merck, NJ, USA), including 1 lM, 5 lM, 10 lM, and 20 lM, were added to the cells and incubated for 48 h.The Oil Red O staining were conducted to observe the difference in lipid droplet size between the treatment groups and the control group.

Protein extraction and Western blot analysis
Samples were homogenised on ice using RIPA lysis buffer (EMD Millipore, Waltham, MA, USA) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA).The lysed and homogenised samples were centrifuged at 10,000 � g for 30 min at 4 � C, and the supernatant fraction was kept as the protein sample.The protein concentration was determined using the BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA).For Western blots, equal amounts of sample proteins were diluted with 40 mM Tris (pH 6.8), 1% dodecyl sodium sulphate, 5% glycerol, 0.0003% bromophenol blue, and 0.05 M DTT.Electrophoresis was performed using 6% to 10% SDS-PAGE with a running buffer composed of 25 mM Tris, 190 mM glycine, and 0.1% SDS at 80 V, until the sample buffer dye reached the bottom of the gel.Prior to transfer, gels were soaked in transfer buffer composed of 25 mM Tris, 190 mM glycine, and 20% methanol for 20 min.Proteins were transferred from gels to methanol-activated PVDF membranes (PerkinElmer, Inc., Waltham, MA, USA) in a transfer buffer at 200 mA for 2 h.After transfer, PVDF membranes were blocked using a solution of 25 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween 20, and 5% skim milk with gentle shaking for 1 h.Membranes were then incubated with primary antibodies overnight at 4 � C, diluted in TBST buffer composed of 25 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween 20.The primary antibodies used were anti-SREBP1 (1/1000, ab28481, Abcam, Cambridge, UK) and antib-actin (1/3000, #4970, Cell Signalling Technology, Inc., Danvers, MA, USA).After incubation with primary antibodies, the PVDF membranes were washed three times for 10 min each with TBST buffer, and then incubated with secondary antibodies diluted in TBST buffer at room temperature for 1 h.The PVDF membranes were washed again with TBST buffer, and then incubated with ClarityTM Western ECL Substrate (Bio-Rad Laboratories, Inc., Santa Clara, CA, USA) before detection using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc., Santa Clara, CA, USA).The quantitation of protein bands was conducted using Image Lab software (Bio-Rad Laboratories, Inc., Santa Clara, CA, USA), and the relative levels of specific proteins in samples were normalised to b-actin.

Statistical analysis
All values were expressed as mean ± SEM. Results involving more than two groups were assessed using a one-way analysis of variance procedure.Tukey's test was used to evaluate differences among means (GraphPad Prism 5, Version 5.01, La Jolla, CA, U.S.A.).A significant difference was considered at p � .05.

Results and discussion
The 16th week is the pre-laying period with a production rate of less than 0%; the 22nd week is the early period with a production rate between 0 and 90%; the 34th week is the peak period with a production rate of 95% or higher; and the 22nd week is the later period with a production rate of less than 85%.The analysis results from Table 5 demonstrate that the total bile acid concentration in the blood of chickens gradually increases with age.After qualitative and quantitative analysis using a mass spectrometer, the concentrations of taurocholic acid, tauro alpha-Muricholic acid sodium salt, taurohyocholic acid sodium salt, taurohyodeoxycholic acid and hyocholic acid in the serum at different ages of laying hens did not show significant differences (Table 6).However, the concentration of TDCA in 74-week-old hens at the end of the laying period was lower compared to that in 34-week-old hens at the peak of egg production.The concentration of TCDCA was significantly higher in 74-week-old hens at the end of the laying period than in 16-week-old hens at the beginning of the laying period.The concentration of TLCA decreased gradually with age, and the concentration in 74-week-old hens at the end of the laying period was significantly lower than that in 34-week-old hens at the peak of egg production.The concentration of GCA was significantly higher in 16-week-old hens at the beginning of the laying period than in other groups.The concentrations of primary bile acids, CA and CDCA, were significantly higher at 16 weeks of age than at 22 weeks at the beginning of egg production and 34 weeks at the peak of egg production.Moreover, the concentration of 5ß-Cholenic acid-7a-ol-3-one was significantly higher in 74-week-old hens at the end of the laying period than in other laying periods.As we observed that taurine-conjugated bile acids were the main components in chicken bile and TLCA showed a significant decreasing trend with increasing age, we further investigated the effects of adding TLCA to chicken liver cells on lipid metabolism.
In Oil Red O staining, it was observed that the group treated with secondary bile acid TLCA showed smaller lipid droplets in liver cells, especially at a concentration of 20 lM, where a significant decrease in lipid droplet accumulation was observed under 40� magnification compared to the control group (Figure 1A).As the concentration of TLCA decreased, the number of lipid droplets increased.In the blank group, which was not induced with oleic acid nor treated with TLCA, no lipid droplet accumulation was observed in chicken liver cells under staining.Furthermore, we extracted the Oil Red O dye for quantitative analysis, which was consistent with the microscopic observation (Figure 1B). Figure 2 shows the measurement of SREBP1 protein expression using Western blotting.The results indicate that the expression of the SREBP1 protein responsible for generating TG decreases as the concentration of TLCA added increases.
It is known that conjugated bile acids can reduce the hydrophobicity of bile acids by binding with    glycine and taurine, preventing the toxic accumulation of bile acids in the liver.Glycine and taurine have lower dissociation constants (pKa) than bile acids, so their conjugation with bile acids can lower the pKa of bile acids, allowing them to be absorbed after dissociation in acidic environments.Many studies have confirmed that different species preferentially conjugate with different amino acids.For example, in humans, rabbits, and guinea pigs, glycine-conjugated bile acids are preferred over taurine-conjugated bile acids, whereas in mice, sheep, and dogs, taurine-conjugated bile acids are preferred over glycine-conjugated bile acids.Experimental results have shown that in chickens and other poultry, taurine-conjugated bile acids are more prevalent.The reasons for the differences in amino acid preferences between species are not yet fully understood.The conjugation and deconjugation of bile acids are related to intestinal microorganisms, and some anaerobic bacterial groups, such as Bacteroides, Lactobacillus, Clostridium, and Bifidobacterium, can express abundant BSH to deconjugate bile acids.However, it is not yet clear whether the bacteria that prefer to use taurine are included in these groups.
Regarding the bile acid-activated receptor pathway, in addition to the bile acid receptors FXR and TGR5, there are many receptors that can be influenced by bile acids or activated by bile acids, including the VDR, PXR, Liver X Receptor a/b (LXR a/b), Constitutive Androstane Receptor, Peroxisome proliferator-activated receptors, and Retinoid X Receptor (Fan et al. 2015).Through the activation of secondary bile acid LCA, PXR can effectively protect the liver from damage, and also further inhibit CYP7A1 from synthesising cholesterol into bile acids, indirectly achieving a negative feedback effect on LCA concentration, and preventing excessive hydrophobic bile acids from accumulating in the liver (Staudinger et al. 2001).It has also been shown that the vitamin receptor VDR can be activated by secondary bile acids to induce the expression of CYP3A in vivo, thereby detoxifying LCA in the liver and intestine, and reducing the risk of colorectal cancer (Makishima et al. 2002).In our study, the addition of different concentrations of the secondary bile acid TLCA was observed to reduce lipid droplets in oil red staining, but no significant differences were found in gene expression analysis by qPCR (data not shown), suggesting that this result may not only be caused by the FXR pathway.
The present study is the first to elucidate the changes in bile acid levels in egg-laying hens during various stages of egg production.The addition of the secondary bile acid TLCA to the liver cells of these hens resulted in a decrease in the size of lipid droplets, as well as a decrease in SREBP1 expression levels, a protein known to be involved in lipid synthesis.Furthermore, bile acid analysis data showed that the chickens tended to conjugate with taurine rather than glycine during bile acid conjugation.These results provide insight into the role of bile acid metabolism in the regulation of liver lipid metabolism pathways.However, there are still many mechanisms that need to be further elucidated, such as whether gut bacteria mediate changes in secondary bile acid concentrations during different egg production stages or the reason why TLCA causes a decrease in lipid droplets in liver cells.

Conclusions
This study revealed age-related variations in bile acid composition in laying hens.TLCA exhibited a decreasing trend with age.TLCA treatment effectively reduced lipid droplet accumulation and SREBP1 protein expression in liver cells.These findings highlight the role of bile acids in lipid metabolism regulation and suggest the potential use of TLCA to improve liver lipid metabolism in chickens.

Figure 1 .
Figure 1.The effect of taurine-conjugated lithocholic acid on fat deposition in LMH cell.(A) Oil Red O staining (B) Quantification of Oil Red O. Data are represented as mean ± SEM, n ¼ 4. LMH refers to a primary hepatocellular carcinoma epithelial cell line established in 1981 by Tomoyuki Kitagawa at the Cancer Institute, Kami-Ikebukuro, Toshima-ku, Tokyo, Japan.

Table 1 .
The feed composition of pre-laying period.

Table 2 .
The feed composition of early period.

Table 4 .
The feed composition of middle to later period.

Table 5 .
The total bile acids concentration at various laying stages.
c Data are represented as mean ± SEM, n ¼ 8. Means within a row with different letters (a,b,c) differ significantly (p < .05).

Table 6 .
The specific bile acids concentration at various laying stages.