Effects of Bile Acids Supplement in High Plant Protein Diet on Common Carp (Cyprinus Carpio) Bile Acids Prole and Hepatopancreas Health

Bile acids (BAs) have considerable importance in the metabolism of glycolipid and cholesterol. BAs profile in mammals has been widely reported, but lacking in fishes. The purpose of the present study is to clarify BAs profiles of common carp and how exogenous additions of BAs could alleviate hepatopancreas injured of common carp under a high plant protein diet. A 11-week feeding trial was conducted with high plant protein diet (18%) (HP) and high plant protein diet (18%) added 600 mg/kg BAs (HP+BAs) for Common carp, and then the UHPLC-MS/MS technology was used to analyze the BAs in the bile and plasma of two groups. HP could induce glycogen hepatopancreas while supplemented BAs to HP could mitigate this symptom. And we determined that the reduction of common carp hepatopancreas glycogen accumulation in the HP+BAs group is importantly related to the change in the endogenous BAs profile after the addition of BAs in HP through an integrated bile acids profile determination by UHPLC-MS/MS. This study has important guiding significance for aquaculture.


Introduction
Bile acids (BAs) are series of amphipathic molecules, that synthesized in the liver from cholesterol and stored in the gallbladder, which synthesized in liver from cholesterol [1]. Most of BAs conjugated with taurine or glycine in liver [2], and then hydrolyzed, dehydroxylated, deconjugated and functioned in gut [3,4]. BAs secret into duodenum to promote lipid digestion and absorption in small intestine and then were reabsorbed in ileum by liver via BAs transporters and portal vein, which is defined as metabolism enterohepatic circulation [5]. BAs have been known to accelerate digestion and absorption of lipids in the gut [6], and regulate cholesterol homeostasis [7]. Moreover, recent years, scientists have found that BAs also acted as various signals receptors to participate in regulations of homeostasis of glucose and energy metabolism [8], as well as in signaling pathways [9,10]. LCA and DCA activate TGR5 to regulate glucose metabolism and anti-inflammatory response [11,12].
TβMCA and CDCA act together on FXR to regulate BA synthesis and glycolipid metabolism [13,14]. These new founding of BAs' functions helped to solve various diseases caused by metabolic disorders.
In order to meet the needs of environmental protection and reduce the cost of feed, plant protein is more and more widely used in aquatic feed. However, the application of plant protein could bring negative effects like liver and intestine damage simultaneously, finally induced BAs enterohepatic circulation disorder and hepatic lesions, together with remarkable reduction of growth performance but increased, inflammatory reaction in Amur sturgeon and Japanse seabass [15,16]. Bile acids were widely used in aquaculture of China, it has a positive effect on fish growth performance, nutrient digestibility and immunity [17,18]

Methods
During the feeding period, the experimental fishes were maintained in compliance with the Laboratory Animal Welfare Guidelines of China

Bile acids quantitative analysis
Plasma and bile samples were prepared following by previous report [32]. The eluted substances of UHPLC-TQqQ-MS/MS were ionized in an electro spray ionization source in negative mode (ESI -).
Both temperatures of ESIsource drying gas and sheath gas were 300°C.
The flow rate of ESI-source drying gas and sheath gas were 5 and 11 L/min, respectively. The pressure of nebulizer was 45 psi, and capillary voltage was 4000 V. The dynamic multiple reaction monitoring (dMRM) was used to acquire data in optimized MRM transition (precursor -> product), fragmentor, and collision energy (CE) as Table 1

Independent-samples T test of variance by the software SPSS
Statistics 20 was used to analyze all data. Homogeneity test of variance (F-test) was also performed for the data between the two groups, log transformation analysis will be executed on the data when variance was irregular. Data were presented as mean ± SEM. Statistically significant results were indicated by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Graphics were dawn using GraphPad Prism 8.0 (GraphPad Software Inc. USA). All BAs unit conversion calculated from ng/mL to mM, and summated individual BAs concentration as total BAs concentration (TBA). The average concentration of individual BAs and the total BAs concentration were normalized to calculate the ratio of individual BAs to the total BAs.

Growth performance
Compared with HP group, growth performance of HP+BAs group

Hepatopancreas histopathological examination
Fish hepatopancreas sections were examined after PAS staining and H & E staining, and 8 samples were selected to be observed and quantified the glycogen granules and effective nucleus in each group.
Two typical phenotypes are shown in Figure 1A. Phenotypes I: Hepatopancreas have obvious glycogen accumulation, vacuolization, blurred cell membrane boundaries, and cell nuclei aggregation.
Phenotypes II: No significant accumulation of glycogen, and cell morphology showed no obvious abnormality, and effective nuclei also increased. Glycogen granules in HP group was significant more than HP+BAs group (p<0.001), while HP+BAs group has more active nucleus.

Plasma biochemical parameters and hepatic glycogen
Plasma biochemical parameters of ALT, AST, TC were listed in Table2. ALT and AST in HP group were apparently higher than HP+BAs (p<0.05). Supplement BAs to high plant protein diet not affected the content of TC in plasma. Plasma glucose and liver glycogen in the HP+BAs group were significantly lower (p<0.05) than those in the HP group ( Figure 1D).

Bile acids profile of in common carp bile and plasma
Ten compounds, including TCA, TβMCA, TωMCA, CA,GLCA, GHCA, GCDCA, HDCA, CDCA and 7,12-KLCA were found quantified in bile samples, whose EIC was show in Figure 4. BAs profiles of common carp bile were summarized in Table 3 and Figure 6, which suggested TCA was the main bile acids in Common Carp, and followed by CA which accounted for 88-92% and 6-7% respectively, CA ranged   44], but which has also been found in humans by some reports [20,45]. Rodents branch off from fish in the evolutionary tree, and humans and rodents are on a small branch, we considered that MCA maybe a common specie of bile acids existed in fish, rodents, and primates. More work should be done in future.  [53]. TGR5 plays an important role in anti-inflammatory and glucose metabolism [54]. TGR5 restrains the activated B cells (NF-κB) to control the proinflammatory factors secretion by mediation of the interaction between IκBα and β-arrestin2

Supplement BAs affected common carp
and thus exert anti-inflammatory effects [55][56][57]. Activating liver TGR5 could reduce blood glucose in mice with a high-fat die [53]. That suggested the potential Hypoglycemic function of TGR5. In present study, plasma CDCA, CA, LCA and GCDCA increased significantly in HP+BAs group, with the assistant of enterohepatic circulation of BAs [58], they will enter the liver and activate TGR5, especially LCA, that cannot be synthesized directly in the liver while can only be recovered from intestinal BAs by blood circulation, thus enhancing the anti-inflammatory ability of the body, regulating glucose metabolism and reducing blood glucose, and decreased the liver inflammation [59]. The expression of TGR5 gene in the liver increase after supplement BAs to HP have been confirmed by Yao et al [29].
In the results of T. IDE et al., it can be found that the content of G-BAs in bile increases with the increase of liver glycine concentration [60]. Kupffer cells in an activated state could release a variety of inflammatory mediators and play a leading role when the liver is invaded, Glycine inactivates Kupffer cells and can protect the liver from inflammation [61]. TβMCA varied quite distinctly in bile between HP group and HP+BAs group. TβMCA is an farnesoid X receptor (FXR) nuclear receptor antagonist [14].FXR is a member of the nuclear receptor superfamily that is primarily expressed in the liver, kidney, and intestine [62].In FXR gene knockout mice, intestinal glucose absorption was delayed, together with blood glucose decreased [63]. TβMCA suppressed the enterohepatic FXR-FGF15 signaling could affect glucose metabolism, reduce blood glucose and treat diabetes [64]. Therefore, the increase of TβMCA in bile after the addition of BAs also plays a certain role in the reduction of plasma glucose.

Conclusion
In summary, HP could induce glycogen accumulation in common

Availability of data and materials
All data generated or analyzed during this study are included in this article.

Figure 3
The work ow of the effect in bile acids supplement to high plant protein diet on Common Carp bile acids pro le and hepatopancreas health.   BAs species composition in bile, A and B for HP and HP+BAs, respectively (n=7).

Figure 7
Supplement BAs to high plant protein feed increases the proportion of Glycine-conjugation BAs, A and B for HP and HP+BAs, respectively (n=7).