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

Abstract


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].But not all BAs have a positive effect, some BAs would bring negative effects， for example, both TCA and bovine bile salt supplementary in a low fishmeal diet to the Atlantic salmon could cause slight or moderate inflammation of the distal intestine.[19].At present, in mammals, profile of BAs has got a lot of results and interesting discoveries have been found [20,21], while there were few reports about concentrated on BAs in fish, since most of them focused on roles of BAs in fish pheromone systems and identification of some new BAs in saltwater fish [22][23][24][25].In general, BAs in fish have less study, and which is relatively one-sided [26][27][28].Previous studies of our team suggested that high plant protein induced common carp liver injury, while BAs supplementary could help to alleviate [29], but which BA played the leading role or how to affect liver health?These are still unknown and BAs profiles offish still kept uncovered.
Common carp [30] is a kind of omnivorous fish, an important economical freshwater fish in China.It needs a certain amount of animal protein, and a high proportion of plant protein may cause intestinal and liver diseases, thereby reducing the benefit of breeding.Therefore, the present study combined UHPLC-MS/MS technology to explore the BAs profile of Common Carp comprehensively and the effect of BAs supplement in high plant protein feed on Common Carp BAs profile and hepatopancreas health.

Methods
During the feeding period, the experimental fishes were maintained in compliance with the Laboratory Animal Welfare Guidelines of China (General Administration of Quality Supervision, Inspection and

Quarantine of the People's Republic of China, Standardization
Administration of China, GB/T 35,892-2018).

Histopathological detections of hepatic tissues
The hepatopancreas tissue fixation, dehydration, embedding, hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) staining procedures are conducted as described by Yu et al. [31].Then the pictures were visualized using TissueGnostics Fluorescence Imaging System (TissueGnostics, Vienna, Austria) and analyzed the glycogen granules and effective nucleus by StrataQuest Analysis Software (TissueGnostics, Vienna, Austria).BAs were extracted and analyzed for the corresponding plasma and bile samples with obvious hepatopancreas damage observed in HP group and no obvious abnormalities in the hepatopancreas observed in HP+BAs group.The graph abstract is shown in Figure 3.

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 ESI -source 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.The total scan time of per cycle was 300 ms.Chromatographic separation was operated on an UPLC BEH C 18 column (100mm×2.1mm,1.7μm).The column temperature was 40°C, and the flow rate was 0.45 mL/min.The mobile phase consisted of water in 0.1% formic acid (A) and acetonitrile in 0.1% formic acid (B).The chromatographic separation was conducted by a gradient elution program as follows: 0.5 min, 15% B; 1 min, 25% B; 3min, 25% B; 5 min, 34% B; 8 min, 40% B; 9 min, 52% B; 10.

Statistical analyses
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 improved significantly (p<0.01)(Figure 2), the final body weight (BW) increased significantly (p<0.01), and hepatosomatic index (HSI) significantly decreased in HP group.The mean of HSI in HP and HP+BAs groups were 0.07 and 0.03, respectively, while the HSI of bony fish is generally 0.007-0.02[33].

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 from 140-170 μM, however, no TCDCA existed.Moreover, eight BAs, including TCDCA, CDCA, CA, LCA, HDCA, GLCA, GCDCA and DCA were detected and quantified from the plasma samples.Table 4showed the detailed BAs in bile and plasma.This was the first time that MCA was found to be exist in fish.So, we confirmed this point.The confirmations of MCA detected were verified by the retention time (RT) and the abundance ratio of MS/MS.

Supplement BAs to high plant protein feed altered the BAs Profile
The ratio of GCDCA, TβMCA, GLCA, HDCA increased significantly in HP+BAs group, while TCA and CDCA decreased (Figure6).An increase of BAs diversity in HP+BAs group could be observed in both bile and plasma.Supplementary BAs to high plant protein diet increased the proportion of G-BAs, which accounted for 0.6% and 1.5% in HP group and HP+BAs group, respectively.

BAs profile changes caused by Supplementary of BAs
TCA was the main BA in common carp bile, which was consistent with the results of the only previously reported about the BAs profile of fish (angelfish (Pterophyllumeimekei)) bile [23].In this study G-BAs such as GCDCA, GLCA, GHCA and GCA were detected.However, this result is different from previous results pointed out that all animals, except for the placental mammals, conjugate their BAs exclusively with taurine [34,35].the reason for this difference is the insufficient coverage of non-mammals in previous studies, only chickens and croaker fish.That conclusion has always been continued use to cause little attention to G-BAs in subsequent studies on fish BAs.The food source of croaker fish in the early stage was mainly fish and shrimp.In this experiment, common carp was an omnivorous fish, and there was a relatively high proportion of plant-based feed in the diet.An animal-based diet significantly increases both BAs tauro-conjugation, and a plant-based diet promotes G-BAs [36].Therefore, the detection of G-BAs in common carp can be reasonably explained.In addition, compared with the HP group, the percentage of G-BA in the HP+BA group increased while the percentage of T-BA decreased that consistent with G-BA and T-BA have a mutual inhibition relationship in previous studies [36,37].Supplementary BAs (mainly HCA, HDCA, CDCA) increased the contents of TβMCA, GLCA, GCDCA, and HDCA in common carp bile, we suppose the following could take responsibility with the help of intestinal microorganisms.HCA transformed into βMCA by 6b-epimerization and further 7b-epimerization [38], then βMCA was reabsorbed back to liver and combined with taurine into TβMCA, while HDCA was directly reabsorbed to the liver.CDCA is dehydroxylated into LCA (Based on KEGG secondary bile acid biosynthesis，map00121), which is partly excreted from the body, and part is reabsorbed to the liver and combined with glycine to form GLCA. The other part of CDCA is reabsorbed to the liver to combine with glycine.
The discovery of TβMCA and TωMCA in Common Carp was a break though since they were thought to be a rodent specific bile acid [38][39][40][41][42], and it not indeed be found in birds and monogastric animal BAs analysis [43,44], 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.

Supplement BAs affected common carp BAs profile to reduced hepatopancreas glycogen accumulation and alleviated hepatopancreas damage with a high plant protein diet
Supplement BAs reduced liver glycogen accumulation and alleviated liver damage in common Carp with a high plant protein diet.
High plant protein could induce fish intestinal and liver damage that has been confirmed in the previous research through our laboratory [15,29,46,47].Intestinal is the organ to digest and absorb nutrients, while damage and functional barriers would lead to nutritional metabolism disorders, especially proteins [48,49],thus affecting the synthesis of key enzymes of other nutrients.In this study, the reason that a high-plant-protein diet cause liver glycogen accumulation and hyperglycemia is the high-plant-protein diet injures the Common carp's intestines and leading to protein digestion and absorption disorders, and then resulting in a lack of phosphorylase (the key enzymes in liver glycogen decomposition) synthesis.Disorders of glucose metabolism can stimulate cell inflammation and apoptosis, which caused liver damage [31,50].
However, supplementary BAs to high plant protein feed could alleviate intestinal and liver damage [29].The saponins of plant protein feed may be the main cause of Common carp intestinal damage [51].BAs can be combined with non-starch polysaccharides and excreted from the body [52].The structure of saponins is similar to that of non-starch polysaccharides.After BAs supplementary could be combined with saponins, thereby reduce the damage of saponins to the intestinal, and then improved protein digestion and absorption, reduced liver glycogen accumulation.
Common carp hepatopancreatic inflammation and glucose metabolism may be regulated by three purposes that were LCA, CDCA and CA to activate liver TGR, increased liver glycine concentration and TβMCA inhibits intestinal FXR.TGR5 could be activated by some BAs, in which LCA is the most potent agonist for TGR5, DCA and the conjugations, CDCA and the conjugations, and CA and the conjugations activate TGR5 effectively simultaneously [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 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 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.

Abbreviations
BAs 2 min, 58% B; 10.21 min, 100% B; 11.2 min, 100% B; 11.21 min, 15% B; 12.5 min, 15% B. The gradient elution was applied and MS detection proceeded in negative mode.Standards for all BAs were used to identify the different BA metabolites detected by UHPLC-MS/MS.The Agilent Mass Hunter software (version B.08.00) was used to control instruments and acquire data.The raw data were processed by Agilent Mass Hunter Workstation Software (version B.08.00) by using the default parameters and assisting manual inspection to ensure the qualitative and quantitative accuracies of each compound.The peak areas of target compounds were integrated and output for quantitative calculation.
carp hepatopancreas while supplemented BAs to HP could mitigate this symptom.BAs supplementary in a high plant protein diet could change the BAs profile of common carp, among them, plasma LCA, CDCA, CA increased significantly, TβMCA and the proportion of G-BAs in bile increased significantly, which might play a leading role in it that reduced accumulation of hepatopancreas glycogen and maintained hepatopancreas health.This study proceeded an integrated bile acids profile determination by UHPLC-MS/MS to identify the effect of exogenous BAs supplementary on the endogenous BAs profile and hepatopancreas health of common carp and discussed how the BAs supplementary is transformed in the body, providing a theoretical basis for the application of BAs products in aquatic animals and a data basis for revealing the mystery of fish BAs.

Figures
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Figure 3 The
Figure 3