Protective Effects of Bile Acids Against Hepatic Lipid Accumulation in Hybrid Grouper Fed a High-Lipid Diet

Bile acids (BAs) usually display growth-promoting and lipid-lowering properties when supplemented to the diet. The effects of a high-lipid diet (HD) and BAs supplementation on growth performance and lipid deposition of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂) was evaluated in this study. Compared to the control diet (CD), the HD did not significantly affect the fish growth performance, but it promoted lipid deposition, as revealed by a significantly higher crude lipid content of the whole body, muscle, and liver. Among the HD supplemented with taurocholic acid (BD) groups, and compared to the HD, fish fed dietary supplementation of BAs at 900 mg kg−1 exhibited the best growth performance and lowest hepatic lipid deposition. In most BD groups, the content of total cholesterol, low-density lipoprotein cholesterol, and triglycerides in serum, as well as the content of total cholesterol in the liver, were decreased, whereas the content of high-density lipoprotein cholesterol in serum was increased. In addition, the most strongly influenced pathways between the control, HD, and B3D groups were fatty acid biosynthesis, insulin signaling pathway, and AMPK signaling pathway. The improvement of lipid metabolism induced by the supplementation of BAs may be attributed to decreased expression of lipogenesis genes and proteins (enzymes), and increased lipolysis. In conclusion, dietary supplementation of BAs at 900 mg kg−1 promoted growth performance and reduced lipid accumulation, whereas BAs supplementation improved the hepatic lipid metabolism by enhancing hepatic lipolysis, inhibiting lipogenesis, and regulating associated transcriptional factors in hybrid grouper.


INTRODUCTION
The hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂) is a popular marine fish in Asia that has great potential in the aquaculture industry due to its rapid growth and popularity with consumers (1). Dietary lipids are considered to be among the most critical nutritional factors that affect fish growth (2). High-lipid diets (HD) for hybrid grouper are favored by the aquaculture industry because they reduce the use of costly protein as an energy source (3). However, excessive dietary lipids can cause lipid metabolism disorders, exacerbate triglyceride (TG) accumulation, increase the prevalence of non-alcoholic fatty liver disease (NAFLD), impair the liver function, and inhibit the growth performance of fish (4)(5)(6).
Bile acids (BAs) are synthesized from cholesterol, produced in the hepatocytes, stored in the gallbladder, and secreted into the intestine to facilitate the digestion of lipids and the absorption of triglycerides (TG), cholesterol, and lipid-soluble vitamins (7,8). Previous studies showed that the addition of BAs improved the growth performance, including the weight gain (WG) and special growth rate (SGR), of largemouth bass (Micropterus salmoides) fed an HD (9) or a high-starch diet (10), of yellow croaker (Larimichthys crocea) fed an HD (11), of grass carp (Ctenopharyngodon idella) fed an HD (12), of tilapia (Oreochromis niloticus) fed a plant protein-based diet (13), of turbot (Scophthalmus maximus) fed a high plant protein diet (14), However, previous studies have not focused on the effects of dietary BAs on growth performance of hybrid grouper.
In addition, BAs play an important role in lipid metabolism (15,16), while the anti-NAFLD effects have been welldocumented by numerous studies in which BAs consumption reduced the weight of liver, serum and hepatic levels of total cholesterol (T-CHO) and TG, and improved morphological conditions of the liver in mice (17)(18)(19), and human subjects (18). Meanwhile, it was reported that BAs could reduce hepatic lipid accumulation in largemouth bass (9) and regulate hepatic lipid homeostasis in the tiger puffer (Takifugu rubripes) (5). However, previous studies have not investigated the effects of dietary BAs on lipid accumulation and metabolism of hybrid grouper.
Although studies have suggested that dietary BAs reduced lipid deposition in the liver of large yellow croaker, which could be attributed to the increased expression of lipid oxidation and decreased expression of lipid synthesis genes (11), it remains unknown whether this regulation pathway also exists in our focus fish, hybrid grouper. In this study, we evaluated the effects of HD and BAs supplementation on growth performance and lipid deposition of hybrid grouper. To gain a more complete picture, we explored the potential mechanisms by which dietary BAs regulated the lipid metabolism and decreased lipid accumulation in the liver. Understanding the functions of BAs may contribute to the development of management strategies for alleviating the Abbreviations: 6pgd, 6-phosphogluconate dehydrogenase; acbp, acyl-CoA binding protein; ACC, acetyl-CoA carboxylase; ATGL, triglyceride lipase; BAs, bile acids; BD, BAs diets; CD, control diet; CPT1, carnitine palmitoyltransferase 1; dgat, acyl CoA diacylglycerol acyltransferase 2; dgka, diacylglycerol kinase alpha; fabp, fatty acid-binding protein; FAS, fatty acid synthase; FCR, feed conversion ratio; FI, feed intake; FXR, farnesoid X receptor; g6pd, 6-phosphate dehydrogenase; HD, high lipid diet; HDL, high-density lipoprotein cholesterol; hl, hepatic lipase; HSI, hepatosomatic index; hsl, hormone-sensitive lipase; IOD, integrated optical density; LDL, low-density lipoprotein cholesterol; LPS, lipase; lxr, liver X receptor alpha; me, malic enzyme; NAFLD, non-alcoholic fatty liver disease; ppara, peroxisome proliferator-activated receptor alpha; pparr, peroxisome proliferatoractivated receptor gamma; SGR, specific growth rate; SHP, small heterodimer partner; SREBP1, sterol responsive element binding protein 1; TG, triglycerides, TGR5, G protein-coupled bile acid receptor 1; T-CHO, total cholesterol; VSI, viscerasomatic index; WGR, weight gain rate. negative impacts of a high-fat diet on growth performance and hepatic lipid accumulation in hybrid grouper.

Animals and Diet Preparation
This study was carried out following the recommendations for the Care and Use of Laboratory Animals in China, Animal Ethical and Welfare Committee of China Experimental Animal Society. The protocol was approved by the Animal Ethical and Welfare Committee of Guangdong Ocean University (Guangdong, China), processing ID: GDOU-AEWC-20180063. The acclimation, feeding and rearing conditions of fish, and diet preparation are described in detail in the Supplementary Material.

Growth Performance Analyses
The growth parameters were calculated: survival rate (SR), weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR), feed intake (FI), viscerasomatic index (VSI), hepatosomatic index (HSI) and condition factor (CF). Full details for all these analyses are provided in the Supplementary Material.

Diets and Body Composition
In brief, crude protein (N × 6.25) was determined following the Kjeldahl method after acid digestion using a Kjeltec system (Kjeltec 2300 Analyzer, Foss Tecator, Sweden); crude fat was evaluated by the ether extraction method using Soxtec System HT (Soxtec System HT6, Tecator, Sweden); moisture was determined by oven drying at 105 • C until constant weight; crude ash was measured using a muffle furnace at 550 • C until constant weight.

Liver Staining Analyses
Samples were flash-frozen in liquid nitrogen, and the frozen tissues were sectioned (9 µm thickness), immersed in 1% oil red O working solution for 10 min, counterstained with hematoxylin, and then rinsed under running tap water for 30 min. Photomicrographs were captured with a light microscope under 200× magnification. Integrated optical density (IOD) of the oil-red O stained areas was analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

The qPCR Analyses
The following genes (Supplementary

Determining the Levels of TCA in Diets
The levels of TCA in diets were determined using highperformance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) on the Ultimate3000-API 3200Q TRAP (USA). The HPLC-MS/MS system consisted of an SRD-3600 Solvent Rack with an analytical 6-channel vacuum degasser, a DGP-3600A pump, WPS-3000TSL analytical autosampler, and a tcc-3200 column compartment. Chromatographic separations were performed on an MSLab C18 column (150 × 4.6 mm, 5 µm). The mobile phase A was 5% acetonitrile (ACN) in water, and the organic mobile phase B was 25% isopropanol and 5% water in ACN. The solvent was delivered to the column at a flow rate of 1 ml min −1 as follows: 0-1 min

The Proteomic Analyses
The peptides were separated using a Shimadzu LC-20AB

Statistical Analysis
Results were presented as means ± standard deviation (SD) and data were tested for normality and homogeneity of variance using the Shapiro-Wilk and Levene's tests, respectively. The data were evaluated by one-way ANOVA and further analyzed by Duncan's multiple range tests. The analyses were performed with SPSS 23.0 (IBM, Armonk, NY, USA). Values of p < 0.05 were considered significant.

Dietary BAs Improved the Growth Performance of Fish Fed an HD
The hybrid groupers were fed CD, HD, and BD (B1D, B2D, B3D, B4D, and B5D) for 8 weeks (unless if stated otherwise, all pairwise comparisons are HD vs. CD and BD vs. HD). In the HD group, the FCR and FI were significantly decreased, and the VSI and HSI were significantly increased (all p < 0.05, Figures 1A-D).
After supplementation with BAs to the HD, the final body weight (BW F ) and WGR in most BD groups (more than three groups) were increased, and the HSI values in all BD groups were decreased (Figures 1E,F). These parameters indicated that dietary BAs improved the fish growth performance. In addition, the WGR and SGR of the B3D group were the highest among the five BD groups, whereas the FCR, FI, and HSI were the lowest (Figures 1G,H). So, when the exogenous dietary BAs level was at 900 mg kg −1 , fish exhibited the best growth performance, with a higher growth rate compared to other levels.

High Dietary Lipids Impaired, While BAs Improved the Fish Lipid Deposition
Furthermore, HD consumption promoted lipid deposition, as revealed by a significantly higher crude lipid content of the whole body, muscle, and liver (all p < 0.05, Table 2). After the dietary supplementation of BAs, the crude lipid content of muscle and liver in most BD groups was decreased. In the BD group, the lowest content of crude lipid of the whole body, muscle, and liver was observed in the B1D, B4D, and B3D groups, respectively. These results implied that BAs intervention obviously reduced the lipid deposition induced by the HD in varying degrees and in different tissues. Lipid accumulation in hepatocytes was elevated in the HD group, as reflected in the larger number of red-stained hepatocytes (lipids stain red by oil-red O), and significantly higher IOD values (Figure 2). The lipid accumulation in all BD groups was significantly decreased, as reflected in the small number of red-stained hepatocytes and lower IOD values (all p < 0.05), especially in the B3D and B5D groups. Overall, these results indicated that HD supplementation with 900 mg kg −1 BAs decreased hepatic lipid accumulation.

High Dietary Lipids Impaired, While BAs Improved the Hepatic Lipid Metabolism
In the HD group, the content of T-CHO, TG, and NEFA in the liver were significantly increased, whereas the content of HDL in serum was significantly decreased (all p < 0.05, Figures 3A-D).
In most of the BD groups, the content of T-CHO, LDL, and TG in serum, as well as the content of T-CHO and NEFA in the liver, were decreased, whereas the content of HDL in serum was increased (Figures 3E-G). These results indicated that the HD diet impaired the lipid metabolism in liver, and these adverse effects were apparently reversed with the addition of BAs.

High Dietary Lipids and BAs Induced Changes of Fatty Acid Biosynthesis in the Proteomic Profile of Liver
Based on the growth performance and lipid deposition of fish, proteomic analyses were performed on liver samples of the CD, HD, and B3D groups to ensure the maximum difference in protein expression between samples. In the Label-free proteome analysis, about 3,331 proteins were unequivocally identified in liver samples (Figures 4A-C). The first two components of the PCA explained 34.2% of the total variance (20.1 and 14.1% for PC1and PC2, respectively), and this analysis clearly separated the three groups along the PC1 (Figure 5A). According to the  abundance of protein in three groups (Figure 4D), the proteins with fold change more than 1.5 and P-value below 0.05 (using the Benjamini-Hochberg multiple testing correction test) were considered to be significantly differentially expressed proteins (DEPs). We found that 59 of DEPs were upregulated and 93 downregulated in the HD compared to CD, whereas 52 were upregulated and 51 downregulated in B3D compared to the HD (Figures 4E-G, 5B). As proteases with similar expression patterns are usually functionally related, we used the heatmap with Euclidean distance to perform hierarchical clustering of the DEPs and samples simultaneously, according to the DEPs in HD group and/or B3D group. Cluster 1 consisted of 47 DEPs, both in the HD group and the B3D group ( Figure 5C). Notably, aconitate hydratase_A0A6I9PAY1 (involved in the pathway: tricarboxylic acid cycle, glyoxylate, and dicarboxylate metabolism) and gamma-enolase-like_A0A6I9PPR6 (glycolysis, gluconeogenesis, and methane metabolism) were downregulated in the HD group and upregulated in the B3D group. Also, flotillin_A0A484D865 (insulin signaling pathway) and protein phosphatase 1 regulatory subunit 1B_A0A4Z2J752 (cAMP signaling pathway) were upregulated in the HD group and downregulated in the B3D group.
By summarizing these pathways, we found that the most strongly influenced pathways between the CD, HD, and B3D groups were fatty acid biosynthesis, insulin signaling pathway, and AMPK signaling pathway (Figures 4H,I, 5F).

Dietary BAs Improved the Lipid Metabolism by Decreasing Lipogenesis and Increasing Lipolysis in the Liver
To further explore the lipid metabolism in response to different diets, we measured the activities of enzymes, expression of genes, and proteins associated with lipid metabolism in liver samples. For ELISA assay, in the HD group, the activities of lipolysis enzymes (LPS and ATGL) were significantly decreased, whereas the activities of lipogenesis enzymes (ACC and FAS) were significantly increased (all p < 0.05, Figures 6A-E). After BAs intervention, the activities of lipolysis enzymes (LPS, CPT1, and ATGL) were increased in most BD groups, and the activities of lipogenesis enzymes (ACC and FAS) were decreased in all BD groups (Figures 6A-E). For Western Blot analyses, in the HD group, the expression of SREBP1 and P-PPARA proteins was significantly increased, while the expression of TGR5 and PPARA was significantly decreased (all p < 0.05, Figures 6F-K). In the B3D group, the expression of FXR, TGR5, PPARA and P-PPARA was significantly increased (Supplementary Table S2).
For qPCR analyses, the expression of il1β, tnfα, cxcl8, lipogenesis genes (6pgd, acc, g6pd, and me) and their transcriptional factors (lxr, pparr, and srebp1) was significantly increased in the HD group (Table 3; Figures 7A-G), whereas the expression of il10, lipolysis genes (cpt1, dgat, hl, and hsl) and their transcriptional factors (ppara), as well as fatty acid uptake genes (fabp), was significantly decreased (all p < 0.05, Figures 7H-L, 8A). After the BAs intervention, expression of il1β, tnfα, cxcl8, lipogenesis genes (acc, fas, g6pd) and their transcriptional factors (pparr) was significantly decreased, whereas the expression of il10, lipolysis (atgl, cpt1, and dgat) and fatty acid uptake (acbp) genes was significantly increased in most BD groups (all p < 0.05, Figures 7A-L, 8A-E). In addition, the expression of BAs receptors tgr5 and fxr genes was significantly decreased in the HD group, and both increased in most BD groups (Figures 8F,G). From the perspective of genes, proteins and enzymes, these results overall indicated that HD impaired the lipid metabolism by increasing lipogenesis and decreasing lipolysis, and BD improved lipid metabolism by decreasing lipogenesis and increasing lipolysis.
FIGURE 6 | SREBP1, PPARA, and P-PPARA proteins normalized to the GAPDH level (n = 3). Values are presented as means with SD, where significant (p < 0.05) differences between groups are indicated by different letters. "*" indicates significant (p < 0.05) differences in the HD group (compared to the CD group), and "#" indicates significant (p < 0.05) differences in the B3D group (compared to the HD group).

DISCUSSION
Firstly, we explored the effects of HD and BAs-supplemented diets on the growth performance of hybrid grouper. Following a previous study (4), the high-lipid diet model of hybrid grouper was established using a diet containing more than 15% fat in this study (HD). Although the FCR and FI were significantly decreased in the HD group, growth rate indicators (BW F , WGR, and SGR) were not affected in hybrid grouper. In this species, one study showed that fish fed a diet containing 10% lipid had the highest WGR, whereas 13% of dietary lipids significantly decreased it (23); another study showed that increasing the dietary lipid level from 7 to 14% did not significantly affect the growth rate indicators (24). In combination with our study, these results indicate that a large number of variables (such as diet composition, duration of the experiment, fish genotype, etc.) influence the lipid tolerance of hybrid grouper. This has important implications for the interpretation of data and comparison of these types of studies. In our study, after administering gradients of BAs under the HD conditions, hybrid grouper first exhibited an increased and then decreased growth rate. The addition of a suitable level of BAs also promoted the growth in other fish species, but the optimal level varied among different fish species: 900 or 475 mg kg −1 in largemouth bass (9,10), 300 mg kg −1 in yellow croaker (11), 80 mg kg −1 in grass carp (12) and 150 mg kg −1 in tilapia (13). These studies also found that high levels of BAs supplementation had negative effects on the fish growth performance. Therefore, we conclude that hybrid grouper fed a high-fat diet responded very well to the 900 mg kg −1 BAs supplementation in terms of growth performance. Following this, we explored the effects of HD on lipid accumulation in hybrid grouper. The HD caused a significant increase in lipid deposition, especially in the liver, which is in agreement with previous studies in hybrid grouper (4), tiger puffer (5), yellow catfish (6), and mice (7). In this study, supplementation of BAs reduced the accumulation of lipids in the liver of hybrid grouper, resulting in better hepatic health, decreased crude lipid levels, and improved biochemical parameters. Correspondingly, supplementation of exogenous BAs also successfully reverted the effects of HD in humans (17), yellow croaker (11) and largemouth bass (10). In agreement with our study, high levels of BAs supplementation increased lipid accumulation in the liver of yellow croaker and largemouth bass (9,11). Our results showed that an HD diet supplemented with 900 mg kg −1 BAs produced the lowest hepatic lipid deposition.
We also explored the effects of HD and BD on the lipid metabolism of fish. In partial agreement with our results, the content of serum HDL was significantly decreased, while LDL was significantly increased, in response to a different hybrid grouper (Epinephelus fuscoguttatus♀ × E. polyphekadion♂) fed an HD (25). However, the BAs supplementation did not affect the content of HDL and LDL in the serum of tiger puffer (5) and largemouth bass (9), nor did it affect the TG and T-CHO content in the liver of largemouth bass (9). These discrepancies among studies could be caused by a number of different variables, such as doses (11,15) and kinds (9) of BAs, as well as the basal dietary content of lipids, carbohydrates and proteins (5,10,26). Further trials are needed to clarify this. Overall, our results indicated that the HD diet impaired the lipid metabolism in the liver, and these adverse effects were apparently reversed with the addition of BAs.
To obtain more precise data, we used the proteome approach to explore the key pathways behind the BAs and lipid metabolism in the liver. The protein profiles differed among the CD, HD and B3D groups, which suggested shifts in the dominant function of organism protein after dietary lipids or BAs supplementation. Previous studies found that supplementation of BAs activated the AMPK pathway in largemouth bass on a high starch diet (10), and improved the insulin sensitivity of mice on an HD diet (17). In line with these observations, in this study we found that among many key functional pathways that were changed, the most strongly influenced pathways were fatty acid biosynthesis, insulin signaling pathway, and the AMPK signaling pathway between three groups. FIGURE 7 | The relative expression of genes associated with lipid metabolism in the liver (part 1). 6pgd, 6-phosphogluconate dehydrogenase; acc, acetyl-CoA carboxylase; g6pd, glucose 6-phosphate dehydrogenase; me, malic enzyme; lxr, liver X receptor alpha; pparr, peroxisome proliferator activated receptor gamma; cpt1, carnitine palmitoyltransferase 1; dgat, acyl CoA diacylglycerol acyltransferase 2; hl, hepatic lipase; hsl, hormone-sensitive lipase. Data are normalized to 18s (18S ribosomal RNA) and β-actin as the reference genes and presented as a fold change in relation to the control group (CD, set as 1) (n = 6). Values are presented as means with SD, where significant (p < 0.05) differences between groups are indicated by different letters.
Moreover, we explored how the BAs supplementation regulated the lipid metabolism. SREBP1 is a transcription factor that regulates multiple genes involved in fatty acid and lipid synthesis, including acc and fas (21). The HD group exhibited increased expression of lipogenesis genes (6pgd, acc, g6pd, and me) and proteins (SREBP1), and activities of lipogenesis enzymes (ACC and FAS) compared to the CD group, while most of the BD groups exhibited an opposite trend. As BAs negatively regulate the gene expression of srebp1 in both fish and mammals (5,16,27), our results showed that supplementation of BAs inhibited the lipogenesis induced by HD. This further confirmed the indications of the proteome analysis, which suggested that lipogenesis was strongly affected. On the other hand, suppression of SREBP1 induces the expression of PPARA and its target genes to promote free fatty acid oxidation and lipolysis (28). In agreement with the downregulated/upregulated FIGURE 8 | The relative expression of genes associated with lipid metabolism in the liver (part 2). fabp, fatty acid-binding protein; fas, fatty acid synthase; atgl, adipose triglyceride lipase; acbp, acyl-CoA binding protein; dgka, diacylglycerol kinase alpha; tgr5, G protein-coupled bile acid receptor 1; fxr, farnesoid X receptor. Data are normalized to 18s and β-actin as the reference genes and presented as a fold change in relation to the CD (n = 6). Values are presented as means with SD, where significant (p < 0.05) differences between groups are indicated by different letters. mRNA expression levels of ppara in large yellow croaker fed high-fat/BAs treatment diets, respectively (11), our study showed that hybrid grouper fed an HD also exhibited reduced expression of lipolysis genes (cpt1, dgat, hl, and hsl) and activities of lipolysis enzymes (LPS and ATGL), while these pathways were enhanced in most BD groups. Comparable to other studies in fish (5, 10-12), we also found that dietary BAs inclusion improved the hepatic lipid metabolism significantly by enhancing the hepatic lipolysis, inhibiting lipogenesis, and regulating associated transcriptional factors.
In addition, BAs are known to regulate lipid and glucose homeostasis through activation of the FXR and TGR5 signaling pathways (29). High hepatic expression of FXR protected against hepatic steatosis and elevated TG through the induction of lipolytic target genes in mice (27,30), while TGR5 activated PPARA to increase mitochondrial oxidative phosphorylation and energy metabolism, as well as reduce obesity in humans (31). In the present study, the low expression of fxr gene, tgr5 gene and protein in the HD group, and high expression of FXR and TGR5 genes and proteins in the B3D group, might indicate that an HD diet impaired, but exogenous BAs activated, the FXR and TGR5 pathways, which altered the hepatic lipid metabolism. Notably, activation of FXR and TGR5 inhibited the level of proinflammatory cytokines and chemokines genes in this study and previous researches (16,32). As a decrease of these inflammatory markers is associated with the improvement of fatty liver disease in mice and humans (33), these findings suggested that activation of FXR and TGR5 signaling might be the key step toward the BAs-induced lipid-lowering outcome (in hybrid grouper). Further studies are needed to elucidate this mechanism.

CONCLUSION
In summary, the present study showed that high dietary lipids induced lipid accumulation and impaired lipid metabolism in the liver of hybrid grouper. Supplementation of BAs promoted growth performance and reduced lipid accumulation in fish. In addition, BAs treatment improved hepatic lipid metabolism by enhancing hepatic lipolysis, inhibiting lipogenesis, and regulating associated transcriptional factors. Meanwhile, the regulatory effects of dietary BAs on lipid metabolism might be achieved through the FXR and TGR5 signaling pathways. The optimal supplementation level of BAs to a high-fat diet is 900 mg kg −1 in hybrid grouper. The findings of present study would help to develop the new feed additives to improve lipid deposition in fish. In addition, these data may also contribute to the understanding of the specific mechanism via which exogenous BAs improve the lipid metabolism in animals. In the future, evaluation of side effects of supplementation of BAs and optimal dosage for animals will require more trials before it can become a routine addition. However, preliminary studies do show a promising efficacy of TCA in the treatment of obesity, and other metabolic disorders such as fatty liver disease.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ETHICS STATEMENT
The protocol was approved by the Animal Ethical and Welfare Committee of Guangdong Ocean University (Guangdong, China), processing ID: GDOU-AEWC-20180063.