Groupers are the important mariculture species in China, with a total output of 192,000 tons in 2020, an increase of 4.87% over 2019 (Food Organization, 2020; China Agriculture Press, 2021). Among them, the hybrid grouper, Epinephelus fuscoguttatus♂ × Epinephelus lanceolatus♀, also known as the pearl gentian grouper, is a popular farmed marine fish in Southeast Asia and China, and is intensively cultured along the coastlines of these areas (Xu et al., 2021). High-lipid diets (>13%) are generally used in this fish species, but it will lead to abnormal lipid metabolism and fat accumulation, which results in the susceptibility of fatty liver, then impair the health of liver, and ultimately damage to the growth of fish (Shapawi et al., 2018; Zou et al., 2019). Therefore, it is of great significance to study the mechanism of lipid deposition in fish, and it is necessary to find effective ways to improve the adverse effects induced by high-fat intake.

Bile acids (BAs) are endocrine molecules with a number of basic functions, such as cholesterol catabolism, intestinal fat emulsification, lipid metabolism, and intestinal microbiota homeostasis (Xie et al., 2021b; Xu et al., 2022a). In fact, BAs have broader biological effects, as they are recognized as regulators of farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5), and therefore, hold important promise for altering lipid content in the treatment of metabolic diseases (Wang et al., 2016; Kumari et al., 2020). Specifically, FXR is a ligand-activated transcription factor that belongs to the nuclear receptor superfamily and plays a vital role in regulating genes network related to the maintenance of lipid homeostasis (Kumari et al., 2020). Many in vitro and in vivo studies have used mouse models to clarify the positive association of FXR with fatty liver disease and its regulatory role on lipid metabolism (Yang et al., 2010; Kumari et al., 2020). In addition, TGR5 is coupled to a stimulatory G-protein and is responsive to various BAs, such as taurolithocholic acid, lithocholic acid, deoxycholic acid, chenodeoxycholic acid, and cholic acid (Reich et al., 2016). In humans, the activation of TGR5 shows promise for the treatment of various metabolic diseases such as type 2 diabetes and obesity (Guo et al., 2016). In mice, TGR5 can prevent oxidative stress and lipid accumulation in kidney (Wang et al., 2016), and the development of steatosis in liver (Pols et al., 2011). Several studies explored the effects of dietary supplementation of BAs on fat metabolism in fish, such as largemouth bass (Micropterus salmoides) (Yu et al., 2019; Yin et al., 2021), tiger puffer (Takifugu rubripes) (Liao et al., 2020), Nile tilapia (Oreochromis niloticus) (Jiang et al., 2018; El-Shenawy et al., 2020), large yellow croaker (Larimichthys crocea) (Ding et al., 2020), grass carp (Ctenopharyngodon idella) (Zhou et al., 2018), and turbot (Scophthalmus maximus) (Huang et al., 2015); however, it is unclear whether the underlying mechanism is through the FXR and TGR5 signaling pathways.

Compared to in vivo experiments, in vitro experiments can be used as auxiliary models for whole-animal assays in vivo to assess the thorough effects of exogenous substances (Zhu et al., 2014). As an excellent in vitro platform, fish cell lines have been widely used in virology, immunology, toxicology, and nutritional analysis (Collet et al., 2018; Li et al., 2022). The liver is a critical organ in metabolic processes and plays a key role in the metabolism of nutrients (Zou et al., 2019). Therefore, fish liver cell lines from various species are commonly used for studying lipid metabolism, such as grass carp (Lu et al., 2012), pearl gentian grouper (Zou et al., 2018, 2019), yellow catfish (Pelteobagrus fulvidraco) (Chen et al., 2014; Song et al., 2015b), large yellow croaker (Lai et al., 2021), and zebrafish (Danio rerio) (Wang et al., 2019). However, so far, no research has focused on the functions of BAs in fish cells and the mechanism behind.

Therefore, the purpose of this study was to evaluate the role of BAs in the regulation of lipid deposition in primary hepatocytes from pearl gentian grouper. First, we explored the effects of BAs on lipid metabolism in a fatty-degenerated hepatocytes model. Then, we separately studied the effects of FXR and TGR5 signaling pathways on lipid metabolism in vitro. This study was firstly investigated the roles of BAs in the fat metabolism of hepatocytes in fish. The findings of the present study would be helpful in developing the new feed additives to improve the lipid deposition in fish.

Firstly, we established the model of fatty-degenerated hepatocytes in pearl gentian grouper. In grass carp, the hepatocytes of fatty degeneration were induced with media containing LE (Lu et al., 2012), which showed a significant accumulation of intracellular TG. Similarly, after exposing to LE in hybrid grouper liver cells, lipid droplets in Oil Red O staining and TG content were increased significantly (Zou et al., 2019). These observations were consistent with our results, that is, BODIPY 493/503 staining showed a large number of lipid droplets accumulated in hepatocytes in V-HL group, which can be proved by the increase of intracellular TG content. Generally, fat accumulation is caused by the imbalance of fatty acids synthesis and fat catabolism, and many key enzymes are involved in these processes (Song et al., 2015a). As providing NADPH essential compounds for fatty acids synthesis in the pentose phosphate pathway, G6PD and 6PGD act important roles in lipogenesis (Song et al., 2015b). As catalyzing the carboxylation of acetyl-CoA to malonyl-CoA, ACC act a rate-limiting enzyme for the synthesis of long-chain fatty acids (Zhu et al., 2014). In this study, the V-HL treatment up-regulated the mRNA level of these genes, as well as fas and me, indicating that the lipogenesis was enhanced in a fatty-degenerated hepatocytes model. Our results are similar to previous study in which LE induced the expression of adipogenesis genes (g6pd, me, and fas) in pearl gentian grouper hepatocytes (Zou et al., 2019). As catalyzing the conversion of fatty acids-CoAs into fatty acids-carnitine, CPT1 is regarded as a main regulatory enzyme in fatty acid oxidation (Xie et al., 2021a; Xu et al., 2022b). The reduced the mRNA level of cpt1 in V-HL group, as well as atgl, dgat, dgka, and hl, suggesting that lipolysis was suppressed in fatty-degenerated hepatocytes model. Similarly, LE co-incubation decreased the expression of lipolytic genes (atgl and lpl) in another reports in pearl gentian grouper hepatocytes (Zou et al., 2019). Furthermore, ppara is a ligand-dependent transcriptional activation of genes related to fatty acids oxidation pathway (Ribet et al., 2010; Zhu et al., 2014). While pparr and srebp1 are the major regulators of fatty acids/lipids biosynthesis genes, both of which promote lipid storage (Minghetti et al., 2011; Zhu et al., 2014). Same LE treatment decreased the expression of ppara (Zou et al., 2019), in the present study, the V-HL treatment significantly enhanced the expression of SREBP1 gene and protein, and repressed the expression of PPARA and P-PPARA proteins. Overall, compared to normal cell, fatty-degenerated hepatocytes increased lipid accumulation, and impaired lipid metabolism by enhancing lipogenesis, repressing lipolysis, and regulating transcriptional factors in pearl gentian grouper.

Then, we explored the effects of BAs on lipid deposition in a model of fatty-degenerated hepatocytes. Unlike 200 μM TCA did not affect the viability of hepatocytes in mice (Allen et al., 2011), the CCK-8 assay showed the TCA concentrations below 100 μM had no significant influence on the viability of hepatocytes in this study. This discrepancy might be due to the different tolerance of animals to BAs (Jia et al., 2018; Kumari et al., 2020). In vivo, exogenous BAs could decrease fat deposition caused by a high-lipid diet and improve the hepatic lipid metabolism in large yellow croaker and largemouth bass (Ding et al., 2020; Yin et al., 2021). Similarly, the V-TCA treatment reduced the lipid deposition in fatty degeneration liver cells. Furthermore, in present study, the increased expression of lipolysis genes, and decreased lipogenesis genes, and associated transcriptional factors, suggesting that TCA co-incubation could improve lipid metabolism by repressing lipogenesis and enhancing lipolysis. Notably, the low expression of fxr and tgr5 genes in the V-HL group, but high expression of both genes and proteins in the V-TCA group, might indicate that the fatty-degenerated hepatocytes inhibited, but TCA co-incubation activated, the FXR and TGR5 signaling pathways, thus altering the lipid metabolism and lipid accumulation in vitro.

Next, we explored whether the effects of BAs on lipid deposition were dependent on the activation of FXR signaling. The INT-747 (10 μM) is a classical FXR agonist that can significantly increase the luciferase activity of FXR reporter gene in HepG2 cells (Gao et al., 2018). Similarly, we also found the INT-747 (5 μM) co-incubation up-regulated the expression of FXR gene and protein in hybrid grouper hepatocytes, accompanied by a reduction in lipid accumulation. Our results are consistent with other studies in which INT-747 (1 μM) treatment significantly reduced TG level in mouse liver (AML12 cells) and human liver (Fa2N-4 cells) (Miyazaki et al., 2018). In human intestinal cells, the activation of FXR inhibited the expression of lxr and srebp1 genes, thereby decreasing the expression of fas gene and lipid synthesis via the SHP pathway (Watanabe et al., 2004; Yang et al., 2010). In addition, the activation of FXR stimulated the fatty acid β-oxidation by inducing the expression of PPARA (Alfaro-Viquez et al., 2018; Kumari et al., 2020). In accordance with this, we also found that the activation of FXR promoted the lipolysis and inhibited the lipogenesis. Same as the present study, the guggulsterone (20 μM) treatment significantly increased the content of TG in primary hepatocytes from yellow catfish (Wu et al., 2020). Thus, antagonizing FXR by guggulsterone, eliminated the lipid-lowering effects of TCA, suggesting that the activation of FXR might be the key step toward TCA-induced lipid-lowering outcome.

Besides, we explored whether the effects of BAs on lipid deposition was dependent on the activation of TGR5 signaling. The INT-777 had been discovered as a specific agonist for TGR5 (Guo et al., 2016). As with the human podocytes (Wang et al., 2016), the results of CCK-8 assay also showed 10 μM INT-777 had no significant effects on the viability of hepatocytes in this study. Since INT-777 treatment enhanced the expression of TGR5 and decreased the level of plasma TG in mice (Bianco et al., 1988; Wang et al., 2016), we also found the activation of TGR5 by INT-777 reduced the lipid deposition in hybrid grouper hepatocytes. In human podocytes cultured with high glucose, INT-777 co-incubation suppressed mRNA level of srebp1, induced the mRNA level of ppara and cpt1, leading to an increase in fatty acid β-oxidation (Wang et al., 2016). Partially agreeing with these, we observed that INT-777 treatment increased the expression P-PPARA protein and cpt1 gene in this study. At the same time, the inhibition of TGR5 by SBI-115 promoted lipid accumulation and impaired lipid metabolism of hybrid grouper hepatocytes. Our results were consistent with the results of another in vivo experiment, the ppara and cpt1 mRNA level were decreased in TGR5^–/– mice (Wakil and Abu-Elheiga, 2009). Since TCA could active TGR5 in various cells (HEK293, Kupffer, and Sinusoidal endothelial cells) (Keitel et al., 2007, 2008), and TGR5 negatively regulated lipid deposition, we inferred that the activation of TGR5 might be a key step toward TCA-induced lipid-lowering outcome.

In pearl gentian grouper, we revealed: (1) a model of fatty degenerated hepatocytes induced by LE is characterized by increasing lipid accumulation and impairing lipid metabolism; (2) BAs co-incubation could improve lipid deposition and metabolism in fatty degenerated hepatocytes model; (3) the inhibition of FXR or TGR5 promoted lipogenesis and inhibited lipolysis in vitro; (4) the activation of FXR and/or TGR5 might be the key step toward the lipid-lowering effects of BAs. Understanding the functions of BAs may help to develop of management strategies to improve lipid metabolism and deposition in the liver of hybrid grouper.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by Animal Ethical and Welfare Committee of Guangdong Ocean University. The permit number for conducting animal experiments is GDOU-AEWC-20180063.

JX: conceptualization, methodology, formal analysis, investigation, data curation, writing – original draft, and writing – review and editing. XY: data curation, investigation, and writing – original draft. XL: investigation, data curation, and visualization. SX: conceptualization, resources, and visualization. SC: methodology and conceptualization. SZ: conceptualization and funding acquisition. JC: methodology, conceptualization, and funding acquisition. BT: methodology, conceptualization, writing – review and editing, and funding acquisition. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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