Taurine (2-aminoethanesulfonic acid), as a free amino acid, does not participate in protein synthesis but is known to play a wide range of key roles in animal physiology, such as immune regulation, osmoregulation, antioxidation, nervous system development and regeneration, and bile acid (BA) conjugation (Huxtable, 1992; Salze and Davis, 2015; Wu, 2020; Xu et al., 2020). BAs are regarded as biosurfactants, which emulsify lipid into micelles to facilitate its digestion and absorption by allowing for more cleavage sites for lipase (Suga et al., 2019; Romano et al., 2020). However, the emulsification of long-chain triglycerides (TGs) is achieved by conjugated BAs, which are better emulsifiers than BAs due to their hydrophilic and lipophilic properties (Gupta and Kim, 2003). It was reported that most primary BAs are linked with taurine or glycine to form conjugated BAs (Chiang and Ferrell, 2020), and taurocholic acid (TCA) or taurodeoxycholic acid (TDCA) formed by taurine conjugating with BAs is predominant in BAs for most teleost fish except common carp (Cyprinus carpio) (Kim et al., 2007, 2015). Previous studies demonstrated that dietary taurine addition increased taurine-conjugated BAs contents in rockfish (Sebastes schlegeli) and tiger puffer (Takifugu rubripes) (Kim et al., 2015; Xu et al., 2020). Thus, taurine may regulate lipid metabolism through accelerating BAs to form taurine-conjugated BAs.

It is clear that dietary taurine supplementation can reduce peripheral cholesterol and visceral fat contents of diabetic rats (Tsuboyama-Kasaoka et al., 2006) and obese humans (Brons et al., 2004; Zhang et al., 2004). The lipid-lowering effect of taurine was also observed in several farmed fish species. For example, dietary taurine addition resulted in a reduction of whole-body lipid content in European seabass (Dicentrarchus labrax) (Martins et al., 2021), serum total cholesterol (TC), and TG contents in yellow catfish (Pelteobagrus fulvidraco) (Li et al., 2016), and liver lipid contents in California yellowtails (Seriola dorsalis) (Garcia-Organista et al., 2019) and rice field eels (Monopterus albus) (Hu et al., 2018). Of particular concern are the liver lipid deposition and even fatty liver syndrome caused by the widespread use of high-lipid feed in intensive aquaculture for protein-sparing and growth-promoting (Du et al., 2005; Li et al., 2012). Moreover, the excessive dietary lipid can also cause poor appetite, growth retardation (Dai et al., 2018, 2019; Nath et al., 2019). It was well-reported that taurine could act as an attractant to increase the feed intake (FI) of fish (Matsunari et al., 2008; Takagi et al., 2008; Qi et al., 2012). In view of the aforementioned lipid-lowering effect of taurine, dietary taurine supplementation may therefore be an effective way to eliminate the negative effects caused by high-lipid feeding. However, it is unclear whether the lipid-lowering effect of taurine is involved in taurine-mediated BA metabolism.

The orange-spotted grouper, Epinephelus coioides, is a commercially important farmed carnivorous fish species in Southeast Asian countries, including China (Shapawi et al., 2019; Feng et al., 2020). The optimal lipid level in feed was reported to be 10% for juvenile E. coioides, and the fish showed a downward growth trend, accompanied by an increasing trend of liver lipid accumulation when the dietary lipid level was increased from 12 to 14% (Luo et al., 2005). However, the visible fatty liver symptom usually occurs in different grouper species such as E. akaara (Ma, 1996), E. awoara (Zhou et al., 1995), E. malabaricus (Tuan and Williams, 2007), and E. bruneus (Yoshii et al., 2010) when they were fed with high-lipid diets, though varied lipid requirements for the fish species. Our latest research results showed that 1% dietary taurine was optimal for the growth of E. coioides and the dietary taurine supplementation can reduce lipid accumulation by decreasing the contents of TGs containing 18:2n-6 at the sn-2 and sn-3 positions at 15% lipid (Bai et al., 2021a,b). In this study, three diets containing 10% lipid, 15% lipid, and 15% lipid with 1% taurine were formulated to investigate how a high lipid diet does affect liver lipid deposition and to explore the lipid-lowering effect of dietary taurine supplementation concerning taurine-mediated changes of conjugated BAs.

Many studies have shown that dietary taurine has a positive effect on the growth performance of fish (de Moura et al., 2018; Liu et al., 2018; Candebat et al., 2020). In this study, compared to groupers fed taurine free diet (15L), fish fed diet containing 1% taurine (diet 15L + T) showed a significantly higher growth rate, as observed in juvenile Cobia (Rachycentron canadum), European sea bass (Dicentrarchus labrax), and parrot fish (Oplegnathus fasciatus) (Lunger et al., 2007; Lim et al., 2013; Martins et al., 2018). Results in yellowtail (Seriola quinqueradiata) and red sea bream (Pagrus major) showed that dietary taurine addition improved the FI (Matsunari et al., 2005; Matsunari et al., 2008). Although FI was not affected by dietary taurine levels at 15% lipid in this study, the trend of FE in response to dietary taurine addition was similar to WG. This finding indicated that taurine promotes the growth rate of grouper via improving feed utilization. In addition, the growth rate and feed utilization were not affected by dietary lipid levels in the absence of taurine. These results disagreed with those that occurred in meager (Argyrosomus regius) and silver barb (Puntius gonionotous), i.e., high lipid diet led to a reduced growth rate (Chatzifotis et al., 2010; Nayak et al., 2018). The contradictory result may be due to different fish species and rearing environments, etc.

Previous studies showed that liver lipid content in Nile tilapia (Oreochromis niloticus) increased with increasing dietary lipid levels (He et al., 2015; Zhou et al., 2019), which was consistent with our current study results. We observed no variations in HSI and plasma TG, HDL-C, and LDL-C contents between groupers fed diet 10L and diet 15L. Plasma components could reflect the absorption, transportation, utilization, and deposition of lipids (Xu et al., 2016). Thus, maybe the grouper has a relatively high tolerance to dietary high lipid under the conditions of this experiment, which supported our result of growth performance. However, decreased HSI and liver lipid content were observed in 15% lipid feed with 1% taurine addition in comparison with 15% lipid feed without taurine addition. This finding supported previous observations on turbot (Scophthalmus maximus), Nile tilapia, and yellowfin seabream (Acanthopagrus latus) (Yun et al., 2012; Michelato et al., 2018; Dehghani et al., 2020), indicating a lipid-lowering effect of taurine in grouper. Furthermore, we also determined plasma hormones contents to find clues to the change of liver lipid content in the presence of taurine in high lipid feeds. Both ADPN and LEP are hormones secreted by adipose tissue and released into the bloodstream. LEP can reduce lipid accumulation by directly inhibiting lipid synthesis in adipocytes (de Gortari et al., 2020). ADPN can enhance the decomposition and energy consumption of fatty acids, and reduce the TG contents in the muscle and liver of large yellow croaker (Larimichthys crocea) (Ji et al., 2020). However, it was also reported that ADPN acts as the feedback signaling regulator of energy expenditure and metabolism (O’Brien et al., 2018). In the current study, groupers fed diet 15L + T showed lower plasma ADPN content than those fed diet 15L, indicating a decrease in liver lipid deposition in groupers. Meanwhile, consistent with our result, the increased plasma LEP content of mice was found to be accompanied by HSI, as a result of the high-lipid FI (Zhao et al., 2020). Dietary taurine reduced the secretion of LEP in rats and plasma LEP content in M. albus (Kim et al., 2012; Li, 2016), which was not observed in this study. However, the associations of dietary taurine with hormone secret and transport were not well-clarified so far, which needs further study.

Cholesterol is an important component of blood lipids. In this study, plasma TC content was upregulated by a 15% lipid diet without taurine addition but downregulated by a 15% lipid diet with taurine addition, as evidenced with increased liver lipid content in this study and previous studies in tiger puffer and European sea bass (Xu et al., 2020; Martins et al., 2021). Thus, taurine could reduce the risk of atherosclerosis by removing cholesterol (Gupta and Kim, 2003). We also observed an upregulation of liver HMGR content of groupers fed a 15% lipid diet with taurine in comparison with a 15% lipid diet without taurine. It was demonstrated that HMGR is the key rate-limiting enzyme that catalyzes the synthesis of a precursor of cholesterol in the regulation of cholesterol synthesis (Mahboobi et al., 2018). Our results may indicate the promotion of cholesterol biosynthesis by taurine via HMGR (Xu et al., 2020). The liver lipid content was found to have a positive association with the plasma content of TC, HMGR, LEP, and ADPN (Figure 6), indicating lipid accumulation in the liver was the result of the combined action of enzymes, hormones, and plasma lipids (Zhao et al., 2020). Furthermore, cholesterol is an integral component of cell membranes and acts as precursors of some hormones and BAs (Abramo, 1989; Suga et al., 2019), which combines with taurine to form taurine-conjugated BAs, the major BAs in most teleost fish (Kim et al., 2015). Thus, The BA profile of groupers was determined in this study.

Bile acids (BAs), amphiphilic (hydrophilic and lipophilic) molecules, are steroids biosynthesized from cholesterol in the liver and stored in the gallbladder, and released into the intestinal lumen after food ingestion (Nguyen et al., 2018; Suga et al., 2019). BAs can effectively emulsify lipids to form small chylous particles, which increase the contact surface between lipid molecules and lipase to accelerate the hydrolysis of lipid molecules in vivo (Ding et al., 2020). The BAs synthesis and its composition vary among animal species (Chiang and Ferrell, 2020). For example, 25 and 75% of primary BAs were conjugated with taurine and glycine in humans, respectively; while 95 and 5% of primary BAs are taurine-conjugated and glycine-conjugated in mice, respectively (Hofmann et al., 2010). However, it was previously believed that almost all BAs secreted by finfish were taurine-conjugated BAs (Kim et al., 2007; Kim et al., 2015). Here, a total of 29 BAs molecules in the bile of the grouper were detected and identified. The majority of them were taurine-conjugated BAs, but a small part of BAs are glycine-conjugated which was consistent with previous studies in other fish species (Garcia-Organista et al., 2019; Xu et al., 2020). It was reported that taurine-conjugated BAs have a high ability to remove cholesterol by enhancing serum HDL and suppressing LDL and very low-density lipoprotein concentration (Gupta et al., 2009). In this study, the three taurine-conjugated BAs such as TCA, TCDCA, and TDCA were the main components of BAs, and the contents of TDCA, GDCA, GLCA, and GUDCA were downregulated by the dietary lipid increasing from 10 to 15%, indicating an inhibiting of cholesterol metabolism, which agreed with plasma TC content discussed before. Interestingly, we also observed downregulation of GDCA, GCA, and GCDCA contents by dietary taurine addition at 15% lipid, which may indicate the higher competition of taurine combining with BAs than glycine in grouper and mice (Hofmann et al., 2010). In a study with Swiss female mice, CA promotes cholesterol absorption, but CDCA does not (Reynier et al., 1981). In our current study, CA content was not affected by dietary lipid level, but CDCA content was promoted by high lipid feed. Similar results were also observed in a recent study (Lu et al., 2018). The findings indicated that cholesterol absorption was limited. Moreover, CA and CDCA are efficacious endogenous ligands, whereas UDCA, MCA, and TMCA are antagonists of farnesoid X receptor (FXR) (Sayin et al., 2013; Su et al., 2019), which could inhibit TG synthesis by inducing small heterodimer partner in mice and grass carp (Ctenopharyngodon idella) (Li et al., 2013; Tian et al., 2021). In this study, β-MCA, γ- and ω-MCA contents were lower in groupers fed diet 15L than that in those fed diet 10L. Moreover, UDCA, β-MCA, and TMCA contents continued to decline when taurine content was increased from 0 to 1% in a 15% lipid diet. This means the activation of FXR via reducing their antagonists could be triggered by taurine too. Notably, compared to taurine free diet at a 15% lipid level, taurine supplementation reduced the contents of THDCA and TUDCA. A previous study in humans demonstrated that the ratio of taurine to glycine-conjugated BAs is about 3:1 and this could be reduced by low taurine supply (Haslewood, 1978). Our results suggested that taurine addition at a 15% lipid diet could promote the taurine-conjugated BAs forming and play more important roles in lipid-lowering effects. Study results showed that most BAs through re-absorption in the intestinal lumen was routed to the liver where they were recycled to the gallbladder (Dawson, 2018). However, the re-absorption of BAs in the intestinal lumen was inhibited by dietary taurine in rats (Chen et al., 2003; Lam et al., 2006; Nishimura et al., 2009). This finding further supported the reduction of BAs caused by dietary taurine addition in our current study. Thus, taking liver lipid content and BAs profile into consideration (Figure 6), we speculate that taurine-conjugated BAs have a stronger ability to emulsify and absorb lipids than glycine-conjugated and other BAs in the fish species, which might be one of the reasons for the growth improvement. Interestingly, DCA and LCA, are hydrophobic cholic acids that have strong toxicity to colonic cells, they induce cell death involving either death receptor or mitochondrial (Rodrigues et al., 2004; Nguyen et al., 2018). The DCA and LCA contents were higher in groupers fed diet 15L than that in those fed diet 10L in this study and were found a positive correlation with liver lipid content (Figure 6), indicating the negative effect of high dietary lipid intake on the lipid metabolism of fish.

The mRNA expression levels of several genes related to both lipid metabolism and lipid accumulation to elucidate the molecular mechanisms underlying the taurine-mediated lipid-lowering effect on groupers. Acyl-CoA carboxylase is a cytosolic enzyme-producing malonyl-CoA, the first step in the biosynthesis of long-chain fatty acids (Qian et al., 2015). G6PD is a key enzyme related to the production of NADPH, and essential for de novo fatty acid biosynthesis catalyzed by FAS (Chen et al., 2013; Zheng et al., 2013). PPARα can modulate the expression of genes encoding several mitochondrial fatty acid-catabolizing enzymes in addition to mediating inducible mitochondrial and peroxisomal fatty acid β-oxidation (Aoyama et al., 1998). In this study, diet 15L upregulated the expression of the pparα gene but downregulated the expression of acc, fas, and 6pgd genes compared with diet 10L. The findings were consistent with previous results in blunt snout bream (Megalobrama amblycephala), hybrid grouper (Epinephelus lanceolatus♀ × Epinephelus fuscoguttatus♂), and large yellow croaker (Yan et al., 2015; Cao et al., 2019; Tan et al., 2019). Moreover, the expression levels of fas and 6pgd genes were decreased, and the expression level of the pparα gene was promoted with the dietary taurine increase. This indicated that liver lipid deposition reduction caused by dietary taurine addition may be the result of lipogenesis inhibition and fatty acid β-oxidation acceleration.

Under the present experimental conditions, the growth performance of the grouper was not affected by dietary lipid level increased from 10 to 15%, but significantly increased by dietary taurine addition at 15% lipid. High lipid diet increased the liver lipid content and decreased the contents of glycine-conjugated BAs and TDCA. Dietary taurine supplementation in a 15% lipid diet could decrease the lipid deposition in the liver of groupers through forming more taurine-conjugated BAs and triggering the activation of FXR, and inhibiting the expression of genes involved in lipid anabolism and accelerating fatty acid β-oxidation (Figure 6). Taurine-conjugated BAs have a higher ability to accelerate lipids emulsification and absorption than glycine-conjugated and other BAs in the fish species.

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.

The animal study was reviewed and approved by Animal Care and Use Committee of Fujian Agriculture and Forestry University.

XW: conceptualization, data curation, formal analysis, investigation, methodology, project administration, validation, visualization, and writing-original draft. FB: data curation, formal analysis, investigation, validation, and methodology. XN: investigation and data curation. YS: supervision and writing-review. JY: conceptualization, formal analysis, funding acquisition, resources, supervision, and writing-review. All authors contributed to the article and approved the submitted version.

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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