BBR has been used in traditional Chinese medicine and Ayurvedic medicine and current research evidences support its use for various therapeutic areas (Singh and Mahajan, 2013). One of its remarkable function is reduction of lipid accumulation, this is conservative from fish to mammals (Hao et al., 2017; Liang and Wang, 2018; Wang et al., 2021; Yang et al., 2019a; Yang et al., 2019b; Zhou et al., 2011). Interestingly, although it has direct role in reducing lipid deposition in hepatocytes or adipocytes (Yang et al., 2019a; Yang et al., 2019b; Zhou et al., 2011), BBR seldom enter into the circulatory system and mainly accumulates in the intestine in vivo (Pan et al., 2019; Sun et al., 2017). Thus, the mechanism of dietary BBR on the lipid metabolism still remain elusive. In the present study, we explored the role of FXR, a modulator of lipid metabolism molecular in the intestine, in this process of grass carp through pharmacological methods. We show that dietary BBR activated the FXR signaling pathway, and inhibition of FXR abolished the fat suppression function, as well as some lipogenic genes induced by dietary BBR in grass carp.
During the feeding experiment, all fish accepted the diets well, and no dead fish was recorded, suggesting that dietary BBR and FXR inhibitor had no negative impact on the experimental fish. Dietary BBR had no impact on the growth of grass carp, which is consistent with previous studies in grass carp (Pan et al., 2019) and black sea bream (Wang et al., 2021). The decreased VI, HI, and IPFI in fish fed BBR are in line with the lipid content of the whole fish, reflecting BBR decreased lipid accumulation in a macroscopic view. On the contrary, dietary BBR + Gly-β-MCA increased the growth, VI, HI, and IPFI in fish compared to the BBR group, which can be explained by the fat accumulation in the vicera. However, the compensatory growth of the fish (such as intestine, hepatopancreas, IPF etc.) in respones to Gly-β-MCA to meet the FXR signalling requirements in tissues cannot be excluded.
The activities of serum ALT and AST are generally important indicators of the liver function, they also reflect the health of other tissues, such as spleen, kidney, etc. (Tian et al., 2014). Our study showed that the activity of serum ALT was decreased in the BBR feeding fish, whereas increased in the BBR + Gly-β-MCA feeding fish. The serum AST also showed similar trends. The changes of these two enyzmes are consistent with the VI, HI, and IPFI, suggesting that dietary BBR may be beneficial for the function of tissues in the viscera of grass carp. However, administration with FXR inhibitor may do harm to the health of the cells in the fish. This is possibly due to the lipid droplets accumualtion in cells that disordered the cellular homeostasis (Hyun et al., 2005). Serum TG and TC are indictaors for the body lipid content both in mammals and fish (De Giorgio et al., 1982; Tian et al., 2019). Our study proved the serum TG and TC were all decreased in fish consuming BBR, which is in line with early study (Pan et al., 2019; Wang et al., 2021). We further proved that administration with Gly-β-MCA rescued the suppresion of these parameters, suggesting that inhibition of FXR could recover the serum fat homeostasis induced by dietary BBR.
By staining with H&E and oil red O, the lipid droplets in the hepatocytes were decreased the accumulation by the dietary BBR (marked in vacuole and red particles, respectively). Because lipid droplets are composed by TG and sterol esters (Lundquist and Susanto, 2020), our quantitative data also showed significantly decreased TG and TC content in BBR feeding fish, suggesting the component of the lipid droplets were also reduced. Our results are in line with previous studies in grass carp (Yang et al., 2019a), black sea bream (Wang et al., 2020), and blunt snout bream (Zhou et al., 2019). As a constrast, BBR + Gly-β-MCA increased the lipid content in the heaptopancreas, suggesting that FXR signalling play a negative role in fat deposition. Our previous study showed that solely treatment with Gly-β-MCA also increased the lipid content in grass carp (Tian et al., 2021), combined with this study, we could conclude that inhibition of FXR could eliminate the fat depression function of dietary BBR. However, it is still not confirmed BBR decreased the lipid accumulation via FXR signalling. We then explored the molecular events in the FXR signalling. In the intestine, activated FXR stimulates the expression of FGF19 hormone, which binds to the liver FGFR4/β-Klotho co-receptor complex to inhibit the rate-limiting enzyme of the bile acid synthesis CYP7A1 after transportation in the portal circulation (Zheng et al., 2017). Intriguingly, dietary BBR increased the mRNA (and protein) expression of FXR and decreased its downstream gene FGF19 in the intestine, as well as decreased the gene (or protein) expression of CYP7A1, CYP8B1, and CYP27A1 in the hepatopancreas, suggesting that BBR did activate the signalling of FXR. Expectedly, the decreased molecular exrpression of FXR combined with the up-regualtion of the downstream of the FXR genes (or protein) by Gly-β-MCA treatment implied that pharmacological treatment succeeded in abolishment in the FXR signaling induced by BBR. These data also indirectly indicate that FXR signalling participated in the fat suppression of dietary BBR in grass carp.
Previous studies have shown that BBR suppresses lipid accumulaiton via inhibiting lipogenesis and promoting lipid oxidative in fish (He et al., 2021; Wang et al., 2021). In the present study, we show that dietary BBR decreased the relative mRNA expreesion of lipogenic genes, SREBP-1c and FAS. Moreover, we also did find the difference of lipid catabolism genes, PPARα, ATGL and CPT-1. These results are consistent with early studies. Importantly, our results provide evidences that inhibition of FXR abolished the down-regulation of lipid anabolism genes, as well as the up-regualtion of lipid catabolism genes induced by dietary BBR, suggesting FXR signalling plays an important role in the modulation of lipid metabolism related transcripts induced by dietary BBR, similar to our early study (Tian et al., 2021).
The results that dietary BBR decreased the total OTUs, ACE index, and Chao 1 index suggest that BBR decreased the richness of gut microbiota in grass carp. This might be due to the antibacterial property of BBR (Zhang et al., 2020). Interestingly, dietary BBR also decreased the simpson index, which implied increased diversity of the gut microbiota in BBR treated fish, in line with early studies (Pan et al., 2019). The total decreased richness but increasd diversity may be due to increased community evenness for the compostion of gut microbita. Furthermore, we also showed that dietary Gly-β-MCA had no obvious differnce of the alpha-diversity of the gut microbiota, indicating that the drug used in this study did not influence the gut microbiota composition. In mammals, the richness and diveristy of gut microbiota are regarded to be a key factor in impacting the body fat accumulation and metabolic disease(Clemente et al., 2012; Turnbaugh et al., 2006). Obesity people have more diversity of the gut microbiota than the thin ones (Turnbaugh et al., 2009). Similarly, the changes of the gut microbiota in fish are also linked to the changes in fat content (Sheng et al., 2018; Tian et al., 2021). It is generally accepted that gut microbiota decogugate and further metabolise primary BAs into secondary BAs through bile salt hydrolase (BSH)(Wahlström et al., 2017). BAs differ widely in their ability to activate FXR (de Boer et al., 2020), the changes in the BAs composition altered by the may altered the signalling of FXR. From this view, BBR may influence the FXR via modulating gut microbiota, which altered the composition of BAs and activated FXR signalling, further decreased the lipid accumulation in grass carp.
It is suggested that obesity mice or hunman have higher content of Firmicutes and lower Bacteroidetes (Kallus and Brandt, 2012). This is possibly due to the differences of these two bacteria in the contents for the enzymes related to lipid/carbonhydrate metabolism (Stephens et al., 2018). In our study, though have no obvious difference of the gut microbiota composition in the phyla level. The concentration of Firmicutes had a decreasd trend in the BBR treatment, whereas had an increased trend in the BBR + Gly-β-MCA treatment, which is in line with the fat content of grass carp. In addition, several other bacteria have similar trends with lipid content, such as acidobacteria and gemmatimonadetes, as well as the genus Phreatobacter, but the relationship between these bacteria and lipid metabolism are scarcely studied.
In conclusion, in this study, we explored the mechanism of dietary BBR on the lipid metabolism by using the FXR inhibitor Gly-β-MCA in grass carp. Dietary BBR modulated gut microbiota composition, activated FXR signalling, and suppressed lipid accumulation. Meanwhile, inhibiton FXR could recover the suppression the fat deposition induced by BBR. It is speculated that FXR signalling play important role in the function of BBR in modulating lipid accumulaiton in grass carp. More studies in related to the gut microbita and BAs composition that altered by BBR are needed to be further addressed.