Carbohydrates are widely used in aquafeeds due to their ability to enhance the binding between feed ingredients, thereby improving their stability in water, and they are one of the cheapest ingredients in compound feeds(Reference Sørensen, Nguyen and Storebakken1,Reference Romano and Kumar2) . According to Rokey et al.(Reference Rokey, Plattner and Souza3), current commercial sinking aquafeeds require a minimum of 10 % carbohydrate, with the requirement for floating aquafeeds being even higher. However, carnivorous fish are generally glucose intolerant, and the recommended carbohydrate level in their diets should not exceed 10 %(Reference Stone4–Reference Li, Li and Zhang7). In practice, the carbohydrate level in commercial feeds often exceeds the tolerance threshold of carnivorous fish, potentially leading to negative impacts, such as liver abnormalities, impaired glucose metabolism, antioxidant damage and inflammatory responses(Reference Polakof, Panserat and Soengas8–Reference Zhang, Yang and Nie10). Therefore, further research is crucial to develop effective strategies that enhance growth and improve metabolism to mitigate the negative effects of high dietary carbohydrates.
Chenodeoxycholic acid (CDCA) is a bile acid that fish can synthesise endogenously(Reference Romano, Kumar and Yang11) and is one of the major endogenous bile acids in some teleost species(Reference Hagey, Møller and Hofmann12). In aquatic animals, dietary CDCA supplementation has been shown to improve growth, metabolism, antioxidant capacity and immune function, as demonstrated in species such as large yellow croaker (Larimichthys crocea)(Reference Du, Xu and Li13), thinlip mullet (Liza ramada)(Reference Abdel-Tawwab, Abdel-Latif and Basuini14) and yellow catfish (Pelteobagrus fulvidraco)(Reference Zheng, Xu and Zhao15), making it as an exogenous non-additive in aquafeeds. CDCA is a potent ligand that directly activates the farnesoid X receptor (FXR), which plays a critical role in regulating glycolipid metabolism and inflammation(Reference Teodoro, Rolo and Palmeira16,Reference Shaik, Prasad and Narala17) . However, Romano et al.(Reference Romano, Kumar and Yang11) reported that excessive CDCA inclusion can disrupt carbohydrate absorption in fish, causing metabolic imbalances and adversely affecting growth performance. Based on previous study on CDCA supplementation in fish diets(Reference Yin, Xie and Zhuang18,Reference Du, Wang and Yu19) (regarding feed formulation and dose-to-weight ratio), this study incorporated CDCA at levels of 0·05 % and 0·10 %. Largemouth bass, known for its limited capacity to utilise carbohydrates, exhibits sustained hepatic glycogen accumulation and compromised health when fed high-carbohydrate diets (≥ 10 %)(Reference Chen, Zhu and Wu20,Reference Xu, Chen and Liu21) , likely due to dysregulation of glycolysis and gluconeogenesis(Reference Polakof, Panserat and Soengas8,Reference Wang, Gong and Li22) . In the liver, the negative regulations of FXR on gluconeogenesis are mediated through the inhibitory effects of a small heterodimer partner (SHP)(Reference Yamagata, Daitoku and Shimamoto23–Reference Zhang, Patil and Chauhan25). Additionally, FXR stimulates the phosphorylation of protein kinase B (AKT1)(Reference Cipriani, Mencarelli and Palladino26,Reference Renga, Mencarelli and Vavassori27) , which is a key factor of the insulin pathway(Reference Caruso and Sheridan28). However, the role of CDCA in glucose metabolism in carnivorous fish remains to be further explored. Furthermore, FXR has been shown to negatively regulate NF-κB mediated liver inflammation(Reference Gai, Visentin and Gui29,Reference Panzitt and Wagner30) . In large yellow croaker (Larimichthys crocea)(Reference Du, Xiang and Li31), CDCA supplementation in high-soyabean oil diets activated intestinal fxr, subsequently downregulated the expression of inflammatory markers, such as tnf-α, il-1β, cyclooxygenase-2 (cox-2) and il-6. These findings suggest that CDCA may regulate inflammation in carnivorous fish through FXR activation.
Largemouth bass is a widely cultured carnivorous freshwater fish species in China, with production reaching 888 030 tons in 2023(32). Commercial feeds often contain high levels of carbohydrates to meet processing performance requirements, which seriously threaten the health of this fish species. Previous studies have primarily focused on the role of CDCA in lipid metabolism in fish(Reference Zheng, Xu and Zhao15,Reference Yin, Xie and Zhuang18,Reference Zhang, Liang and He33) , but its involvement in regulating glucose metabolism remains unclear. Therefore, this study aimed to elucidate the influence of dietary CDCA inclusion on carbohydrate metabolism and the inflammatory response in largemouth bass.
Materials and methods
Experimental diets
Four isonitrogenous (52·74 %) and isolipidic (11·38 %) diets were formulated, including a positive control group (5 % α-starch content, LC) and a negative control group (10 % α-starch content, HC). Another two diets were formulated by supplementing 500 mg/kg (HCC-0·05) and 1000 mg/kg (HCC-0·10) CDCA to the HC group, respectively (Table 1). All low-fat ingredients were crushed to a particle size of less than 178 μm and mixed stepwise according to the recipe before adding the lipid component. Then, all feed ingredients were ground to a particle size of less than 178 μm. An appropriate amount of water (20 % diet weight) was added to the thoroughly mixed feed ingredients, followed by thorough mixing and subsequent extrusion. Complete starch gelatinisation was achieved at 120°C, followed by drying the feed at 60°C and storage at −20 °C until use.
Formulation and chemical composition of experimental diets (% DM)
* Supplied by Xinxin Tian’en Aquatic Feed Co., Ltd (Zhejiang, China).
† Vitamin Premix (mg/kg diet): vitamin A, 16 000 IU; vitamin D3, 8000 IU; vitamin K3, 14·72; vitamin B1, 17·80; vitamin B2, 48; vitamin B6, 29·52; vitamin B12, 0·24; vitamin E, 160; vitamin C, 800; niacinamide, 79·20; calcium-pantothenate, 73·60; folic acid, 6·40; biotin, 0·64; inositol, 320; choline chloride, 1500; L-carnitine, 100.
‡ Mineral Premix (mg/kg diet): Cu (CuSO4), 2·00; Zn (ZnSO4), 34·4; Mn (MnSO4), 6·20; Fe (FeSO4), 21·10; I (Ca (IO3)2), 1·63; Se (Na2SeO3), 0·18; Co (COCl2), 0·24; Mg (MgSO4·H2O), 52·7.
§ Supplied by Meryer (Shanghai) Biochemical Technology Co., Ltd. (Shanghai, China).
Experimental procedure
Juvenile fish were obtained from a commercial aquaculture farm (Zhejiang, China) and acclimatised in the culture system for 2 weeks before the commencement of the formal experiments. The culture system comprised a culture module (24 buckets; Volume 800 L), a filtration module (coral stones and mesh sponges), a disinfection module (UV irradiation) and an aeration module with a pipe network. After that, twelve buckets, each containing 360 healthy, morphologically similar fish (5·46 ± 0·03 g) were randomly assigned. The same experimental diet was fed to each of the three buckets twice daily until apparent saturation (08.00 and 16.00), and the feed consumption was recorded for 8 weeks. The trial was conducted in a recirculating aquaculture system with a daily water exchange rate of 10 %. All tanks were supplied with a continuous flow of aerated water (dissolved oxygen ≥ 6 mg/L). The water temperature and pH were maintained at 27 ± 1°C and 7·2 ± 0·2, respectively.
Sample collection
After the culture procedure was completed, fish were anesthetised with eugenol (1:1000; Shanghai Reagent Corp., Shanghai, China) following 24 h of starvation for sampling. All experimental fish in each group were counted and weighed to calculate the survival rate and specific growth rate (SGR). Body composition analysis was then performed on five randomly selected fish per tank. In addition, twelve fish were selected to measure body weight, length and condition factor. The viscera and liver from six of these fish were collected for the calculation of viscerosomatic index and hepatosomatic index (HSI). Furthermore, a 1 cm3 sample of liver apex was collected for gene expression and protein quantification analysis. The livers of all dissected fish were photographed for morphological assessment.
Chemical analysis
Moisture content was determined by drying the sample in an oven at 105 °C until a stable weight was achieved. Crude protein content was measured using the Kjeldahl nitrogen method (N × 6·25) with a Kjeldahl nitrogen analyser. Crude lipid content was determined following the method of Folch et al. (1957)(Reference Folch, Lees and Stanley34), with modifications as described by Peng et al. (2014)(Reference Peng, Xu and Mai35). Ash content was determined by placing the sample in a muffle furnace at 550 °C until complete combustion, after which the residue was weighed. Crude fibre content was determined from the defatted powdered sample using the AOAC method (2023)(36). Nitrogen-free extract was calculated as DM minus the sum of crude protein, crude lipid, crude ash and crude fibre. Glycogen content was determined using the KOH/anthrone method as described by Seifter et al. (1950)(Reference Seifter, Dayton and Novic37), which converted glycogen into a detectable form, with results obtained through spectrophotometric analysis.
Hepatic histopathology
Fresh liver tissue was fixed in Bouin’s fixative and sent to Servicebio in Shanghai, China for paraffin section preparation. Briefly, the tissue was immersed in Bouin’s fixative at a 1:10 volume ratio for 24 h, then dehydrated using increasing concentrations of ethanol (75 % to 100 %). Ethanol was removed with xylene, and the tissue was embedded in paraffin wax. The embedded tissue was sectioned into 5 μm thick slices using a rotary microtome (340E, Thermo Scientific). Sections were stained with hematoxylin and eosin for nuclear and cytoplasmic visualisation, then dehydrated, blocked and images were acquired using a fully automated pathology slide scanner (WS-10, ZhiYue).
Measurement of hepatic biochemical parameters
The liver samples were homogenised with phosphate buffer (1:9, w/w) and centrifuged at 3500 rpm to obtain the supernatant for further testing. The total protein concentration was quantified using Coomassie Brilliant Blue dye, as described by Bradford (1976)(Reference Bradford38), with a commercial kit (A045-2-2). The malondialdehyde (MDA) content was determined using the TBARS test(Reference Buege and Aust39) with a commercial kit (A003-1-2). The hydroxylamine technique was used to measure total superoxide dismutase (T-SOD) activity(Reference McCord and Fridovich40) with a commercial kit (A001-1-1). The rate of H2O2 breakdown was estimated to determine catalase (CAT) activity(Reference Aebi41) using a commercial kit (A007-1-1). Ferrous ion concentration was used to measure total antioxidant capacity (T-AOC)(Reference Benzie and Strain42) with a commercial kit (A015-1-2). The activity of lysozyme (LZM) was measured using a commercial kit (A050-1-1). All commercial kits were supplied by Nanjing Jiancheng Bioengineering Institute.
RNA extraction and real-time quantitative PCR
Total RNA was extracted from liver samples using Trizol Reagent (TransGen Biotech, China) and reverse transcribed to cDNA using the Prime Script™ RT Reagent Kit (Takara, Japan). The primers used for quantifying gene expression are listed in Table 2, with β-actin selected as the reference gene. Real-time quantitative PCR was performed using a quantitative thermal cycler with the following procedure: 95 °C, 2 min; 40 cycles of 95 °C,10 s; 57 °C, 10 s and 72 °C, 20 s. The relative expression was calculated using the 2–ΔΔCt method(Reference Livak and Schmittgen43).
Sequences of the primers used in this study *
* fxr, farnesoid X receptor; shp, small heterodimer partner; irs, insulin receptor substrate; ira, insulin receptor a; pi3kr1, phosphoinositide 3-kinase regulatory subunit 1; akt1, protein kinase B; g6pc, Glucose-6-phosphatase; pepck, phosphoenolpyruvate carboxykinase; fbp1, fructose bisphosphatase 1; pk, pyruvate kinase; pfkl, phosphofructokinase liver type; gk, glucokinase; tlr2, toll-like receptor 2; tgf-β, transforming growth factor β.
Western blot
A 100 mg liver sample was treated with Radioimmunoprecipitation Assay (RIPA) lysis buffer (Beyotime P0013B, China) and incubated on ice for 40 min to lysis the tissue and extract the proteins. The homogenate was then centrifuged (4 °C, 12 000 rpm, 10 min) to obtain the supernatant. Total protein extraction was repeated twice, and the protein concentration was determined using the Bicinchoninic Acid Assay (BCA) method (Beyotime P0009, China). The proteins were then treated with SDS-PAGE loading buffer and denatured at 95 °C for 10 min. Electrophoresis was performed on a 10 % polyacrylamide gel, followed by protein transfer to a polyvinylidene fluoride membrane (Millipore, USA), which was then blocked with Tris-Buffered Saline with Tween 20 (TBST) containing 5 % non-fat milk at room temperature for 2 h. The blocked membrane was incubated overnight at 4 °C with primary antibodies (FXR, AKT1, p-AKT1 and β-ACTIN, all from Cell Signaling Technology, USA). After primary antibody incubation, the membrane was washed five times with TBST buffer, followed by incubation with an enzyme-labeled secondary antibody (Sigma, USA) for 1·5 h and then washed again. Finally, the results were analysed using a ChemiDoc MP Imaging System (Bio-Rad, USA) and Image Lab software (Bio-Rad, USA).
Statistical methods
All data were presented as mean ± sem. Statistical analysis was performed using ANOVA with SPSS 26·0 software, followed by Duncan’s multiple range test. Statistical evaluations were performed after testing the data for normality and homoscedasticity. Dunnett’s test was applied to compare the data from the HC, HCC-0·05 and HCC-0·10 groups to the LC group. The significant level was set at 5 %.
Results
Growth performance
No significant differences were observed in initial body weight, survival rate and condition factor across all groups (P > 0·05) (Table 3). However, increased dietary carbohydrate levels significantly reduced the final body weight (FBW), SGR, FI and feed conversion ratio of cultured fish (P < 0·05) while significantly increasing the HSI and viscerosomatic index (P < 0·05) (Table 3). Compared with the NC group, the addition of 0·05 % CDCA had no significant effect on FBW, SGR, HSI, or viscerosomatic index (P > 0·05) but significantly increased the FI and feed conversion ratio (P < 0·05) (Table 3). In contrast, the HCC-0.10 group showed significant increases in FBW and SGR (P < 0·05), while HSI and VSI were significantly decreased (Table 3).
Growth performance of largemouth bass fed the experimental diets for 8 weeks
Values (means ± sem (Standard Error of Mean), n= 3) within a row with a common superscript letter are not significantly different from the other dietary groups (P > 0·05), with the asterisks being significantly different compared with that of the LC group (Dunnett’s test, P < 0·05).
Survival rate (SR, %) = final fish number/initial fish number × 100.
Specific growth rate (SGR, %/day) = (Ln final body weight − Ln initial body weight) × 100/days.
Feed intake (FI, %/day) = feed consumption (g)/[(initial weight + final weight)/2 × final number of individuals] × 100/days.
Condition factor (CF) = final body weight (g)/length (cm)3 × 100.
Feed conversion ratio (FCR) = Total feed consumption (g DM)/(Final biomass – Initial biomass) (g wet weight).
Hepatosomatic index (HSI, %) = liver weight/final body weight × 100.
Viscerosomatic index (VSI, %) = viscera weight/final body weight × 100.
Body composition
The high-carbohydrate diet increased moisture and crude lipid content and decreased the ash content in the whole body of largemouth bass (P < 0·05) (Table 4). Compared to both LC and HC groups, the addition of CDCA significantly reduced the crude lipid content in the whole body (P < 0·05) (Table 4). Furthermore, compared with the LC group, the high-carbohydrate diet significantly increased both crude lipid and glycogen content in the liver (P < 0·05) (Table 4). However, the inclusion of CDCA in the diet significantly reduced lipid and glycogen accumulation in the liver compared with the HC group (P < 0·05) (Table 4). The high-carbohydrate diet significantly reduced liver crude protein content compared to the LC group (P < 0·05), but this reduction was significantly mitigated in the HCC-0·10 groups compared with the HC group. However, the liver moisture content was not affected by the addition of CDCA (P > 0·05). Additionally, no significant differences in muscle moisture content and crude protein content were observed across all experimental groups (P > 0·05) (Table 4). The trend in muscle crude lipid content was consistent with that observed in the whole body (P < 0·05) (Table 4).
The whole body, liver proximate composition and muscle proximate composition (wet weight basis) of largemouth bass fed the experimental diets for 8 weeks
Values (means ± sem (Standard Error of Mean), n= 3) within a row with a common superscript letter are not significantly different from the other dietary groups (P > 0·05), with the asterisks being significantly different compared with that of the LC group (Dunnett’s test, P < 0·05).
Hepatic histopathology analysis
Simple macroscopic observations of the livers of experimental fish revealed visible differences (Figure 1). Notably, the livers from the HC group were significantly hypertrophied compared with the LC group, which improved progressively with the addition of CDCA, consistent with the HSI values obtained previously. Additionally, the high-carbohydrate diet caused a slight yellow colouration in the liver, which progressively turned reddish with increasing levels of CDCA inclusion (Figure 1).
The livers of largemouth bass fed the experimental diets for 8 weeks.
As shown in Figure 2, the hepatocytes in the LC group were regularly arranged, exhibiting normal cell morphology with only occasional small lipid droplets. In contrast, hepatocytes in the HC group were significantly enlarged, with unclear cell margins, severe swelling, vacuolisation, displaced or absent nuclei and abundant round lipid droplets and inflammatory infiltrates (Figure 2(a) and (b)). Following the addition of 0·05 % CDCA, hepatocyte swelling and vacuolisation were reduced, and the number of lipid droplets and inflammatory infiltrates decreased. However, the nuclei remained displaced or absent (Figure 2(c)). After the addition of 0·10 % CDCA, hepatocyte morphology returned to normal, with only a few swollen cells remaining. The nuclei were largely intact, and lipid droplets nearly disappeared, although a few inflammatory infiltrates were still observed (Figure 2(d)).
The morphology analysis of the liver (bar = 50 μm) of largemouth bass from LC (a), HC (b), HCC-0·05 (c) and HCC-0·10 (d) groups fed the experimental diets for 8 weeks.
Hepatic biochemical parameters
Hepatic total protein content exhibited a significant decrease in response to elevated dietary carbohydrate levels, whereas CDCA supplementation significantly increased total protein content (P < 0·05) (Table 5). Conversely, hepatic MDA content was significantly higher in the HC group compared to the LC group, showing a dose-dependent decrease with CDCA supplementation (Table 5). Hepatic T-SOD and CAT activities and T-AOC were all significantly lower in the HC group compared to the LC group (P < 0·05) (Table 5). Notably, 0·05 % CDCA supplementation did not affect CAT activity but significantly increased T-SOD and T-AOC activities (P < 0·05) (Table 5). However, 0·10 % CDCA supplementation significantly increased all antioxidant enzyme activities (P < 0·05) (Table 5). Additionally, the high dietary carbohydrate significantly depressed LZM activity (P < 0·05) (Table 5), while 0·10 % CDCA supplementation significantly enhanced LZM activity (P < 0·05) (Table 5), restoring it to levels comparable to the positive control group (P > 0·05) (Table 5).
Hepatic biochemical parameters of largemouth bass fed the experimental diets for 8 weeks
Values (means ± sem (Standard Error of Mean), n = 3) within a row with a common superscript letter are not significantly different from the other dietary groups (P > 0·05), with the asterisks being significantly different compared with that of the LC group (Dunnett’s test, P < 0·05). TP, total protein; MDA, malondialdehyde; CAT, catalase; T-SOD, total superoxide dismutase; T-AOC, total antioxidant capacity; LZM, lysozyme.
Expression of hepatic insulin signalling involved genes and proteins
The results indicated that, compared to the LC group, the expressions of fxr, shp, insulin receptor substrate (irs), phosphatidylinositol 3-kinase 1 (pi3kr1) and protein kinase b (akt1) were significantly reduced in the HC group (P < 0·05) (Figure 3). The addition of CDCA significantly increased the expression of these genes compared with the HC group (P < 0·05) (Figure 3). Furthermore, the high-carbohydrate diet significantly inhibited the expression of FXR and p-AKT1, with no effect on total AKT1 expression (P > 0·05) (Figure 4). Compared with the HC group, the addition of CDCA significantly activated the expression of FXR and p-AKT1 (P < 0·05).
The expression of genes related to insulin signalling pathway, fxr (a), shp (b), irs (c), ira (d), pi3kr1 (e), akt1 (f) in the liver of largemouth bass fed the experimental diets for 8 weeks. Values (mean ± standard error of the mean, sem) in bars that have the same letter are not significantly different between treatments (P > 0·05; Duncan’s test, n = 3), with the asterisks are significantly different compared with that of the LC group (Dunnett’s test, P < 0·05).
The expression of FXR, p-AKT1, AKT1 and β-ACTIN protein in the liver of largemouth bass fed the experimental diets for 8 weeks. Values (mean ± standard error of the mean, sem) in bars that have the same letter are not significantly different between treatments (P > 0·05; Duncan’s test, n = 3), with the asterisks are significantly different compared with that of the LC group (Dunnett’s test, P < 0·05).
Expression of hepatic glucose metabolism involved genes
As shown in Figure 5, compared with the LC group, the expressions of phosphofructokinase (pfkl), phosphoenolpyruvate carboxylase (pepck), glycogen phosphorylase (g6pc), glucokinase (gk) and phosphorylase (pk) were significantly reduced in the HC group (P < 0·05) (Figure 5), while fructose-1,6-bisphosphatase (fbp1) expression was significantly elevated (P < 0·05) (Figure 5). Compared with the HC group, the addition of 0·05 % and 0·10 % CDCA significantly increased the gene expression of gk, pk and pfkl (P < 0·05) (Figure 5) while significantly decreasing the gene expression of fbp1 (P < 0·05) (Figure 5). However, the expression of g6pc and pepck was not affected by CDCA inclusion in the high-carbohydrate group (P > 0·05) (Figure 5).
The expression of genes related to glucose metabolism, g6pc (a), fbp1 (b), pepck (c), gk (d), pk (e) and pfkl (f) in the liver of largemouth bass fed the experimental diets for 8 weeks. Values (mean ± standard error of the mean, sem) in bars that have the same letter are not significantly different between treatments (P > 0·05; Duncan’s test, n = 3), with the asterisks are significantly different compared with that of the LC group (Dunnett’s test, P < 0·05).
Expression of hepatic inflammation response involved genes
With the increase of dietary carbohydrate level, the expression of tlr2, nf-κb and il-1β was significantly increased (P < 0·05) (Figure 6), while the expression of iκb and il-10 was significantly decreased (P < 0·05) (Figure 6). Dietary CDCA inclusion significantly reduced the expression of nf-κb and il-1β (P < 0·05) (Figure 6), while significantly enhancing iκb, il-10 and tgf-β expression (P < 0·05) (Figure 6).
The expression of genes related to inflammatory response, tlr2 (a), nf-κb (b), il-1β (c), iκb (d), il-10 (e) and tgf-β (f) in the liver of largemouth bass fed the experimental diets for 8 weeks. Values (mean ± standard error of the mean, sem) in bars that have the same letter are not significantly different between treatments (P > 0·05; Duncan’s test, n = 3), with the asterisks are significantly different compared with that of the LC group (Dunnett’s test, P < 0·05).
Discussion
High-carbohydrate diets have significant negative effects on largemouth bass, including reduced growth performance, hepatic glycogen and lipid accumulation, as well as subsequent hepatic inflammation(Reference Zhang, Yang and Nie10,Reference Hemre, Mommsen and Krogdahl48) . Therefore, this experiment aimed to investigate the effects of dietary CDCA inclusion on growth performance, antioxidant capacity, insulin signalling, glucose metabolism and the inflammatory response in largemouth bass fed a high-carbohydrate diet. The results showed that a high-carbohydrate diet significantly decreased FI, feed conversion ratio, FBW and SGR of largemouth bass, which is consistent with previous studies on largemouth bass(Reference Liu, Liu and Wang45,Reference Tao, Gong and Chen49) . However, the inclusion of exogenous bile acids has been demonstrated to improve the feed intake in thin lip mullet (Liza ramada)(Reference Abdel-Tawwab, Abdel-Latif and Basuini14). Similarly, in this study, the addition of 0·05 % CDCA significantly improved the feed intake, which was partly related to the potential role of CDCA in elevating carbohydrate utilisation in largemouth bass. Additionally, as previously reported by Zeng et al. (2015)(Reference Zeng, Lei and Ai50), diets with higher carbohydrate content exhibited greater total energy content, which may induce a protein-sparing effect in fish. It is therefore plausible that the HC group resulted in a lower feed conversion ratio in largemouth bass compared with the LC group in this experiment. Additionally, feeding largemouth bass a diet including 0·10 % CDCA significantly increased FBW and SGR, suggesting that CDCA inclusion enhances their carbohydrate tolerance. Meanwhile, a high-carbohydrate diet increased the lipid content in the liver, muscle and whole body of the cultured fish, as evidenced by the numerous aggregated lipid droplets observed in the hepatic histopathology of the HC group. This suggests that largemouth bass cope with a high-carbohydrate diet by converting excess carbohydrates into lipids. CDCA has been shown to improve lipid metabolism(Reference Zheng, Xu and Zhao15,Reference Liu, Zhao and Wang51,Reference Li, Yao and Zhang52) . Consistently, we found that adding CDCA to the high-carbohydrate diet significantly reduced lipid content in the liver, muscle and whole body of largemouth bass.
Considerable studies have demonstrated that a high-carbohydrate diet can induce oxidative stress and cellular damage in carnivorous fish(Reference Li, Wang and Li53–Reference Xu, Liu and Huang55). The primary cause of oxidative stress is the production of excessive oxygen-free radicals, which interact with various cellular components, damaging cell structure and function. An important target of these oxygen-free radicals is PUFA in biological membranes, which can trigger lipid peroxidation, producing lipid peroxides, such as reactive chemical MDA. This process further amplifies the damaging effects of oxygen-free radicals and leads to further cellular damage(Reference Gaweł, Wardas and Niedworok56). Thus, MDA levels reflect the extent of lipid peroxidation and the severity of cellular damage. To counteract oxidative stress, organisms rely on several antioxidant systems, including two key antioxidant enzymes: SOD, which scavenges superoxide radicals and CAT, which breaks down hydrogen peroxide, thereby reducing the production of hydroxyl radicals(Reference Kanter, Coskun and Korkmaz57). T-AOC reflects the overall level of antioxidant macromolecules, small molecules and enzymes in the system(Reference Ghiselli, Serafini and Natella58). In this study, it was found that a high-carbohydrate diet increased liver MDA content while decreasing T-SOD and CAT activities, as well as T-AOC. However, the inclusion of CDCA in the diet had the opposite effect, decreasing MDA levels and increasing T-AOC, T-SOD and CAT activities. This suggests that CDCA effectively alleviates oxidative stress induced by a high-carbohydrate diet, thereby protecting the liver. Consequently, fewer abnormal hepatocytes were observed in the hepatic histopathology of the HCC-0·10 group.
FXR has been proven to play a direct role in liver glucose homeostasis(Reference Duran-Sandoval, Cariou and Percevault59). In carnivorous fish, excessive carbohydrate intake leads to a sharp increase in postprandial glucose levels(Reference Wilson60), but sustained high-carbohydrate consumption has been reported to have no effect on fxr expression(Reference Chen, Zhu and Wu20,Reference Ge, Chen and Sun61) . In primary rat hepatocytes, D-glucose has been found to increase fxr mRNA in a dose and time-dependent manner(Reference Duran-Sandoval, Mautino and Martin62). However, in the present experiment, a significant decrease in fxr expression was observed, which requires further investigation. CDCA, a natural and potent activator of FXR(Reference Song, Zhang and Klaassen63), has the potential to increase the fxr expression included in a high-carbohydrate diet. Activation of shp by fxr leads to the repression of hepatic gluconeogenesis genes such as fbp1, pepck and g6pc (Reference Yamagata, Daitoku and Shimamoto23,Reference Zhang, Lee and Barrera24) . However, in this study, fbp1 expression was significantly up-regulated in the HC group compared to the LC group. This upregulation may be due to the high-carbohydrate diet providing specific substrates that promote fbp1 synthesis, facilitating cellular adaptation to elevated glucose concentrations, while gluconeogenesis (pepck, g6pc) is suppressed. The addition of 0·10 % CDCA significantly reduced FBP1 expression but had no significant effects on pepck and g6pc expression, indicating an inhibition of gluconeogenesis and supporting the regulatory role of fbp1. However, the precise mechanisms underlying the high-carbohydrate-induced upregulation of fbp1 require further investigation. Additionally, as tauroursodeoxycholic acid has been shown to improve liver and muscle insulin sensitivity and enhance muscle insulin signalling in obese humans(Reference Kars, Yang and Klein64), this study found CDCA supplementation significantly increased the expression of ira, irs, pi3kr1and akt1, which are key genes in the insulin pathway(Reference Caruso and Sheridan28). Moreover, the expression of glycolysis-related genes, including gk, pk and pfkl, was significantly elevated compared with the negative group. These results indicate that CDCA supplementation plays a beneficial role in regulating insulin and glycogen metabolism via fxr, enhancing the utilisation of dietary carbohydrate by largemouth bass. Moreover, protein expression analysis of FXR, AKT1 and p-AKT1 confirms that CDCA inclusion in the high-carbohydrate diet activates FXR and promotes AKT1 phosphorylation, which may contribute to the regulation of insulin secretion.
The innate immune system in fish serves as the first line of defense against a broad spectrum of pathogens, with LZM activity being a key indicator of immune function(Reference Saurabh and Sahoo65). Our results confirmed the detrimental effect of a high-carbohydrate diet on liver LZM activity in largemouth bass, but the addition of CDCA ameliorated this negative effect. Furthermore, tlr2 is a member of the toll receptor family and functions as a pattern-recognition receptor that plays a crucial role in innate immunity(Reference Zarubin and Han66). Histopathological analysis of liver tissue revealed significant inflammatory infiltration induced by the high-carbohydrate diets, which was consistent with the results observed for tlr2 in this study. Meanwhile, NF-κB is a central transcriptional regulator of inflammatory responses and cell proliferation and is involved in the inflammatory response induced by high-carbohydrate diets, similar results were obtained in the present experiment(Reference Liu, Liu and Wang45,Reference Karin and Greten67,Reference Tao, Wang and Qiu68) . Abundant evidence has suggested that FXR can interact with NF-κB to modulate the inflammatory response(Reference Wang, Chen and Wang69–Reference Bijsmans, Guercini and Ramos Pittol71). In this study, the addition of CDCA significantly activated the expression of fxr and inhibited the expression of nf-κb, which suggested that fxr might act on nf-κb to inhibit its expression and reduce the inflammatory response. Moreover, dietary CDCA supplementation restored the expression of the anti-inflammatory genes il-10 and iκb which were suppressed by the high dietary carbohydrate diet. The expression of another anti-inflammatory gene, tgf-β, was significantly increased with the addition of CDCA, regardless of the dietary carbohydrate level(Reference Letterio and Roberts72–Reference Wang and Secombes74), suggesting that CDCA has the potential to restore and enhance the anti-inflammatory response in the liver of carnivorous fish.
Conclusions
In conclusion, the addition of CDCA improved growth performance and liver health, alleviated hepatic glycogen accumulation through FXR-mediated activation of the insulin pathway and inhibition of gluconeogenesis and reduced inflammatory responses in largemouth bass fed high-carbohydrate diets (Figure 7).
Schematic overview of the mechanisms by which CDCA regulates the hepatic insulin pathway, glucose metabolism and immune metabolism in largemouth bass via fxr. CDCA, chenodeoxycholic acid.
Acknowledgements
This work was financially supported by the Shanghai Rising-Star Program (Grant 24QA2703500) and the National Natural Science Foundation of China (31802308).
W. L.: Investigation, Data curation, Formal analysis, Writing – Original Draft; N. Z.: Investigation, Methodology, Data curation; N. L.; Investigation, Methodology; S. C.: Investigation; Y. G.: Investigation, Methodology, Writing – Review & Editing; N. C.: Conceptualisation; Project administration; S. L.: Conceptualisation, Supervision, Writing – Review & Editing, Funding acquisition.
The authors declare that there are no conflicts of interest.
The present experiment strictly followed the requirements of the Animal Care and Use Committee of Shanghai Ocean University.
