Effects of dietary berberine on growth performance, lipid metabolism, antioxidant capacity and lipometabolism-related genes expression of AMPK signaling pathway in juvenile black carp (Mylopharyngodon piceus) fed high-fat diets

This study aimed to investigate the effects of high-fat diet (HFD) supplemented with berberine on growth, lipid metabolism, antioxidant capacity and lipometabolism-related genes expression of AMPK signaling pathway in juvenile black carp (Mylopharyngodon piceus). Five hundred and forty healthy fish (4.04 ± 0.01 g) were randomly distributed into six groups, and fed six experimental diets: normal-fat diet (NFD, 5% fat), HFD (15% fat), and four HFDs supplemented with graded levels of berberine, respectively. The results showed that, compared with fish fed NFD, HFD had no effects on the growth of fish except for reducing survival rate, whereas HFD caused extensive lipid accumulation, oxidative stress injury and hepatic abnormalities. However, compared with the HFD group, fish fed HFD containing an appropriate berberine (98.26 or 196.21 mg/kg) improved the growth performance, increased hepatic lipid metabolism and antioxidant enzymes activities, and up-regulated the mRNA expression levels of ampk subunits and lipolysis genes such as pparα, cpt-1, acox, atgl and hsl (P < 0.05). Meanwhile, HFD supplemented with an appropriate berberine reduced crude lipid contents in liver and whole-body, decreased serum lipid contents, and ALT and AST activities, and down-regulated the mRNA expression levels of lipogenesis genes such as srebp-1, acc1, gpat, fas and pparγ, and lipid transporter genes such as fatp, fabp and fat/cd36 (P < 0.05). Thus, HFD supplemented with an appropriate berberine could improve growth of black carp, promote lipid metabolism and enhance antioxidant capacity. The lipid-lowering mechanism of berberine might be mediated by activating AMPK pathway, up-regulating lipolysis genes expression, and down-regulating lipogenesis and transport genes expression.


Introduction
Lipids play a crucial role in fish nutrition due to its high energy-density and providing essential fatty acids, cholesterol and phospholipids, which are efficiently utilized by most fish species (Du et al. 2005;Lu et al. 2014;Wang et al. 2019;Yan et al. 2015).Therefore, the application of high-fat diets (HFD) has become a trend in intensive aquaculture, which is conducive to the protein-sparing effect of dietary lipid, improving feed efficiency, and reducing feed cost and nitrogen emission (Li et al. 2012;Wang et al. 2019).However, HFD often lead to excessive lipid accumulation in liver or visceral fat tissue in farmed fish, causing obesity and nutritional fatty liver, resulting in liver injury and lipid metabolism disorders (Gao et al. 2011;Lu et al. 2014).Excessive deposition of liver fat also induces oxidative stress, immune suppression, growth retardation and even death of fish, resulting in large economic losses (Tang et al. 2019;Yan et al. 2015).
AMP-activated protein kinase (AMPK) universally exists in eukaryotic cells and is known as "a nutrient and energy sensor", which plays a key role in regulating energy homeostasis (Garcia et al. 2017;Hardie et al. 2012).AMPK is a heterotrimeric complex composed of catalytic α-subunit (α1 and α2) and regulatory β-and γ-subunits (β1 and β2, and γ1, γ2 and γ3) (Davies et al. 1994;Nie et al. 2020).Activation of AMPK suppresses anabolism such as fatty acid and cholesterol synthesis, and promotes catabolism like fatty acid oxidation, reducing lipid deposition (Hardie et al. 2012;Nie et al. 2020).AMPK regulates lipid metabolism by up-regulating the expression of lipolysis pathway genes such as peroxisome proliferator-activated receptor α (pparα), carnitine palmitoyltransferase 1 (cpt-1), acyl-coenzyme A oxidase (acox), adipose triglyceride lipase (atgl) and hormone sensitive lipase (hsl), and/or down-regulating the expression of lipogenesis pathway genes such as sterol regulatory element-binding protein-1 (srebp-1), acetyl-CoA carboxylase 1 (acc1), glycerol-3-phosphate acyltransferase (gpat) and fatty acid synthetase (fas) (Hardie et al. 2012;Ran et al. 2021).Knockout of ampk gene in mouse is linked to higher incidence of obesity and fatty liver (Viollet et al. 2003).Therefore, AMPK has become a novel target for the prevention and treatment of metabolic diseases such as fatty liver and hyperlipidemia (Garcia and Shaw 2017;Hardie et al. 2012).Although it has been reported that AMPK activation could regulate lipid degradation in zebrafish (Ran et al. 2021) and energy homeostasis in olive flounder (Paralichthys olivaceus) (Nie et al. 2020), there are few studies on the regulation mechanism of AMPK pathway in lipid metabolism of fish.
Berberine is an isoquinoline alkaloid isolated from the stems and roots of various Berberis species such as Coptis Chinensis, Hydrastis canadensis and Berberis aristate without side effects and toxicity (Gaba et al. 2021;Wang and Zidichouski 2018).It has been commonly used in traditional Chinese medicine for its antibacterial, anti-inflammatory, antioxidant, and antiapoptotic activities (Choi 2016;Doan et al. 2020;Ji et al. 2012;Lu et al. 2017).Berberine is also used as a new lipid-lowering drug in treating fatty liver, obesity and hyperlipidemia (Brusq et al. 2006;Gaba et al. 2021;Kong et al. 2004).In aquaculture, berberine has been used as a functional feed additive for the prevention and treatment of hepatic lipid accumulation and oxidative stress injury (Chen et al. 2020;Lu et al. 2017;Zhou et al. 2019).Many in vivo and in vitro studies showed that berberine could promote lipolysis and suppress lipogenesis in liver cells or adipocytes through AMPK activation (Kim et al. 2009;Ren et al. 2020;Zhu et al. 2019).However, as far as we know, the mechanism of dietary berberine Vol.: (0123456789) regulates lipid metabolism of fish by activating AMPK pathway has been rarely reported.
Black carp (Mylopharyngodon piceus) is a carnivorous freshwater fish native to China, and has been a favored aquaculture species in China due to its fast growth, high yield and tender meat.The production of black carp reached about 694.53 thousand tons in 2020 (Ministry of Agriculture and Rural Affairs of China 2021), its aquaculture output is still increasing year by year in China.However, black carps fed HFD lead to anomalous lipid deposition in intensive aquaculture, resulting in increasingly prominent problems such as obesity and fatty liver.Although the causes of fish fatty liver are complex, the nutritional fatty liver accounts for a large proportion in the production practice, and excessive lipid accumulation leads to lipid metabolism disorders and oxidative stress injury (Du et al. 2005;Lu et al. 2014;Tang et al. 2019).This kind of fish not only grows slowly and has a high feed coefficient, but also has reduced disease resistance and stress resistance, and increased mortality, which has caused huge economic losses to fishery production.Some previous studies showed that berberine could improve lipid metabolic disorders and revert hepatic lipid accumulation by activating AMPK pathway (Kim et al. 2009;Zhu et al. 2019).Therefore, the present study aimed to investigate the effects of dietary berberine on growth performance, lipid metabolism, antioxidant capacity and lipometabolism-related genes expression of AMPK signaling pathway in black carp fed high-fat diets.The research result will provide a scientific basis for preventing or alleviating lipid metabolism disorders and excessive fat deposition in fish.

Diet preparation
Formulation and proximate composition of the experimental diets are shown in Table 1.Fish meal, casein and soybean meal served as dietary protein sources.Fish oil and soybean oil were used as lipid sources.
Corn starch was the main carbohydrate source.Berberine (HPLC ≥ 98%) was purchased from Beijing Soleibao Technology Co., Ltd (Beijing, China).The experimental diets were designed as two control diets, namely the normal-fat diet (NFD, 5% fat) and high-fat diet (HFD, 15% fat), and four HFDs supplemented with berberine 50, 100, 200 and 400 mg/ kg, respectively.The corresponding berberine levels in the six kinds of diets were analyzed using a highperformance liquid chromatography (HPLC) to be 0, 0, 49.13, 98.26, 196.21 and 392.07 mg/kg diet, respectively.Dietary ingredients were ground through a 60-mesh sieve, weighed accurately, mixed thoroughly, and blended with oil (fish oil and soybean oil), then an appropriate distilled water was added to form soft dough, which was then pelleted into a 2-mm diameter granular feed using a pellet mill, and dried for about 18 h in a ventilated oven at 40 ℃.All dry diets were stored in sealed plastic bags at − 20 ℃.

Fish and feeding trial
Black carps were purchased from a local fish hatchery (Huzhou, China).Before the experiment, fish were acclimated to indoor recirculating aquaculture system and fed with the NFD for two weeks.After the acclimation, five hundred and forty healthy fish with initial mean body weight of (4.04 ± 0.01) g were randomly allocated to 18 cylindrical tanks (500-L) with 30 fish per tank.Fish tanks were randomly divided into six groups, each group had three replications and fed with one of the six experimental diets.Fish were hand-fed twice daily at 7:30 and 16:30 to apparent satiation for 60 days.During the feeding trial, fish were reared under a natural photoperiod and the following conditions: water temperature, 26-29 ℃; pH, 7.2-7.8;dissolved oxygen (DO) > 5.0 mg/L; NH3-N < 0.01 mg/L.

Sample collection
At the end of the feeding trial, fish were fasted for 24 h before harvest.Then all fish were anesthetized with diluted MS-222 (tricaine methanesulfonate, Sigma, USA) at a concentration of 100 mg/L.The fish per tank were weighed and counted to determine the growth performance.Three fish in each tank were randomly sampled (3 samples/tank, 9 samples/ group).Nine blood samples per group were extracted from fish caudal vein using 1-mL syringe, and centrifuged at 3000 g for 10 min after 4 h clot at 4 ℃, the serum was collected and stored at − 80 ℃ until use.Then, nine liver samples in each group were quickly dissected out and frozen at − 80 ℃ until analysis.In addition, the liver samples for the histology observation were fixed in the relevant buffer.Dorsal muscle samples (9 samples/group) were obtained from the back of the fish after removing scales and skin, and then kept at − 80 ℃ for further analysis.

Liver ultrastructure
In order to make transmission electron microscopy (TEM) sections, the fish liver samples were fixed in 2.5% glutaraldehyde (pH 7.2) for 24 h, and then fixed by 1% OsO 4 for 2 h and dried in a graded sequence of ethanol.Then, the samples were embedded in epoxy resin Epon812, cut into 60-nm thick sections with a PowerTome-XL microtome (RMC, USA), stained with uranyl acetate and lead citrate, and observed in a Hitachi H7650 TEM (Tokyo, Japan).

Growth performance analysis
W 0 is initial body weight (g), W t is final body weight (g) and t is the feeding time in days.

Proximate composition assay
The proximate compositions of experimental diets were analyzed following the methods of the Association of Official Analytical Chemists (AOAC (Association of Official Analytical Chemists) 2005).
The lipid contents of whole fish body, muscle and liver were determined by Soxhlet extraction method.

Serum biochemical parameters analysis
The triglyceride (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C), and non-esterified fatty acid (NEFA) in the serum were measured by colorimetric methods using commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).Activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured according to the method of Reitman and Frankel (1957).

Liver lipid metabolism enzymes activities and antioxidant indices measurement
Liver samples were homogenized and then centrifuged at 3000 g for 10 min (4 ℃).The supernatant was collected and determined the protein concentration by Coomassie brilliant blue method for further analysis (Ming et al., 2020).The activities of hepatic lipase (HL), lipoprotein lipase (LPL) and malate dehydrogenase (MDH) were determined by colorimetry, total lipase (TL) = HL + LPL.The hormonesensitive lipase (HSL) and fatty acid synthase (FAS) activities were tested with the ELISA kits.The above detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as total antioxidant capacity (T-AOC) were determined according to the methods described by Ming et al. (2015).GSH content was measured by the modified method of Vardi et al. (2008).Malondialdehyde (MDA) level was tested using thiobarbituric acid colorimetry (Drape et al. 1993).

Real-time quantitative PCR analysis
Total RNA was extracted from the liver of black carp using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions followed by RNasefree DNase I treatment.The quality and quantity of isolated RNA were assessed by electrophoresis in 1.0% denaturing agarose gel and spectrophotometer of NanoDrop ND-2000 (Thermo Fisher Scientific, USA), respectively.The cDNA was generated from 500 ng of total RNA and synthesized using a Prime-Script™ RT reagent Kit (Takara, Japan) following the protocols.Specific primers for the candidate genes were designed by Primer Premier 5.0 according to the sequences of black carp in GenBank.The housekeeping gene β-actin was chosen as the reference gene after confirming its stability across the experimental treatments.As shown in Table 2, these primers were synthesized by Shanghai Invitrogen Lifetechnologies Co., Ltd.
Real-time quantitative PCR assays were performed in a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA).The reaction mixture was 20 μL, containing 1 μL of each primer (10 μmol/L), 10 μL of 2 × SYBR Green I Master (Takara, Japan), 1.5 μL of the diluted cDNA and 6.5 μL of diethyl pyrocarbonate (DEPC) water.The amplification protocol of 21 genes was set as follows: 95 ℃ for 1 min; followed by 40 cycles of 95 ℃ for 5 s, at their optimal anneal temperature (Table 2) for 15 s, and 72 ℃ for 25 s.Each amplicon specificity was checked by analysis of melting curve and confirmed by electrophoresis of 1% agarose gel.The amplification efficiencies of all genes were approximately equal and ranged from 98 to 101%.The expression data of all genes were presented relative to the expression of the NFD control group, the relative expression levels of target genes were calculated by 2 −ΔΔC T method (Livak and Schmittgen 2001).

Statistical analysis
All data are presented as means ± SD (standard deviation).Statistical analysis was conducted by SPSS 25.0 for Windows (SPSS Inc., USA).Shapiro-Wilk and Levene's tests were applied to determine normality Vol:.( 1234567890) and homogeneity of all data, respectively.Data between the NFD and HFD control groups were analyzed by independent sample t-test.The data among the HFD group and HFD berberine-supplemented groups were subjected to one-way analysis of variance (ANOVA), followed by Turkey's multiple range test, and P < 0.05 was considered to be statistically significant.

Growth performance
As shown in Table 3, there was no significant difference in the weight gain (WG), specific growth rate (SGR) and feed conversion rate (FCR) between the normal-fat diet (NFD) and high-fat diet (HFD) groups (P > 0.05), while the survival rate of NFD group was significantly higher than that of HFD group (P < 0.05).
WG, SGR and survival rate were improved with HFD berberine content increasing up to 98.26 mg/ kg diet, and reduced thereafter (P < 0.05).However, FCR first decreased and then increased, which had the lower levels when dietary berberine contents were at 98.26 and 196.21 mg/kg diets (P < 0.05).

Lipid deposition
As can be seen in Table 4, the crude lipids of the whole body and liver in the HFD group were significantly higher than those in the NFD group (P < 0.05), while there was no significant difference in the muscle lipid level between the NFD and HFD groups (P > 0.05).
Compared with HFD control group, the crude lipids of the whole body and liver significantly decreased when dietary berberine contents increasing up to 98.26 and 196.21 mg/kg diets (P < 0.05).However, the muscle crude lipid was not affected by different doses of berberine treatment (P > 0.05).Vol:.( 1234567890) Liver ultrastructure The liver of fish fed NFD showed normal hepatocyte structure with few lipid droplets (Fig. 1A), the nucleus was round and the nucleolus was clearly visible.However, fish fed the HFD showed the hypertrophied liver cells had many lipid droplets, which took up the main space of cytoplasm, and nucleus was translocated to the cell edge (Fig. 1B).In contrast, fish fed HFD supplemented with different doses of berberine exhibited healthy hepatocyte structure, which had relatively fewer lipid droplets than that of fish fed HFD (Fig. 1C, 1D, 1E, 1F).According to the ultrastructural images, liver lipid accumulation could be significantly alleviated by fish fed diets containing berberine of 98.26 and 196.21 mg/kg diet.

Serum biochemical parameters
As shown in Table 5, compared with the NFD group, serum TG, TC, NEFA and LDL-C contents, and activities of ALT and AST in the HFD group were significantly increased, while the HDL-C level was significantly reduced (P < 0.05).
Compared with HFD control group, serum TG, TC, NEFA and LDL-C contents, and activities of ALT and AST in the groups of HFD supplemented with different doses of berberine first decreased and then increased, which had the lower levels when dietary berberine levels were at 98.26 and 196.21 mg/kg diets (P < 0.05).However, serum HDL-C content increased with dietary berberine content increasing up to 98.26 mg/kg diet (P < 0.05), and then maintained a higher level (P > 0.05).

Liver lipid metabolism enzymes activities
As shown in Table 6, liver MDH activity in HFD group significantly decreased in comparison with NFD group, while liver FAS activity was the opposite (P < 0.05).However, there were no significant differences in the activities of HL, LPL, TL and HSL between the NFD and HFD groups (P > 0.05).
Compared with HFD control group, the activities of liver HL, LPL, TL, MDH and HSL in the groups of HFD supplemented with different doses of berberine first increased and then decreased, which had the higher levels when dietary berberine levels were at 98.26 and 196.21 mg/kg diets (P < 0.05).However, liver FAS activity decreased with dietary berberine content up to 98.26 mg/kg diet (P < 0.05), and then increased, but was still lower than that of the HFD control group (P < 0.05).

Liver antioxidant-related parameters
As shown in Table 7, The liver oxidative status parameters such as SOD, CAT, GPx and GR activities, and T-AOC and GSH levels in the HFD group were significantly lower than those of the NFD group, while MDA content in the HFD group exhibited an opposite result (P < 0.05).
Compared with HFD control group, the activities of liver SOD, CAT, GPx and GR, and T-AOC and GSH levels in the HFD supplemented with different doses of berberine increased with dietary berberine  content increasing up to 98.26 or 196.21 mg /kg diet, and then declined thereafter (P < 0.05), but above parameters showed no significant difference between the two groups (P > 0.05).However, liver MDA content decreased with dietary berberine content up to 98.26 mg /kg diet, then increased, but still significantly lower than that of the HFD control group (P < 0.05).

Liver lipometabolism-related genes expression of AMPK signaling pathway
As shown in Figs. 2 and 3, the mRNA expression levels of ampk subunits (ampkα1, ampkα2, ampkβ1, ampkβ2, ampkγ1, ampkγ2 and ampkγ3) and partial lipolysis genes (pparα, cpt-1 and acox) in the HFD group were significantly lower than those in the NFD group, while the opposite was true for hsl gene (P < 0.05).However, there was no significant difference in atgl mRNA expression level between the NFD and HFD groups (P > 0.05).Compared with HFD control group, ampk subunits and lipolysis genes mRNA expression levels in the groups of HFD supplemented with different doses of berberine first increased and then decreased, which had the higher levels when dietary berberine contents were at 98.26 or 196.21 mg/kg diets (P < 0.05).
As shown in Figs. 4 and 5, the mRNA expression levels of lipogenesis genes (srebp-1, acc1, gpat, fas and pparγ) and lipid transporter genes (fatp, fabp and fat/cd36) in the HFD control group were significantly higher than those in the NFD group (P < 0.05).Compared with HFD control group, the mRNA expression levels of lipogenesis and lipid transporter genes in the groups of HFD supplemented with different doses of berberine first decreased and then increased, which had the lower levels when dietary berberine levels were at 98.26 and 196.21 mg/kg diets (P < 0.05).

Discussion
In the present study, the WG, SGR and FCR were not significantly different between the NFD and HFD groups, which were in line with some previous studies in large yellow croaker (Larimichthys crocea) (Yan et al. 2015) and grass carp (Ctenopharyngodon idella) (Tang et al. 2019).However, several studies have shown that HFD could promote growth in white seabass (Atractoscion nobilis) (López et al. 2009) and blunt snout bream (Megalobrama amblycephala) (Li et al. 2012), or suppress growth in grass carp (Du et al. 2005), blunt snout bream (Zhou et al. 2019) and black sea bream (Acanthopagrus schlegelii) (Wang et al. 2019).Furthermore, it was also observed in this study that HFD supplemented with berberine of 98.26 mg/kg could promote the growth of black carp, dietary excess berberine had negative effect.Zhou et al. (2019) also reported that HFD supplemented with berberine of 50 mg/kg improved the growth performance of blunt snout bream while berberine of 100 mg/kg inhibited the growth.Like some Chinese herbs have hepatoprotective effect (Zhou et al. 2015), berberine could attenuate the adverse effects caused by HFD such as fatty liver, oxidative stress injury, hepatocyte apoptosis and immunosuppression (Xu et al. 2017;Zhou et al. 2019), which were beneficial to growth of black carp.However, zebrafish fed highcholesterol diet were exposed to berberine at doses of 5 and 25 mM for 10 days, which prevented liver lipid accumulation and decreased body weight (Chen et al. 2020).Thus, the relationship between growth performance and berberine supplementation is complex, it might vary with fish species, growth stages, feed lipid levels and feeding strategies.
Several reports have shown that HFD caused excessive lipid deposition in fish liver and whole body (Regost et al. 2001;Yan et al. 2015), but muscle lipid content was not affected by dietary lipid levels (Regost et al. 2001), which was in agreement with our results.The liver is considered to be the main site of lipid deposition in fish, so fish liver appears to be more sensitive and prone to steatosis induced by HFD (Du et al. 2005;Yan et al. 2015;Zhou et al. 2019).
In the present study, fish fed HFD supplemented with different doses of berberine, especially at 98.26 and 196.21 mg/kg diets, decreased lipid deposition in whole body and liver in comparison with those fed the HFD.Berberine is considered as a natural lipidlowering drug, and its effect might be attributed to inhibiting lipogenesis and increasing lipolysis in mammals by AMPK activation (Kim et al. 2009).Our results showed that berberine also decreased lipid accumulation in fish.
In the present study, histological ultrastructural examination showed HFD-induced liver cell damage, such as excessive lipid droplets accumulation, nuclear polarization and other pathologies.These abnormalities might also inhibit lipoprotein secretion and fatty acid oxidation, thereby resulting in a vicious cycle (Du et al. 2008;Lu et al. 2014).However, these pathologies could be alleviated through dietary berberine supplementation, especially at 98.26 and 196.21 mg/kg diets.The protective effect of berberine against the impact of HFD was reported in blunt snout bream (Lu et al. 2017;Zhou et al. 2019), and berberine also attenuated hepatic steatosis, liver mitochondrial damage in zebrafish fed high-cholesterol diet (Chen et al. 2020).Increased plasma TG, TC, NEFA and LDL-C contents are regarded as a risk sign of steatosis, lipid accumulation and liver dysfunction (Du et al. 2008;Yan et al. 2015).In addition, serum ALT and AST activities are common indicators of liver function (Lu et al. 2017;Wang et al. 2019).In the present study, compared with the NFD group, serum TG, TC, NEFA and LDL-C contents, as well as ALT and AST activities in the HFD group increased while the HDL-C level reduced.These evaluated serum parameters caused by the HFD were consistent with their histological results and some other studies in grass carp (Du et al. 2008), large yellow croaker (Yan et al. 2015), blunt snout bream (Lu et al. 2017) and black sea bream (Wang et al. 2019).Conversely, black carp fed HFD containing berberine of 98.26 and 196.21 mg/kg reduced serum TG, TC, NEFA, LDL-C, ALT and AST levels, and increased serum HDL-C content, which suggested that impairment of lipid metabolism and liver function induced by the HFD could be alleviated by dietary berberine supplementation.Similarly, the lower levels of plasma TG, AST and ALT appeared in blunt snout bream fed HFD supplemented with berberine 50 mg/ kg diet (Lu et al. 2017).Xu et al. (2017) also reported that berberine supplementation decreased plasma TG and TC contents in blunt snout bream, but had no effect on plasma HDL-C and LDL-C levels.Hepatic TG and TC contents decreased by berberine exposure in zebrafish pretreated with high-cholesterol diet (Chen et al. 2020).This study also indicated that HFD supplemented with excess berberine had negative effects on lipid-lowering effect, and the reason needs further study.
LPL mainly catalyzes the hydrolysis of TG in plasma chylomicrons (CM) and very low density lipoproteins (VLDL) into fatty acids, which then enters cells (Goldberg 1996;Lindberg and Olivecrona 2002).HL, synthesized and secreted by the liver, is a key ratelimiting enzyme of TG hydrolyzation (Liu et al. 2020).Together with LPL, HL is responsible for the clearance of TG in the circulation.Malate dehydrogenase (MDH) activity is directly related to the production of reduced nicotinamide purine dinucleotide phosphate (NADPH), which in turn affects the lipid synthesis Vol.: (0123456789) (Arnesen et al. 1993).Wang et al. (2005) reported that MDH activities decreased with the increase of dietary lipid level in the liver of cobia (Rachycentron canadum).Hormone-sensitive lipase (HSL) is an important enzyme involved in lipolysis (Ma et al. 2013), whereas FAS is involved in de novo fatty acid synthesis (Jia et al. 2020), which are closely related to the lipid oxidation or storage (Lu et al. 2014).In the present study, HFD had no effect on hepatic HL, LPL, TL and HSL activities of black carp in comparison to NFD.But HFD inhibited liver MDH activity, which was consistent with the report of Wang et al. (2005).Meanwhile HFD boosted liver FAS levels, the increased activity of FAS was also observed in medaka fed HFD (Matsumoto et al. 2010).In this study, HFD supplemented with berberine of 98.26 and 196.21 mg/kg increased liver lipid-metabolizing enzymes activities, suggesting that berberine could enhance lipid lipolysis and fatty acid β-oxidation in liver.Previous studies have shown that HFD supplemented with an appropriate berberine improved fatty acid oxidation and reduced lipid deposition in blunt snout bream (Lu et al. 2017;Zhou et al. 2019) and obese mice (Kim et al. 2009).
According to two-hit hypothesis, increased intrahepatic triglyceride accumulation leads to lipid metabolism disorders, breaking the balance of hepatic lipid metabolism, which is the first hit; the second hit is mainly oxidative stress caused by excessive reactive oxygen species (ROS) generated by lipid deposition, causing lipid peroxidation, resulting in damage to cell structure and function (Day and James 1998;Tessari et al. 2009).In order to resist oxidative stress damage, fish have evolved an antioxidant defense system that includes antioxidant enzymes, such as SOD, CAT, GPx and GR, and non-enzyme antioxidant as GSH (Lu et al. 2017;Ming et al. 2020).MDA is one of the stable end products of lipid peroxidation, so it is also used to assess oxidative stress and lipid peroxidation in aquatic animals (Drape et al. 1993;Ming et al. 2015).
In the present study, liver antioxidant enzymes activities, as well as T-AOC and GSH levels in the HFD group were significantly lower than those of the NFD group, while MDA content in the HFD group exhibited an opposite result.These results are similar to the reports in the liver of blunt snout bream (Xu et al. 2017)  salmoides) (Guo et al. 2019) fed high-fat diets.The elevated MDA level of fish fed HFD indicated an imbalance between the generation and removal of ROS, and GSH also played an important role in eliminating ROS.Our results showed that HFD supplemented with an appropriate berberine increased the antioxidases activities of SOD, CAT, GPx and GR, and T-AOC and GSH levels, while decreased MDA content in the liver of black carp.Berberine has a strong free radical scavenging ability to quench superoxide anions and nitric oxide (Chen et al. 2020;Shan et al. 2011), and induces antioxidant defenses by increasing the levels of antioxidases and nonenzymatic antioxidants (Lee et al. 2003;Lu et al. 2017).Previous studies also reported that HFD supplemented with berberine 50 or 100 mg/kg increased the antioxidative enzymes activities and GSH content, and reduced the MDA levels in the liver of blunt snout bream (Lu et al. 2017;Xu et al. 2017).
AMPK plays a key role in regulating lipid metabolism, sensing the AMP/ATP ratio and regulating energy homeostasis (Garcia and Shaw 2017;Hardie et al. 2012).Activated AMPK regulates lipid metabolism through inhibiting lipogenesis and promoting fatty acid oxidation (Nie et al. 2020;Ran et al. 2021).In the current study, the mRNA expression levels of ampk subunits (ampkα1, ampkα2, ampkβ1, ampkβ2, ampkγ1, ampkγ2 and ampkγ3) in the HFD group were significantly lower than those in the NFD group, which indicated that HFD inhibited ampk mRNA expression.HFD supplemented with berberine of 98.26 or 196.21 mg/kg diet significantly increased mRNA expression of ampk subunits.Some previous studies suggested that berberine could promote lipolysis and suppress lipogenesis in liver cells or adipocytes through the activation of AMPK pathway (Kim et al. 2009;Ren et al. 2020;Zhu et al. 2019).
In order to further explore the lipid-lowering mechanism of dietary berberine, we analyzed the relative expression levels of ampk downstream lipometabolism-related genes involved in lipolysis (pparα, cpt-1, acox, atgl and hsl), lipogenesis (srebp-1, acc1, gpat, fas and pparγ) and transporter (fatp, fabp and fat/ cd36) pathways.It is believed that pparα could activate lipid catabolism by regulating the expression of target genes encoding enzymes including mitochondrial and peroxisomal β-oxidation, such as cpt-1 and acox (Yoon 2009).cpt-1 and acox are the rate-limiting enzymes for fatty acid β-oxidation, in which cpt-1 is located in the outer membrane of mitochondria and controls the entry of long-chain fatty acids into mitochondria (Kerner and Hoppel 2000;Lu et al. 2016), while acox specifically catalyzes the β-oxidation of long-chain and very longchain fatty acids in peroxisomes (Morais et al. 2007).atgl and hsl are the major rate-limiting enzymes regulating triglyceride hydrolysis (Kim et al. 2016).In this study, the mRNA expression levels of pparα, cpt-1 and acox were down-regulated in the liver of fish fed the HFD, suggesting that HFD caused impaired fatty acids β-oxidation.Similar results were described in HFD-fed medaka (Matsumoto et al. 2010), blunt snout bream (Lu et al. 2014(Lu et al. , 2016) ) and tilapia (Oreochromis niloticus) (Jia et al. 2020).The current study showed that the mRNA expression levels of pparα, cpt-1, acox, atgl and hsl were up-regulated in the liver of fish fed HFD containing berberine of 98.26 or 196.21 mg/kg diet compared with fish fed HFD.This indicated that dietary optimal berberine could improve lipolysis and fatty acid β-oxidation by up-regulating these key genes expression in AMPK pathway.Previous studies also reported that dietary berberine supplementation could enhance fatty acid oxidation and lipolysis in blunt snout bream (Lu et al. 2016;Zhou et al. 2019), obese mice (Kim et al. 2009) and in vitro (Brusq et al. 2006;Ren et al. 2020).
Activation of AMPK also inhibits lipogenesis (Hardie et al. 2012).srebp-1 is a key transcription factor that regulates cholesterol, fatty acid, triacylglycerol and phospholipid synthesis pathways (Minghetti et al. 2011).acc1 is a cytosolic enzyme responsible for the production of malonyl-CoA and plays an important role in the biosynthesis of long-chain fatty acids (Hardie et al. 2012;Wakil and Abu-Elheiga 2009).Activated AMPK could directly phosphorylate GPAT, reducing its activity and inhibiting the synthesis of diglycerides and triglycerides (Hardie et al. 2012;Park et al. 2002).pparγ generally binds to the promoter regions of lipogenic genes to regulate lipogenesis and promote lipid storage (Oku and Umino 2008;Tang et al. 2019), thus it is a master regulator of adipogenesis, especially for hepatic lipogenesis (Gavrilova et al. 2003).In the current study, the mRNA expression levels of srebp-1, acc1, gpat, fas and pparγ were up-regulated in the liver of fish fed the HFD, indicating that HFD enhanced lipogenesis.Tang et al. (2019) also reported that HFD up-regulated the mRNA expression levels of srebp-1 and pparγ in the liver of grass carp.The mRNA expression levels of acc1 and fas were upregulated in the liver of HFD-fed medaka (Matsumoto et al. 2010).Conversely, HFD down-regulated these two genes mRNA expression levels in the liver of tilapia (Jia et al. 2020).The reason needs further study.In the present study, compared with fish fed the HFD, the mRNA expression levels of srebp-1, acc1, gpat, fas and pparγ were down-regulated in the liver of fish fed HFD containing berberine of 98.26 and 196.21 mg/ kg diets.These results were consistent with previous reports in black sea bream (Wang et al. 2021) and blunt snout bream (Zhou et al. 2019).
The uptake of long-chain fatty acids by cells is mainly mediated by transporters, such as FATP, FABP and FAT/CD36, which could contribute to the uptake and transport of fatty acids throughout the cytoplasm (Lu et al. 2014;Yan et al. 2015).In this study, the mRNA expression levels of these three transporters were up-regulated in the liver of fish fed HFD, which indicated the increase of fatty acids uptake and transport, accelerating fatty acids influx into liver and increasing hepatic lipid accumulation (Lu et al. 2014;Yan et al. 2015).Compared with fish fed the HFD, the mRNA expression levels of fatp, fabp and fat/cd36 were down-regulated in the liver of fish fed HFD containing berberine of 98.26 and 196.21 mg/kg diets, which showed that dietary an appropriate berberine could decrease the uptake and transport of fatty acids by down-regulating these transporter genes expression in AMPK pathway, improving hepatic lipid ectopic deposition.Similar result was also reported in the liver of blunt snout bream fed HFD supplemented with berberine of 50 mg/kg diet (Zhou et al. 2019).
In summary, black carps fed HFD resulted in lipid metabolism disorders, extensive lipid accumulation, oxidative stress injury and hepatic histopathological abnormalities, and disrupted expression of lipometabolism-related genes.However, HFD supplemented with an appropriate amount of berberine (98.26 mg/kg or 196.21 mg/kg) could improve growth of juvenile black carp, promote lipid metabolism, enhance antioxidant capacity and reduce excessive lipid deposition of fish.The lipid-lowering mechanism of berberine might be mediated by activating AMPK signaling pathway through upregulating lipolysis genes expression, and down-regulating lipogenesis and transport genes expression.However, dietary excess berberine had negative effect on fish, the reason remains to be further studied.

Fig. 1
Fig. 1 Transmission electron microscope (TEM) images of the liver in juvenile black carp fed the different diets for 60 days.(A) Normal-fat diet (NFD) group; (B) High-fat diet (HFD)

Fig. 2
Fig. 2 Effects of dietary berberine on relative mRNA expression levels of ampkα1, ampkα2, ampkβ1, ampkβ2, ampkγ1, ampkγ2 and ampkγ3 in the liver of black carp fed the different diets for 60 days.Bars represent the means ± SD (n = 3).* indicates a significant difference between NFD and HFD groups in t-test (P < 0.05); Different small letters above the bars indi-

Fig. 3
Fig. 3 Effects of dietary berberine on relative mRNA expression levels of pparα, cpt-1, acox, atgl and hsl in the liver of black carp fed the different diets for 60 days.Bars represent the means ± SD (n = 3).* indicates a significant difference between NFD and HFD groups in t-test (P < 0.05); Different small letters above the bars indicate significant differences among the HFD group and HFD Fig. 4 Effects of dietary berberine on relative mRNA expression levels of srebp-1, acc1, gpat, fas and pparγ in the liver of black carp fed the different diets for 60 days.Bars represent the means ± SD (n = 3).* indicates a significant difference between NFD and HFD groups in t-test (P < 0.05); Different small letters above the bars indicate significant differences among the

Fig. 5
Fig. 5 Effects of dietary berberine on relative mRNA expression levels of fatp, fabp and fat/cd36 in the liver of black carp fed the different diets for 60 days.Bars represent the means ± SD (n = 3).* indicates a significant difference between NFD and HFD groups in t-test (P < 0.05); Different small let-

Table 1
Formulation and proximate composition of the experimental diets a Vitamin premix and mineral premix were purchased from Zhejiang Jingbao Feed Co., Ltd b The following values were measured according to the methods of AOAC Vol:.(1234567890)

Table 3
Effects of dietary berberine on growth performance of black carpValues are expressed as means ± SD (n = 3).* indicates a significant difference between NFD and HFD control groups in t-test (P < 0.05); Values with different superscript letters in the same row are significantly different among the HFD group and HFD berberine-supplemented groups in Turkey's multiple range test (P < 0.05).IBW, initial body weight (g); FBW, final body weight (g); WGR , weight gain rate; SGR, specific growth rate; FCR, feed conversion ratio

Table 4
Effects of dietary berberine on lipid deposition of black carp (%) Values are expressed as means ± SD (n = 3).* indicates a significant difference between NFD and HFD control groups in t-test (P < 0.05); Values with different superscript letters in the same row are significantly different among the HFD group and HFD berberine-supplemented groups in Turkey's multiple range test (P < 0.05)

Table 5
Effects of dietary berberine on serum biochemical parameters of black carpValues are expressed as means ± SD (n = 3).* indicates a significant difference between NFD and HFD control groups in t-test (P < 0.05); Values with different superscript letters in the same row are significantly different among the HFD group and HFD berberine-supplemented groups in Turkey's multiple range test (P < 0.05).TG, triglyceride; TC, total cholesterol; NEFA, non-esterified fatty acid; LDL-C, low density lipoprotein-cholesterol; HDL-C, high density lipoprotein-cholesterol; ALT, alanine aminotransferase;

Table 6
Effects of dietary berberine on liver lipid metabolism enzymes activities of black carp Values are expressed as means ± SD (n = 3).* indicates a significant difference between NFD and HFD control groups in t-test (P < 0.05); Values with different superscript letters in the same row are significantly different among the HFD group and HFD berberine-supplemented groups in Turkey's multiple range test (P < 0.05).LPL, lipoprotein lipase; HL, hepatic lipase; TL, total lipase; TL = LPL + HL; MDH, malate dehydrogenase; HSL, hormone-sensitive lipase; FAS, fatty acid synthase Vol:.(1234567890)

Table 7
Effects of dietary berberine on liver antioxidant-related parameters of black carp Values are expressed as means ± SD (n = 3).* indicates a significant difference between NFD and HFD control groups in t-test (P < 0.05); Values with different superscript letters in the same row are significantly different among the HFD group and HFD berberine-supplemented groups in Turkey's multiple range test (P < 0.05).SOD, superoxide dismutase; CAT , catalase; GPx, glutathione peroxidase; GR, glutathione reductase; T-AOC, total antioxidant capacity; GSH, reduced glutathione; MDA, malondialdehyde bVol.: (0123456789)