Effects of berberine on growth performance, lipid deposition and liver ultrastructure of black carp fed high-fat diets
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 the WG and SGR of fish increased with HFD berberine content up to 98.26 mg/kg diet, and then declined. 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 high-cholesterol diet were exposed to berberine at doses of 5 and 25 mM for 10 d, 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 lipid-lowering 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).
Effects of berberine on lipometabolism-related biochemical parameters in the serum and liver of black carp fed high-fat diets
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 inclusion different doses of berberine, especially at 98.26 and 196.21 mg/kg diets, 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 were 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 rate-limiting 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 (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). The activities of liver HL, LPL, TL, MDH and HSL increased in the HFD supplemented with berberine of 98.26 and 196.21 mg/kg diets, while FAS activity reduced, suggesting that berberine could enhance lipid lipolysis and fatty acid β-oxidation in liver. In line with the present study, previous studies have shown that HFD supplemented with 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).
Effects of berberine on antioxidant-related parameters in the liver of black carp fed high-fat diets
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. 2019). 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 SOD, CAT, GPx and GR 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) and largemouth bass (Micropterus 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 (98.26 or 196.21 mg/kg diet) 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 (Shan et al. 2011, Chen et al. 2020), and induces antioxidant defenses by increasing the levels of antioxidases and non-enzymatic 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).
Effects of berberine on lipometabolism-related genes expression of AMPK signaling pathway in the liver of black carp fed high-fat diets
AMPK plays a key role in regulating lipid metabolism, sensing the AMP/ATP ratio and regulating energy homeostasis (Hardie et al. 2012, Garcia et al. 2017). 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, Zhu et al. 2019, Ren et al. 2020).
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 long-chain 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, 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 (Wakil and Abu-Elheiga 2009, Hardie et al. 2012). Activated AMPK could directly phosphorylate GPAT, reducing its activity and inhibiting the synthesis of diglycerides and triglycerides (Park et al. 2002, Hardie et al. 2012). 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 up-regulated in the liver of HFD-fed medaka (Matsumoto et al. 2010). Conversely, HFD down-regulated the 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).
Therefore, we conclude that dietary an appropriate berberine supplementation could decrease lipid deposition and alleviate hepatic steatosis by activating AMPK signaling pathway, up-regulating lipolysis genes expression, and down-regulating lipogenesis and transport genes expression.