Nutrient metabolism in the liver and muscle of juvenile blunt snout bream (Megalobrama amblycephala) in response to dietary methionine levels

A 75-day rearing trial was designed to study the response of juvenile Megalobrama amblycephala to dietary methionine (Met) levels. Three practical diets with graded Met levels (0.40%, 0.84% and 1.28% dry matter) were prepared to feed the juvenile fish. The results showed that the 0.84% Met diet significantly improved the growth compared with 0.40% diets. Compared with 0.84% and 1.28% Met, 0.40% Met significantly increased the hepatic lipid content, while decreasing the muscular lipid and glycogen contents. 0.40% Met decreased the protein levels of phospho-Eukaryotic initiation factor 4E binding protein-1 (p-4e-bp1), 4e-bp1 and Ribosomal protein S6 kinase 1 in the liver, compared with 0.84% diet, while an increasing trend was observed in the muscle. Met supplementation tended to decrease and increase lipid synthesis in the liver and muscle, respectively, via changing mRNA levels of sterol regulatory element-binding protein 1, fatty acid synthetase and acetyl-CoA carboxylase. 1.28% dietary Met promoted fatty acid β-oxidation and lipolysis in both the liver and muscle by increasing carnitine palmitoyl transferase 1, peroxisome proliferator activated receptor alpha, lipoprotein lipase and lipase mRNA levels. Compared with 0.40% and 0.84% dietary Met, 1.28% Met enhanced the mRNA levels of hepatic gluconeogenesis related genes phosphoenolpyruvate carboxykinase (pepck), and glucose-6-phosphatase, and muscular glycolysis related genes phosphofructokinase (pfk), and pyruvate kinase (pk). The mRNA levels of hepatic pfk, pk and glucokinase were markedly downregulated by 1.28% Met compared with 0.84% level. Muscular pepck, glycogen synthase, and hepatic glucose transporters 2 mRNA levels were induced by 1.28% Met. Generally, deficient Met level decreased the growth of juvenile Megalobrama amblycephala, and the different nutrient metabolism responses to dietary Met were revealed in the liver and muscle.

Blunt snout bream, Megalobrama amblycephala, is the main freshwater aquaculture species in China. Our previous studies have confirmed that dietary methionine (Met) is important for the growth performance of blunt snout bream, and optimal dietary Met could improve the immunity and antioxidant capacity of dietary Met of blunt snout bream [1][2][3] . In addition, Met has been shown to regulate glucose and lipid metabolism in fish. The primary muscle cells of turbot (Scophthalmus maximus L.) under Met deprivation showed inhibited expression of the key target of rapamycin (TOR) pathway elements, and genes related to glycolysis and fatty acid synthesis, while inducing fatty acid β-oxidation 4 . In cobia (Rachycentron canadum), Met deficiency suppressed hepatic lipogenesis related gene (sterol regulatory element-binding protein (srebp1) and fatty acid synthetase (fas)) mRNA expressions and upregulated fatty acid oxidation-related gene (carnitine palmitoyl transferase 1 (cpt1), peroxisome proliferator activated receptor alpha (pparα), and lipoprotein lipase (lpl)), phosphoenolpyruvate carboxykinase (pepck) relative mRNA expression levels 5,6 . Moreover, the nutrient metabolism response to dietary Met showed species-specific responses. Rainbow trout (Oncorhynchus mykiss) fed Met-deficient diets showed a positive relationship with hepatic fas mRNA expression, and negative with cpt1 and fructose-1,6-biphosphatase (fbp) relative mRNA expression levels 7 . While Met deficiency and excess diet induced hepatic lipid accumulation in yellow Table 1. The effects of dietary methionine levels on the growth performance of juvenile blunt snout bream (Megalobrama amblycephala) (means ± SEM) 1 . 1 All data are mean value of three replicates ± SEM (n = 3). Means in the same column with different superscripts "a, b, c" are significantly different (P < 0.05). 2 IBW: initial body weight. 3 FBW: final body weight. 4 Feed conversion ratio (FCR). 5 Specific growth rate (SGR, %/ day). 6 Weight gain rate (WGR, %).
Dietary Met levels (%) IBW 2 (g) FBW 3 (g) FCR 4 SGR 5 (%/day) WGR 6 (%) The protein and gene expression levels of the TOR signaling pathway in the liver and muscle. As shown in Fig. 3a,b, the protein levels of p-4e-bp1, 4e-bp1, S6k1 and Pi3k in the liver of fish fed the 0.84% Met diet were higher than those in fish fed the 0.40% diet. The hepatic protein levels of p-PI3K and Akt in the 0.40% Met group were higher than those in the 0.84% group (Fig. 3b). Contrary to the liver, the protein levels of p-4e-bp1, 4e-bp1, S6k1, Pi3k in the muscle of fish fed the 0.40% Met diet were higher than those in fish fed 0.84% diet; and the p-Pi3k and Akt protein levels in 0.40% and 0.84% diets were similar, and lower than those in the liver (Fig. 3c). The mRNA expression levels were shown in Fig. 3d. Compared with 0.84% dietary Met, 0.40% Met significantly promoted hepatic tor mRNA levels, while hepatic 4e-bp1 mRNA levels were significantly upregulated by 1.28% Met level (P < 0.05). The relative mRNA levels of s6k1 in both the liver and muscle were markedly downregulated by 0.40% dietary Met compared with 0.84% Met level (P < 0.05). Dietary Met levels had no significant effects on muscular tor or 4e-bp1 mRNA expressions (P > 0.05).
The expression of lipid metabolism related genes in the liver and muscle. As presented in Fig. 4a, hepatic srebp1, fas and acetyl-CoA carboxylase (acc) mRNA levels were induced by 0.40% dietary Met level (P < 0.05). Hepatic cpt1 and pparα mRNA levels were increased with increasing dietary Met levels (P < 0.05). Dietary Met levels of 1.28% significantly reduced hepatic lipase (lp) mRNA expression levels compared with 0.84% Met level (P < 0.05). While in the muscle, the mRNA levels of srebp1, fas, acc, cpt1, pparα and lpl were significantly upregulated in fish fed 1.28% Met diet, compared with 0.40% or 0.84% Met diet (P < 0.05) (Fig. 4b).   www.nature.com/scientificreports/ There were no significant differences in the mRNA expression levels of muscular lp and hepatic lpl in fish fed the graded Met level diets (P > 0.05) (Fig. 4a,b). And the mRNA levels of glucose-6-phosphate dehydrogenase (g6pd) in both the liver and muscle were not significantly affected by dietary Met levels (P > 0.05).
The expression of glucose metabolism related genes in the liver and muscle. As presented in Fig. 5a,b, the mRNA expression levels of hepatic pepck and glucose-6-phosphatase (g6pase), and muscular pepck, phosphofructokinase (pfk), pyruvate kinase (pk) and glycogen synthase (gs) were markedly increased by 1.28% Met diet compared with the 0.40% diet (P < 0.05). gk, pfk and pk mRNA levels in the liver were significantly suppressed by 1.28% diet compared with 0.84% diets (P < 0.05). Glucose transporters 2 (glut2) mRNA levels in the liver were significantly induced by 1.28% Met diet compared to 0.84% diet (P < 0.05); while muscular glut4 and fbp, and hepatic gs and fbp mRNA levels were not markedly affected by dietary Met levels (P > 0.05).

Discussion
Optimal dietary Met levels improved the growth performance of juvenile Megalobrama amblycephala. The optimal Met levels in diets could improve the growth performance of fish, which has been proven in many fish species, such as large yellow croaker, Pseudosciaena crocea R 21 ; juvenile humpback   6,25 . In other related studies, some studies revealed that optimal dietary Met levels markedly increased crude protein and decreased crude lipid content in whole body composition such as in Chinese sucker and Indian major carp (Cirrhinus mrigala) 1,26 . Whereas Yan et al. 27 reported that whole body protein and lipid contents were significantly increased with increasing dietary Met up to 1.58% in juvenile rockfish (Sebastes schlegeli). These results suggested that the various change of whole body composition of fish species in response to dietary Met maybe species-specific, due to the different metabolic systems of fish species. However, the mechanism of the specific-    www.nature.com/scientificreports/ metabolism response to dietary Met is unclear, and needs to be further investigated. In addition, 0.40% Met level significantly increased the lipid content in the liver compared with high Met diets. A Met-deficient diet was also found to increase the hepatic lipid content of broiler 28 and P. fulvidraco 8 . In the present study, it was observed that the lipid content in the muscle was very low (< 1%), and 0.40% dietary Met level as well decreased lipid content in muscle. Therefore, the lack of dramatic difference in crude lipid content of body composition might be related to the differences in lipid accumulation in tissues, which also implied that the tissue-specific metabolism in blunt snout bream fed graded methionine diets.
0.40% dietary Met decreased hepatic TOR signaling, while improved muscular TOR signaling in juvenile Megalobrama amblycephala. The present study also investigated the response of TOR pathway related protein synthesis to dietary Met. 0.40% dietary Met level decreased TOR signaling in the liver of blunt snout bream; this was evidenced by reduced protein levels of hepatic S6k1 and p-4e-bp1, the downstream of TOR that regulate protein synthesis 29 , in the 0.40% diet group. This result indicated that the liver of blunt snout bream is sensitive to Met via TOR pathway, and 0.84% Met diet could promote hepatic protein synthesis compared with the 0.40% diet. Before this study, Dai et al. 30 reported that trout hepatocytes treated with four-fold amino acids (4 × AA) combined with insulin significantly activated the TOR pathway compared with the control. The present study was also consistent with the finding that TOR pathway key genes in porcine mammary epithelial cells were significantly increased by a mix of d-and l-Met compared with no Met 31 . However, TOR pathway response to dietary Met in blunt snout bream showed a tissue-specific and dose-dependent response in this study. Increased protein levels of S6k1, 4e-bp1 and p-4e-bp1 in the muscle of blunt snout bream were observed in the 0.40% Met diet but not in the 0.84% diet that is similar to the liver. Additionally, the trends in the mRNA expression levels of TOR pathway key genes were different from the trends at the protein level. Similar phenomena were also observed in the study by Zeitz et al. 32 , which may be due to the temporal and spatial differences between transcription and translation.

0.40% dietary Met increased hepatic lipid accumulation related genes expression, while suppressed lipogenesis in the muscle of juvenile Megalobrama amblycephala. Hepatic srebp1, acc
and fas mRNA expression levels were markedly induced by 0.40% dietary Met compared with 0.84% or 1.28% Met levels, which was consistent with the result in lipid content of liver. SREBP1 is an important nuclear transcription factor in lipid synthesis, that controls the synthesis of enzymes involved in ACC and FAS 33,34 . Met restriction enhanced whole lipogenic capacities of growing pigs 35,36 . The current results might indicate that low Met (0.40%) promoted liver lipid synthesis via increasing related genes in blunt snout bream. Similar results were also found in other fish species. In Atlantic salmon (Salmo salar), Met deficiency contributed to high Fas activity and triglyceride accumulation in the liver 37 . Met deficiency also induced fas and srebp1 expression in rainbow trout 38 . Recent studies report that PI3K/Akt activates SREBPs, major transcriptional regulators of lipid metabolism 39,40 . Akt activation was reported to be the necessary and sufficient factor for the increase of SREBP1C and lipid accumulation in the liver 41,42 . Yecies et al. 43 found that Akt could induce hepatic SREBP1C and lipogenesis via parallel mTORC1-dependent and mTORC1-independent pathways. In the current study, the protein levels of p-Pi3k and Akt in the liver were increased by 0.40% Met, consistent with srebp1 but not tor, which might imply that low dietary Met level (0.40%) potentially increased hepatic lipid accumulation in a Pi3k/ Akt-srebp1 independent TOR manner. In contrast to the lipid synthesis promoted by 0.40% Met in the liver, high dietary Met levels (0.84% and 1.28%) tended to promote lipogenesis in the muscle in this study. The evidence was that the mRNA levels of muscular lipogenesis genes including srebp1, acc, and fas, as well as the lipid content in the muscle, were markedly induced by 0.84% and 1.28% dietary Met. The results were in line with turbot primary muscle cells treated with Met deprivation, which significantly reduced the relative mRNA expression of fas and srebp1 compared to those in the control group 4 . Latimer et al. 13 demonstrated similar results: rainbow trout fed Met restricted diets for 8 weeks showed increased fat accumulation in the liver and decreased fat accumulation in the muscle. Meanwhile, compared with the elevated Pi3k/Akt in the liver induced by the 0.40% diet, the protein levels of p-Pi3k and Akt in the muscle were both very low compared with those in the liver in the 0.40% and 0.84% Met dets. The results indicated that Met regulated lipogenesis was species-dependent in fish.
Higher dietary Met levels (0.84% and 1.28%) induced fatty acid β-oxidation in both the liver and muscle of juvenile Megalobrama amblycephala than 0.40% diet. Unlike lipogenesis, β-oxidation is a process of fatty acid degradation, which supplies energy for the body. In the present study, higher dietary Met levels (0.84% and 1.28%) induced fatty acid β-oxidation in both the liver and muscle of Megalobrama amblycephala, which was demonstrated by the expression levels of pparα (except in muscle) and its downstream: cpt1 44 , were significantly upregulated by 0.84% and 1.28% Met compared with the 0.40% diet. Induced muscular pparα mRNA levels were found in the fish fed the 1.28% Met diet, higher than that in fish fed the 0.40% diet. Rolland et al. 7 reported similar results in rainbow trout that hepatic cpt1 expression levels in the low Met group were lower than those in the high Met group. In juvenile tiger puffer (Takifugu rubripes), lipolytic gene (acox1 and hsl) expression levels were significantly induced by high dietary Met 9 . In the present study, 0.84% dietary Met increased hepatic lp mRNA levels compared with the 1.28% diet, and could catalyze triglyceride 45 . High Met preferentially improved muscular lipolysis, as evidenced by the muscular lpl mRNA level being induced by 1.28% Met in this study. The results of the present study implied that high dietary Met levels (0.84% or 1.28%) were more conducive to promoting lipolysis in the liver and muscle than the 0. www.nature.com/scientificreports/ juvenile blunt snout bream but also may partly contribute to the plasma TG and TC contents that did not show significant differences among the experimental groups. Similar results were also reported in juvenile silver pompano, Trachinotus blochii (Lacepede, 1801) 46 .

Changes in glucose metabolism in the liver and muscle of Megalobrama amblycephala in response to dietary Met were dose-dependent.
The liver, as the main tissue responsible for glucose homeostasis and plays a key role in regulating intermediary metabolism in response to nutritional status 47,48 . In the present study, the highest mRNA levels of glut2 were found in the 1.28% Met diet, which promoted glucose transfer between blood and liver and glucose metabolism, which might be helpful for stable plasma glucose content 49 . Hepatic gk, pfk and pk relative mRNA expression levels were significantly induced by dietary 0.40% and/or 0.84% Met levels, suppressed by 1.28% dietary Met; while pfk and pk mRNA levels in the muscle were increased by the 1.28% diet compared with the control diet (0.84%). The present data about glycolysis revealed that lower dietary Met (0.40-0.84%) potentially promoted hepatic glucose utilization while muscular glucose utilization was enhanced by 1.28% dietary Met. Similar results were observed in cobia, in which 1.24% dietary Met enhanced hepatic glycolysis by increasing pk mRNA levels compared with an 0.70% diet 5 . Primary muscle cells of turbot treated with Met deprivation exhibited decreased gk and pk expression levels compared with the control 4 . The energy released by enhanced glycolysis in both the liver and muscle may contribute to the growth of blunt snout bream. In addition, the increased muscular glycolysis in the 1.28% diet may provide a substrate for lipid synthesis as shown in this study 50 . Additionally, in the current study, 0.40% dietary Met significantly induced hepatic gk and pfk expression levels compared with 1.28% Met level, while pk was not impacted. This result indicated that 0.40% Met potentially promoted the preparation stage of glycolysis but did not promote entry into the energy release stage 50 , which may be part of the reason that low dietary Met led to poor growth. Regarding gluconeogenesis, another way of glucose metabolism, juvenile blunt snout bream fed 1.28% Met diet showed marked mRNA levels of the rate-limiting enzymes: pepck and g6pase in the liver. Also, the study was in line with the findings of Skiba-Cassy et al. 51 , who equally found that feeding rainbow trout with a high Met diet significantly enhanced the expression of hepatic g6pase2 and pepck 2 h after a meal. And the results were also in agreement with Dai et al. 30 and Lansard et al. 52 , also reported that high levels of amino acid could markedly up-regulate hepatic gluconeogenic gene mRNA levels in trout compared with those in fish treated with one-fold amino acid. Interestingly, combined with the hypothesis about glycolysis in the muscle described above, Megalobrama amblycephala fed 1.28% diet may activate the Cori cycle, that increased gluconeogenesis in the liver and resulting glucose is transported through the blood to the muscle where it is either utilized through glycolysis to supply energy demands of muscle contraction or build up muscle glycogen stores through glycogenesis 53,54 . The muscular glycogen synthesis was promoted by dietary Met supplementation (0.84% and 1.28%) that significantly increased muscular gs expression and glycogen contents in the present study. Higher dietary Met tended to enhance glucose and glycogen synthesis, which might be partly due to Met being a glucogenic amino acid 7 . In the muscle, 1.28% dietary Met markedly increased pepck mRNA relative expression levels in the current study to promote the production of phosphoenolpyruvate, which might help to activate PK and potentially link with lipid metabolism 50,53,55 .

Conclusions
In summary, this study revealed that 0.84% dietary Met could the enhance growth performance of juvenile blunt snout bream. Dietary Met levels had no significant effect on the body composition and plasma parameters of the fish. However, 0.40% Met level markedly increased hepatic lipid content in a Pi3k/Akt-srebp1 independent TOR manner. 0.84-1.28% Met markedly increased the contents of lipid and glycogen in muscle by increasing related genes' expression levels. The 0.40% dietary Met downregulated hepatic key TOR signaling genes, while improved muscular TOR signaling. The influence of nutrient metabolism in blunt snout bream in response to dietary Met levels in a tissue-specific and dose-specific manner (Fig. 6). Experimental design and diets. Given the dietary Met requirement of juvenile blunt snout bream was determined by Liao et al. 1 , three isonitrogenous (35% protein) and isoenergetic (18 kJ/g) feeds with the followed graded dietary Met levels were formulated: 0.40% (deficient), 0.84% (optimal, control) and 1.28% (excess). The composition of the basal feed was shown in Table 3. The composition and amino acid contents of the experimental diets (Table 4) are the same as those shown in our previous study 3 . It is worth noting that blunt snout bream, as an herbivorous fish, can digest plant protein well as fish meal 56,57 . Therefore, we used rapeseed meal and soybean meal as the main protein source in this study, which was to obtain more knowledge in practical situation, and to provide data support for practical production. As described in our previous study 1 , the pellet diets were processed by F-26 (II) (South China University of Technology, China), air-dried, and finally stored in a refrigerator at − 20 °C until feeding. Experimental fish and feeding. As mentioned in our previous study 3  www.nature.com/scientificreports/ ery Sciences (Wuxi, Jiangsu, China). After a 15-day acclimation period, the juvenile fish consistent with health and specification (initial weight 4.37 ± 0.01 g) were randomly distributed into nine nylon cages in the pond, and every cage (1 m × 1 m × 1 m) with 20 fish. Each diet was randomly assigned to triplicate cages. Fish were hand-fed three times daily at 7:30, 12:00 and 16:30 for 75 days, until apparent satiation based on visual observation of fish feeding behavior. The water quality was tested weekly (ProDSS Multiparameter Water Quality Meter, YSI, USA), the water temperature was maintained at 28 to 31 °C, pH was maintained from 7.0 to 7.8, ammonia nitrogen was not higher than 0.05 mg/L and dissolved oxygen was higher than 6.0 mg/L. And the photoperiod was the same as natural light (12 h:12 h).
After drawing blood, the abdominal cavity of the fish was cut from the cloaca along the lateral line, and then the liver was sampled. And muscle was taken from the dorsal white muscle. Plasma was obtained by centrifugation of blood samples (3500 × g, 10 min, 4 °C). The other two fish from each cage were collected to test whole body composition. The samples were stored at -80 °C until analyzed.
Analyses of composition and amino acids. The content of moisture, crude protein and lipid, and ash of feeds, whole body and ingredients were analyzed according to the methods described in AOAC 58 . The lipid contents in the liver and muscle were extracted by using chloroform: methanol (C-M) (2:1, v/v) according to the methods described in Peng et al. 59 . The concentrations of amino acid in feeds and ingredients were analyzed by using an amino acid analyzer (SYKAM S-433D, Sykam GmbH, Munich, Germany).  (Table 5) were designed according to the partial cDNA sequences showed in Gao et al. 60 . The assay used β-actin as the reference gene, and the target gene expression levels were analyzed using the 2 −∆∆ct model.

Analyses of plasma parameters and glycogen contents.
Western blot analysis. 50  www.nature.com/scientificreports/ incubation of the membrane and primary antibodies, the membrane was incubated for 1 h with appropriate secondary antibodies. A Beyo ECL Star kit (Beyotime Biotechnology) was used to develop the signal. The bends were scanned and quantified using a chemiluminescence imaging system (Clinx, Shanghai, China). Antibodies against the following proteins were used: phospho-phosphatidylinositol 3-kinase (p-Pi3k, Tyr458/Tyr199) (Cat# 4228), phospho-eukaryotic initiation factor 4E binding protein-1 (p-4e-bp1, Thr37/46) (Cat# 9459), and 4e-bp1 (Cat# 9452) were purchased from Cell Signaling Technology Inc. Pi3k (Cat# 20,584-1-AP), ribosomal protein S6 kinase 1 (S6k1, Cat# 14,485-1-AP) and protein kinase B (Akt, Cat# 10,176-2-AP) were purchased from Proteintech Group, Inc. And β-actin (Cat# AY0573) was purchased from Abways Technology. Among them, antibodies Pi3k, S6k1 and Akt were successfully used in our previous study 16 . The densities of the protein bands were normalized to that of β-actin, which served as an internal control. In addition, the need is, the blots were cut prior to hybridization with antibodies, therefore, original images of full-length blots cannot be provided.
Statistical analysis. Parameters were calculated as followed: Data were analyzed by one-way analysis of variance (ANOVA) and Tukey's multiple comparisons with SPSS 16.0 software. The results are presented as the means with SEM and P < 0.05 indicates a statistical significance. Figures 1, 2, 3b,c,d, 4 and 5 were produced using Prism 5 software.  Weight gain rate (WGR) = 100 × final weight (g) − initial weight (g) initial weight (g)  TTT ACA CGA GCA AGT CTA CGGA  CTT CAT CTT GGC TCA GCT CTCT   4e-bp1 b  GCT GGC TGA GTT TGT GGT TG  CGA GTC GTG CTA AAA AGG GTC   s6k1 c  GGT GCA TGT CAC CTT ATG GG  AGC TGG CAG CAC TTC TAG TC   gk d  GCT TCC ACT GGG ATT CAC CT  CGA CGT TAT TGC CTT CAG