Optimal dietary methionine levels improved the growth performance of juvenileMegalobrama amblycephala
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 ; grass carp, Ctenopharyngodon idella ; blunt snout bream ; Chinese sucker, Myxocyprinus asiaticus ; and juvenile cobia . Similarly, in the present study, the data related to growth performance indicated that Met supplementation (0.84% and 1.28% dietary Met) significantly improved WGR, SGR and FBW of juvenile blunt snout bream compared with the diet without Met supplementation (0.40% methionine diet). This result was consistent with the conclusion in Liao et al.  that dietary methionine played important roles in the growth performance of juvenile Megalobrama amblycephala. In our present study, juveniles fed diets containing graded dietary methionine levels showed no significant difference in whole body composition. Similar results were also reported in large yellow croaker and juvenile cobia [5, 32]. Meanwhile, the present data showed that whole body protein contents in 0.84% and 1.28% groups were slightly higher than that in the 0.40% group, while the lipid contents were slightly lower. Studies in Chinese sucker, blunt snout bream and Indian major carp (Cirrhinus mrigala) also revealed that optimal dietary methionine levels markedly increased crude protein and decreased crude lipid content in whole body composition [27, 31, 33]. However, Yan et al.  reported that whole body protein and lipid contents were significantly increased with increasing dietary methionine up to 1.58% in juvenile rockfish (Sebastes schlegeli). These results suggested that the whole body composition of different fish species in response to dietary methionine varies and might reflect changes in nutrient metabolism. However, the mechanism of the specific-catabolism response to dietary methionine is unclear, and needs to be further investigated.
0.40% dietary methionine impaired hepatic TOR signaling, while improved muscular TOR signaling in juvenileMegalobrama amblycephala
The present study investigated the response of TOR pathway related protein synthesis to dietary methionine. 0.40% dietary Met level inhibited TOR signaling, while 0.84% Met activated TOR signaling in the liver of blunt snout bream; this was evidenced by reduced protein levels of hepatic S6K1 and p-4E-BP1, which are the downstream of TOR that regulate protein synthesis , in the 0.40% diet group. The 0.84% dietary Met induced hepatic S6K1 and p-4E-BP1 protein levels, indicating 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. Prior to this study, Dai et al.  reported that trout hepatocytes treated with amino acids (4*AA) combined with insulin significantly activated 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 methionine . However, TOR pathway response to dietary Met in blunt snout bream showed a tissue-specific and dose-dependent response in this study. Increasing protein levels of S6K1, 4E-BP1 and p-4E-BP1 in the muscle of blunt snot bream were observed in the 0.40% Met diet but not in the 0.84% diet that 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 also were observed in the study by Zeitz et al. , which may be due to the temporal and spatial differences between transcription and translation.
0.40% dietary methionine increased hepatic lipid accumulation, while suppressed lipogenesis in muscle in juvenileMegalobrama amblycephala
In the present study, hepatic SREBP1, ACC and FAS mRNA levels were markedly induced by 0.40% dietary Met compared with 0.84% or 1.28% Met level. SREBP1 is an important nuclear transcription factor in lipid synthesis, that controls the synthesis of enzymes involved in ACC and FAS [38, 39]. These results indicated that low Met (0.40%) may promote liver lipid synthesis via increasing related genes in blunt snout bream. In Atlantic salmon (Salmo salar), methionine deficiency contributes to high FAS activity and triglyceride accumulation in the liver . Met deficiency also induced FAS and SREBP1 expression in rainbow trout . In growing pigs, Met restriction enhanced FAS gene expression and whole lipogenic capacities [4, 8]. Recent studies reports that PI3K/AKT activates SREBPs, major transcriptional regulators of lipid metabolism [42, 43]. AKT activation was reported to be the necessary and sufficient factor for the increase of SREBP1c and lipid accumulation in liver [44, 45]. Yecies et al.  found AKT induces 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%) 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 level (0.84% and1.28%) tended to promote lipogenesis in the muscle in this study. The evidence was that the relative expression levels of lipogenesis genes including SREBP1, ACC, and FAS, were markedly induced by 0.84% and 1.28% dietary Met in the muscle of blunt snout bream. 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 than those in the control group . Latimer et al.  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, different with 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 methionine levels (0.84% and 1.28%) induced fatty acid β-oxidation in both the liver and muscle of juvenileMegalobrama amblycephalathan 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 , were significantly upregulated by 0.84% and 1.28% Met compared with the 0.40% diet. Induced muscular PPARα mRNA levels were found in fish fed the 1.28% Met diet, higher than that in fish fed the 0.40% diet. Rolland et al.  reported the 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 . In the present study, 0.84% dietary Met increased hepatic lipase mRNA expression levels compared with the 1.28% diet, and could catalyze triglyceride . High Met preferentially improved muscular lipolysis, as evidenced by the muscular LPL mRNA level being induced by 1.28% methionine in this study. The results of our present study implied that high dietary Met levels (0.84% or 1.28%) were more conductive to promoting lipolysis in the liver and muscle than the 0.40% dietary Met. These results also revealed the different lipolysis responses to dietary Met in the liver and muscle of juvenile blunt snout bream. The induced lipolysis and β-oxidation not only provide energy for the growth and Met of 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 shown in juvenile silver pompano, Trachinotus blochii (Lacepede, 1801) .
Changes in glucose metabolism in the liver and muscle ofMegalobrama amblycephalain response to dietary methionine were dose dependent
The liver, as the main tissue of glucose homeostasis, plays a key role in regulating intermediary metabolism in response to nutritional status [51, 52]. In the present study, the highest expression levels of GLUT2 were found in the 1.28% Met diet, which promoted glucose transfer between blood and liver and glucose catabolism and might be helpful for stable plasma glucose content . Hepatic GK, PFK and PK expression levels were significantly induced by dietary 0.84% Met level, suppressed by 1.28% dietary Met; while PFK and PK expression in the muscle were increased by the 1.28% diet compared with the control diet (0.40%). The present data about glycolysis revealed that 0.84% dietary Met 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 via increased PK mRNA levels compared with an 0.70% diet . Primary muscle cells of turbot treated with Met deprivation exhibited decreased GK and PK expression levels compared with the control . 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 . 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 , 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 upregulation of the rate-limiting enzymes: PEPCK and G6Pase in the liver. In addition, the study was in accordance with Skiba-Cassy et al. , who found that feeding rainbow trout a high Met diet significantly enhanced the expression of hepatic G6Pase2 and PEPCK 2 h after a meal. And the results also in agreement with Dai et al.  and Lansard et al. , who reported that high levels of amino acid markedly up-regulated 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 muscle described above, Megalobrama amblycephala fed 1.28% diet may activate the Cori cycle between liver and muscle, which can effectively reuse lactic acid, and supply energy and glucose to other tissues . In addition, dietary Met supplementation (0.84% and 1.28%) significantly increased hepatic GS expression to promote hepatic glycogen synthesis. Higher dietary Met tended to enhance glucose and glycogen synthesis, which might be partly due to Met being a glucogenic amino acid . In the muscle, 1.28% dietary Met markedly increased PEPCK mRNA levels in the current study to promote the production of phosphoenolpyruvate, which might help to activate PK and potentially link with lipid catabolism [54, 57, 58].