Leucine promotes protein synthesis of juvenile white shrimp Litopenaeus vannamei through TOR signaling pathway

Leucine is an essential amino acid for animal growth and development. However, the role of leucine in protein and glucose metabolism in crustaceans remains unclear. In this study, the effects of leucine on TOR pathway and glucose metabolism of Pacific white shrimp (Litopenaeus vannamei) were investigated by a feeding trial and an in vitro experiment. A total of 540 juvenile shrimps (0.67 +/- 0.00 g) were randomly distributed into 18 tanks with 30 shrimps per tank. Shrimp were fed 6 diets with leucine levels of 2.10, 2.30, 2.50, 2.70, 2.90 and 3.10% respectively for 56 days (n = 3). The results showed that 2.50% leucine promoted muscle crude protein content, hepatopancreatic fibrillar cell number and area, and decreased serum and hepatopancreas glucose content. It was found that 2.50% leucine significantly up-regulated the expression levels of key genes in the TOR signaling pathway, including protein kinase B (akt), ras homologue enriched in brain (rheb), solute carrier family 3 member 2 (slc3a2), target of rapamycin (tor), regulatory-associated protein of mTOR (raptor), RPTOR independent companion of mTOR, Complex 2 (rictor), rhodopsin (rho), ribosomal protein S6 kinase (s6k) and eukaryotic translation initiation factor 4E 2 (eif4e2), but down-regulated the expression of tuberous sclerosis 2 (tsc2) and 4E-Binding protein (4e-bp) in hepatopancreas, which suggests that leucine may regulate protein deposition through TOR pathway. Similarly, the protein expression of Akt was also increased at 2.50% dietary leucine level. In addition, 2.50% dietary leucine level significantly increased the gene expressions of hexokinase (hk), pyruvate kinase (pk) and glucose transporter 2 (glut2), and down-regulated the gene expressions of pyruvate carboxylase (pc) and phosphoenolpyruvate carboxy kinase (pepck), which indicates appropriate leucine lowers glucose through glycolysis and gluconeogenesis pathway. Furthermore, the in vitro study showedthat 250 mg/L leucine supplement significantly increased hepatopancreas cell viability and up-regulated the expression levels of tor, akt, slc3a2, s6k, eif4e2, rictor, rho, hk, pk, pepck and glut2 compared with the control group (without leucine). The expression level of Sestrin (sesn) in hepatopancreas cells was down-regulated. These results suggest that appropriate dietary leucine supplementation can promote protein deposition of white shrimp, which may be attributed to the fact that leucine can maintain the structural integrity of hepatopancreas and improve protein synthesis and glucose utilization through TOR signaling pathway. The leucine requirement of Litopenaeus vannamei is estimated to be 2.60%-2.67% of the diet (6.67%-6.88% of dietary protein) according to the polynomial regression analysis of whole-body protein, muscle protein, hepatopancreas glucose and hepatopancreatic fibrillar cell number. These results will help us further understand the effects of leucine on growth and protein deposition in crustaceans.