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The mammalian target of rapamycin (mTOR) has been shown to play a central role in regulating cell growth and metabolism. However, little is known about the function of mTOR in nutrient metabolism in bivalve mollusks. In this study, the role of mTOR in the regulation of nutrient metabolism was investigated in Sinonovacula constricta. First, the activation of mTOR was assayed after starvation and refeeding. Afterwards, the role of mTOR in the regulation of nutrient metabolism was investigated using an activator (MHY1485) or inhibitor (rapamycin) of mTOR. The open reading frame of the S. constricta mTOR is 7416 bp in length and encodes a polypeptide consisting of 2471 amino acids. The mTOR amino acid sequence of S. constricta was highly conserved when compared with other species and had a close evolutionary relationship with the TOR proteins of Crassostrea gigas and Lingula anatine. mTOR was expressed in the intestine, exhalent siphon, labial palppus, muscle, inhalent siphon, gill, mantle, digestive land, and gonad tissue of S. constricta, with the highest expression in muscle. During starvation, the level of phosphorylated mTOR protein was relatively low, and the ratio of LC3II/LC3I protein and the AMPKα mRNA level significantly increased with the increase in starvation time. After feeding, the level of phosphorylated mTOR protein increased from 0.13 to 0.56, and the ratio of LC3II/I protein and AMPKα mRNA level decreased from 1.17 to 0.38. MHY1485 significantly increased the level of phosphorylated 4E-BP1 and significantly decreased the ratio of LC3II/I proteins. Furthermore, MHY1485 significantly increased the mRNA level of the glucose metabolism-related gene glucokinase (GK), significantly decreased the mRNA expression of the G6P gene, and significantly increased the mRNA expression of the lipid synthesis-related genes sterol-regulatory element-binding protein (SREBP) and stearoyl-CoA desaturase (SCD). Rapamycin significantly reduced the level of phosphorylated 4E-BP1 and the mRNA expression of mTOR, and the expression level of phosphorylated 4EBP1 decreased from 0.97 to 0.28. Meanwhile, it also significantly reduced the mRNA expression of glucose metabolism-related genes GK, pyruvate kinase (PK), glucose transporter 1 (GLUT1), and G6P, as well as lipid synthesis-related genes SCD and acetyl-CoA carboxylase (ACC). These results indicate a conserved role of mTOR in regulating nutritional metabolism, including glucose metabolism, lipid synthesis, and autophagy in S. constricta. [ABSTRACT FROM AUTHOR]

The razor clam, Sinonovacula constricta, contains high levels of long-chain PUFA (LC-PUFA), which are critical for human health. In addition, S. constricta is the first marine mollusc demonstrated to possess Δ6 fatty acyl desaturase (Fad) and complete LC-PUFA biosynthetic ability, providing a good representative to investigate the molecular mechanism of sterol regulatory element binding proteins (SREBP) in regulating Δ6 Fad for LC-PUFA biosynthesis in marine molluscs. Herein, S. constricta SREBP and Δ6 Fad promoter were cloned and characterised. Subsequently, dual luciferase and electrophoretic mobility shift assays were conducted to explore the SREBP binding elements in the core regulatory region of S. constricta Δ6 Fad promoter. Results showed that S. constricta SREBP had a very conservative basic helix-loop-helix-leucine zipper motif, while S. constricta Δ6 Fad promoter exhibited very poor identity with teleost Fads2 promoters, indicating their differentiation during evolution. A 454 bp region harbouring a core sequence in S. constricta Δ6 Fad promoter was predicted to be essential for the transcriptional activation by SREBP. This was the first report on the regulatory mechanism of LC-PUFA biosynthesis in marine molluscs, which would facilitate optimising the LC-PUFA biosynthetic pathway of bivalves in further studies. [ABSTRACT FROM AUTHOR]