dl-Methionine supplementation in a low-fishmeal diet affects the TOR/S6K pathway by stimulating ASCT2 amino acid transporter and insulin-like growth factor-I in the dorsal muscle of juvenile cobia (Rachycentron canadum)

Abstract

An 8-week feeding experiment was conducted to investigate the effects of dl-methionine (Met) supplementation in a low-fishmeal diet on growth, key gene expressions of amino acid transporters and target of rapamycin (TOR) pathway in juvenile cobia, Rachycentron canadum. Seven isonitrogenous and isolipidic diets were formulated, containing 0·72, 0·90, 1·00, 1·24, 1·41, 1·63 and 1·86 % Met. Weight gain and specific growth rates increased gradually with Met levels of up to 1·24 % and then decreased gradually. In dorsal muscle, mRNA levels of ASCT2 in the 1·00 % Met group were significantly up-regulated compared with 0·72, 1·63, and 1·86 %. The insulin-like growth factor-I (IGF-I) mRNA levels in the dorsal muscle of fish fed 1·00 and 1·24 % Met were higher than those in fish fed other Met levels. In addition, fish fed 1·24 % Met showed the highest mRNA levels of TOR and phosphorylation of TOR on Ser2448. The phosphorylation of ribosomal p70-S6 kinase (S6K) on Ser371 in the dorsal muscle of fish fed 1·86 % Met was higher than those in the 0·72 % group. In conclusion, straight broken-line analysis of weight gain rate against dietary Met level indicates that the optimal Met requirement for juvenile cobia is 1·24 % (of DM, or 2·71 % dietary protein). Met supplementation in a low-fishmeal diet increased cobia growth via a mechanism that can partly be attributed to Met’s ability to affect the TOR/S6K signalling pathway by enhancing ASCT2 and IGF-I transcription in cobia dorsal muscle.

Effects of dietary methionine on growth performance, muscle nutritive deposition, muscle fibre growth and type I collagen synthesis of on-growing grass carp (Ctenopharyngodon idella)

“…Our current observations indicated that optimal Met level elevated muscle TOR mRNA abundance, the total and phosphorylation of TOR protein levels of on-growing grass carp. These results were consistent with reports in cobia Rachycentron canadum (56) , songpu mirror carp (57) and broiler (58) demonstrating that Met could increase TOR (mTOR) mRNA abundance in the muscle, as well as in grass carp (59) , showing that Met could advance the protein levels of total and phosphorylated TOR in the intestine. Also, we found that optimal Met level reduced the ratio of p-TOR Ser2448 /t-TOR in the current experiment, which was inconsistent with that in cobia (56) and turbot Scophthalmus maximus L. (60) .…”

“…That result might be due to the fact that the effect of methionine on translation was greater than activation to TOR in our result, and similar result could be found in the study of bovine mammary epithelial cells (61) . In addition, optimal methionine level enhanced the protein level of p-S6K1 Ser389 and the ratio of p-S6K1 Ser389 : t-S6K1, while we failed to test any obvious change in the level of p-4E-BP1 Thr37/46 and the ratio of p-4E-BP1 Thr37/46 : t-4E-BP1, as the similar results in cobia (56) . Such a discrepancy between the phosphorylation status of these two proteins (S6 and 4E-BP1) has already been discovered in the muscle of rainbow trout (17) and in the liver of rats (62) in response to dietary methionine intake and highlights the complexity of the signaling network associated with the regulation of the activation of these translation initiation factors.…”

Changes in the PI3K/Akt/TOR signalling pathway after methionine treatment in the primary muscle cells of cobia ( rachycentron canadum )

“…These results indicate that the ability to regulate cell growth in vitro after Met treatment is related to the PI3K/Akt/TOR pathway, and these changes, which regulate cell growth, affect protein synthesis. We previously reported that dietary Met can affect the TOR pathway by stimulating the ASCT2 AA transporter and IGF‐I in cobia dorsal muscle (He et al, 2019). In this study, there were no significant differences in IGF‐I gene expression, indicating that Met might directly affect the PI3K/Akt/TOR pathway by stimulating ASCT2 in cobia muscle cells.…”

“…Western blot protocols were conducted based on our earlier article (He et al, 2019). Cells were lysed using Enhanced RIPA Lysis Buffer (Biosharp Biotech).…”

“…Since mammalian antibodies were used, the AA sequences of the studied cobia proteins were aligned in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to check the identities of the antibodies. All antigenic regions of these antibodies have been successfully used in cobia (He et al, 2019), turbot (Wang, He, et al, 2016; Wang, Mai, et al, 2016), rainbow trout (Dai et al, 2013) and zebrafish ( Danio rerio ; Tian et al, 2018). Next, membranes were incubated with HRP‐labelled goat anti‐rabbit IgG (H + L) secondary antibody (Beijing Ray Antibody Biotech) and developed using Chemiluminescent HRP Substrate (Millipore).…”