PT - JOURNAL ARTICLE AU - Bijal Patel AU - Martina Pauk AU - Miryam Amigo-Benavent AU - Alice B. Nongonierma AU - Richard J. Fitzgerald AU - Philip M. Jakeman AU - Brian P. Carson TI - A cell-based evaluation of a non-essential amino acid formulation as a non-bioactive control for activation and stimulation of muscle protein synthesis using <em>ex vivo</em> human serum AID - 10.1101/713768 DP - 2019 Jan 01 TA - bioRxiv PG - 713768 4099 - http://biorxiv.org/content/early/2019/07/24/713768.short 4100 - http://biorxiv.org/content/early/2019/07/24/713768.full AB - Purpose The purpose of this study was to compare the effect of treating skeletal muscle cells with media conditioned by postprandial ex vivo human serum fed with either isonitrogenous NEAA or a whey protein hydrolysate (WPH) on stimulating MPS in C2C12 skeletal muscle cells.Methods Blood was taken from six young healthy males following overnight fast (fasted) and 60 min postprandial (fed) ingestion of either WPH or NEAA (0.33 g.kg-1 Body Mass). C2C12 myotubes were treated with media conditioned by ex vivo human serum (20%) for 4 h. Activation of MPS signalling (phosphorylation of mTOR, P70S6K and 4E-BP1) were determined in vitro by Western Blot and subsequent de novo MPS were determined in vitro by Western Blot and surface sensing of translation technique (SUnSET) techniques, respectively.Results Media conditioned by NEAA fed serum had no effect on protein signalling or MPS compared to fasted, whereas media conditioned by WPH fed serum significantly increased mTOR, P70S6K and 4E-BP1 phosphorylation (p&lt;0.01, p&lt;0.05) compared to fasted serum. Furthermore, the effect of media conditioned by WPH fed serum on protein signalling and MPS was significantly increased (p&lt;0.01, p&lt;0.05) compared to NEAA fed serum.Conclusion In summary, media conditioned by NEAA fed serum did not result in activation of MPS. Therefore, these in vitro findings suggest the use of isonitrogenous NEAA acts as an effective control for comparing bioactivity of different proteins on activation of MPS. These findings also confirm that activation of MPS in C2C12 myotubes treated with media conditioned by WPH-fed serum is primarily due to circulating EAA.