Abstract
PurposeThe 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 Non-Essential Amino Acid (NEAA) or a whey protein hydrolysate (WPH) on stimulating Muscle Protein Synthesis (MPS) in C2C12 skeletal muscle cells.MethodsBlood 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 MPS were determined in vitro by Western Blot and surface sensing of translation technique (SUnSET) techniques, respectively.ResultsMedia 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 (Ser2448), P70S6K and 4E-BP1 phosphorylation (p<0.01, p<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<0.01, p<0.05) compared to NEAA fed serum.ConclusionIn 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.
Highlights
Muscle protein synthesis (MPS) is integral to the repair, growth and maintenance of skeletal muscle and sensitive to nutrient ingestion
Media conditioned by NonEssential Amino Acid (NEAA) fed serum had no effect on protein signalling or MPS compared to fasted, whereas media conditioned by whey protein hydrolysate (WPH) fed serum significantly increased mTOR (Ser2448), P70S6K and 4E-BP1 phosphorylation (p
The European Food Safety Authority (EFSA) advises “human intervention studies assessing the effect of a specific protein source/constituent against another isonitrogenous protein source/constituent were considered as pertinent to the claim, whereas studies controlling for energy only could not be used for the scientific substantiation of these claims" [9] as comparisons of a test protein to isoenergetic, but not isonitrogenous carbohydrate control is more likely to show an effect of protein supplementation [6]
Summary
Muscle protein synthesis (MPS) is integral to the repair, growth and maintenance of skeletal muscle and sensitive to nutrient ingestion. Several studies have assessed the role of protein and amino acids in the regulation of MPS [1,2,3,4]. The importance of appropriate controls in establishing the bioactivity of compounds in human MPS studies has been emphasised in recent reviews by Morton et al [5] and Phillips [6]. These meta-analyses include many studies reporting on the effects of amino acid and protein supplementation on MPS which use either a less appropriate (including carbohydrate and collagen) or no feeding control/placebo [5,7,8]. Validation of appropriate non-bioactive isonitrogenous controls is important for the future evaluation of bioactivity of protein formulations
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