Background:
In vitro models provide an important platform for the investigation of skeletal muscle growth to inform and extend mechanistic insights in invasive and often logistically challenging human trials. Although these models allow for greater understanding of the mechanistic underpinning of adaptation in skeletal muscle, many models involve supraphysiological dosages and non-physiological conditions which limit translation of findings to humans. The aim of this research was the development and validation of a translational model for the evaluation of sustainable protein sources in stimulating muscle protein synthesis (MPS) and skeletal muscle anabolism using ex vivo human serum. To achieve this overall aim, three primary objectives had to be realised: (i) Development of an in vitro skeletal muscle cell bioassay to measure muscle growth and MPS; (ii) Development of an ex vivo model to evaluate the humoral effect on MPS in response to protein feeding; (iii) Use of a stable isotope technique to evaluate MPS in response to protein feeding in vivo.
Methods:
Changes in cell behavior and adhesion properties were monitored by measuring impedance via interdigitated microelectrodes using the xCELLigence system. MPS was measured by puromycin incorporation using the SUnSET technique, intracellular signalling measured by western blot, and myotube thickness by microscopy. To establish the ability of the bioassay to measure the humoral effect of MPS in response to protein feeding, media was conditioned by ex vivo human serum from fasted, and protein-fed conditions. To evaluate MPS in response to protein feeding in vivo, acute MPS (5 h) was assessed by measuring stable isotope deuterium oxide (D2O) incorporation into m. vastus lateralis skeletal muscle following consumption of sustainable proteins compared with a non-essential amino acid (NEAA) formulation.
Results:
In this presentation we will demonstrate the ability to monitor changes in cell behaviour, cell size and intracellular signalling when conditioning media with ex vivo human serum in response to feeding with alternative proteins. Proteins containing essential amino acids, known regulators of MPS and muscle anabolism, display greater anabolic qualities than isonitrogenous NEAA formulations. We also confirm translation of this in a human in vivo model using stable isotope tracers.
Conclusion:
We have developed a translational model of muscle protein synthetic bioactivity using ex vivo and in vivo methodologies. We have shown that we can impact MPS in vitro using ex vivo human serum to condition media, that MPS in vitro is differentially regulated by ex vivo serum from alternative proteins compared with an isonitrogenous NEAA control, as well as translation of these findings in vivo using stable isotope technology.