FHI2 identified skeletal muscle metabolism as common to Healthy Ageing and Performance Nutrition (HAPN) and Glycaemic Management (GM) and coordinated action to validate a ‘translational’ muscle cell model was a key objective. Innovative and productive cross-institutional collaboration between researchers based in HAPN (Phil Jakeman, Brian Carson, Miryam AmigoBenavent & Bijal Patel, UL) and GM (Brendan Egan & Karl Cogan, UCD) sought to evaluate the bioactive potential of milk-based proteins and hydrolysates to regulate skeletal muscle metabolism, specifically muscle protein synthesis and glucose uptake.
Muscle cell culture was established early in the FHI2 programme and mature myotubes subjected to conditioned media known to elicit MPS (Murphy et al 2016) and GLUT4 translocation to validate this approach. The in vitro muscle cell model was then used to challenge the key premise that humoral bioactivity in a non-fed state is elevated in a temporal manner following ingestion of a milk protein and the magnitude and/or temporal response in humoral bioactivity is modified by specific milk protein hydrolysates. To do this the cell culture media was ‘conditioned’ by addition of serum sampled serially from a cohort of subjects sampled from specific populations when fasted and post-ingestion of the milk protein. Difference in cell response to the ‘fed’ vs. ‘fasted’ state provided the criteria for evaluation of beneficial effects between native and hydrolysed milk proteins. The use of human serum, sampled ex-vivo, for subsequent evaluation in cell-based assay in vitro has been applied to representative cohorts of human subjects spanning the HAPN and GM health pillars.
The nature of traditional approaches to cell culture lack direct translation to in vivo whole body physiology. When protein hydrolysates are ingested, for example, a plethora of hormones from several different tissues are released, which stimulate cross-talk between multiple organs to coordinate an integrated systemic response. Due to these hugely dynamic multi-organ responses, direct presentation of a protein to a cell in culture, very rarely replicates the nutrient and humoral bioactives the cell will encounter in vivo. In an attempt to develop an innovative platform to assess bioactive protein hydrolysates under more physiologically relevant conditions, the two work packages have combined human samples and traditional cell culture experimental models to address this problem. The innovation is provided in the conditioning of media using ex vivo postprandial human serum.
Researchers in UCD, led by Dr. Brendan Egan, established an in vitro cell based model to screen hydrolysates on their potential to regulate skeletal muscle function. Specifically, the model focused on investigating the potential bioactivity of protein hydrolysates to regulate glucose transporter protein isoform 4 (GLUT4), an important protein that facilitates glucose uptake in skeletal muscle. This protein is particularly sensitive to changes in hormone secretion after feeding, and represented an ideal functional readout to investigate the bioactivity of hydrolysate-conditioned human serum on GLUT4 translocation. Following productive meetings and interaction between group members on the potential application of combining human samples collected during the HAPN feeding trial with Glycaemic Management bioassays, Prof. Phil Jakeman made the serum samples available for investigation leading to the identification of whey protein hydrolysates as bioactive for GLUT4 translocation. Simultaneously, the HAPN team developed a muscle bioassay investigating the effects of ex vivo human serum on MPS in skeletal muscle cells in vitro. Interestingly, one of the two protein hydrolysates identified in the GLUT4 assay, also displayed bioactive properties above its parent compound in regulating muscle protein synthesis (MPS) using this ex-vivo/in-vitro approach. Dr. Brendan Egan and Karl Cogan, designed and implemented the in-vitro based experiments investigating protein hydrolysate conditioned human serum on GLUT4 translocation while Prof. Phil Jakeman and Dr. Brian Carson designed the experiments investigating the effects of media conditioned by ex vivo human serum on MPS.
Although the ex-vivo/in-vitro approach to screening was initially proposed as part of the HAPN work package, through debate and collaborative discussion at FHI meetings, this innovative platform has spread to inform the work of collaborators within FHI. This extremely productive collaborative endeavour has helped to harmonise work packages and provides an additional approach to the identification, and application, of bioactive peptides to improve human health.
This research has directly contributed to the identification of two protein hydrolysates, currently being investigated in three human trials. Furthermore, this work has directly contributed to two conference presentations entitled ‘An Ex-Vivo/In-Vitro Approach to the Identification of Milk-Derived Peptides with Potential for Glycaemic Control’ (Copenhagen Bioscience Conference: Metabolism in Action; Copenhagen, Denamrk 2017) and ‘Regulation of muscle protein synthesis in vitro using postprandial ex vivo human serum’ (The Physiological Society, H3 Muscle Physiology and Metabolism Symposium, London, UK, 30th November)…..oh, and a small award for “best cross-institutional initiative” within the FHI national technology centre!
Dr. Brian Carson is a Lecturer in Exercise Physiology in the department of Physical Education and Sport Sciences. He is Course Director for the BSc. Sport and Exercise Sciences. View Brian’s profile here. Contact Brian on firstname.lastname@example.org
Phil Jakeman is a Professor of Sport and Exercise Sciences in the department of Physical Education and Sport Sciences at the University of Limerick. View Phil’s profile Here! Contact Phil on: email@example.com