Abstract
Background/aimsMethodological challenges have been associated with the dynamic measurement of muscle protein breakdown (MPB), as have the measurement of both muscle protein synthesis (MPS) and MPB within the same experiment. Our aim was to use the transmethylation properties of methionine as proof-of-concept to measure rates of MPB via its methylation of histidine within skeletal muscle myofibrillar proteins, whilst simultaneously utilising methionine incorporation into bound protein to measure MPS. ResultsDuring the synthesis measurement period, incorporation of methyl[D3]-13C-methionine into cellular protein in C2C12 myotubes was observed (representative of MPS), alongside an increase in the appearance of methyl[D3]-methylhistidine into the media following methylation of histidine (representative of MPB). For further validation of this approach, fractional synthetic rates (FSR) of muscle protein were increased following treatment of the cells with the anabolic factors insulin-like growth factor-1 (IGF-1) and insulin, while dexamethasone expectedly reduced MPS. Conversely, rates of MPB were reduced with IGF-1 and insulin treatments, whereas dexamethasone accelerated MPB. ConclusionsThis is a novel stable isotope tracer approach that permits the dual assessment of muscle cellular protein synthesis and breakdown rates, through the provision of a single methionine amino acid tracer that could be utilised in a wide range of biological settings.
Highlights
The use of stable isotope labelled compounds in musculoskeletal research, through dynamic measurement of rates of metabolism and turnover of proteins across a range of conditions and interventions, has allowed us to gain crucial insights into fuel and protein metabolism in health and disease [1,2]
Incorporation of methyl[D3]-13C-methionine into protein in C2C12 myotubes was observed over time in a non-linear manner (Fig. 2A), and by 48 h had reached an enrichment in cellular protein of 8.5 ± 0.1%
During initial development of the method, we were unable to detect the generation of a fragment containing the 13C mass only via GC-MS, fractional synthetic rates (FSR) calculations were made using the enrichment from the 13C and [D3]-containing compound
Summary
The use of stable isotope labelled compounds in musculoskeletal research, through dynamic measurement of rates of metabolism and turnover of proteins across a range of conditions and interventions, has allowed us to gain crucial insights into fuel and protein metabolism in health and disease [1,2]. One primary method for assessing dynamic MPB involves infusion/incubation of a stable isotope labelled amino acid and measuring its rate of appearance (via dilution of the tracer) from tissues or cells [5,6]. These models have provided important physiological insights into musculoskeletal protein metabolism, there are certain limitations associated with the technique, such as the assumption of steady state, and underestimation of MPB due to the possibility of reutilization of amino acids for MPS [7]
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