Some of these findings have been supported by mechanistic studies in various muscle cell cultures, where IGF-1 , myogenesis  and protein synthesis [10, 12, 13] were increased, and also a more explorative approach using microarrays on muscle biopsies from creatine supplemented individuals revealed cytoskeleton remodelling, protein and glycogen synthesis regulation, as well as cell proliferation and differentiation . Other techniques such as proteomics and metabonomics may reveal additional insight into some of the biochemical effects of creatine supplementation at the protein and metabolite level. see more High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy is
JNK inhibitors high throughput screening a well-established analytical technique for metabolic fingerprinting of biofluids and various tissues and has also been used for elucidating the metabolic effects of dietary factors in both humans [14–17], animals [18–20], and also in cell cultures . These studies have demonstrated that NMR-based metabonomics is extremely efficient in detecting endogenous and exogeneous metabolic perturbations. However, while being capable of identifying biomarkers and
metabolic perturbations, the metabolic network responsible for the perturbations can only be hypothesised. Proteomics displays protein products as a result of gene expression and efficiency of translation, and has been used to separate and identify differentially regulated proteins from in response to various treatments of cultured cells [22, 23] and muscles . Linking information obtained from metabolic fingerprinting with proteomics would pave the way for obtaining a better understanding of the primary pathways
involved in perturbations associated with CMH supplementation. In this study we have for the first time examined and integrated the NMR metabolite profile and the proteomic profile of myotubes in the presence and absence of creatine supplementation in a systems biology approach. Methods Muscle Cell Culture Myotube cultures were established from a mouse myoblast line (C2C12) originally derived from a thigh muscle  (American Type Culture Collection, Manassas, VA). A clone from this cell line, which effectively fused and formed myotubes, was isolated . The clone was grown in 80 cm2 culture flask in 10 mL of medium consisting of Dulbecco’s modified Eagle’s medium (DMEM), 10% (vol/vol) fetal calf serum (FCS), and supplemented with 1% antibiotics giving 100 IU/mL penicillin, 100 μg/mL streptomycin sulfate, 3 μg/mL amphotericin B, and 20 μg/mL gentamycin (growth medium). Cells were maintained in an atmosphere of 95% air and 5% CO2 at 37°C. Prior to confluence, cells were harvested in 0.25% trypsin and sub-cultured into 80 cm2 culture flasks or 96 well plates.