Studies of skeletal muscle disuse, either in patients on bed rest or experimentally
in animals (immobilization), have demonstrated that decreased protein synthesis is
common, with transient parallel increases in protein degradation. Muscle disuse atrophy
involves a process of transition from slow to fast myosin fiber types. A shift toward
glycolysis, decreased capacity for fat oxidation, and substrate accumulation in atrophied
muscles have been reported, as has accommodation of the liver with an increased gluconeogenic
capacity. Recent studies have modeled skeletal muscle disuse by using cyclic stretch
of differentiated myotubes (C2C12), which mimics the loading pattern of mature skeletal
muscle, followed by cessation of stretch. We utilized this model to determine the
metabolic changes using non-targeted metabolomics analysis of the media. We identified
increases in amino acids resulting from muscle atrophy-induced protein degradation
(largely sarcomere) that occurs with muscle atrophy that are involved in feeding the
Kreb's cycle through anaplerosis. Specifically, we identified increased alanine/proline
metabolism (significantly elevated proline, alanine, glutamine, and asparagine) and
increased α-ketoglutaric acid, the proposed Kreb's cycle intermediate being fed by
the alanine/proline metabolic anaplerotic mechanism. Additionally, several unique
pathways not clearly delineated in previous studies of muscle unloading were seen,
including: (1) elevated keto-acids derived from branched chain amino acids (i.e. 2-ketoleucine
and 2-keovaline), which feed into a metabolic pathway supplying acetyl-CoA and 2-hydroxybutyrate
(also significantly increased); and (2) elevated guanine, an intermediate of purine
metabolism, was seen at 12h unloading. Given the interest in targeting different aspects
of the ubiquitin proteasome system to inhibit protein degradation, this C2C12 system
may allow the identification of direct and indirect alterations in metabolism due
to anaplerosis or through other yet to be identified mechanisms using a non-targeted
metabolomics approach.