Cessation of biomechanical stretch model of C2C12 cells models myocyte atrophy and anaplerotic changes in metabolism using non-targeted metabolomics analysis

Abstract

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 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 aicds (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 12 hours 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.

Increased plasma proline concentrations are associated with sarcopenia in the elderly

“…Ribel-Madsen et al found that young, healthy low-birth-weight men showed high plasma concentrations of proline and alanine after high-fat overfeeding, probably due to an increase in insulin-resistance and proteolysis in skeletal muscle [ 31 ]. Ilaiwy et al observed that muscle cell atrophy was associated with increased concentrations of proline and alanine together with other metabolites including glutamine in culture media in vitro [ 32 ], again agreeing with our findings.…”

Identification of a Circulating Amino Acid Signature in Frail Older Persons with Type 2 Diabetes Mellitus: Results from the Metabofrail Study

“…Alanine and glutamic acid are crucial intermediates of muscle energy metabolism and liver-muscle metabolic interchange under both physiologic and pathologic conditions [51][52][53]. Perturbations in alanine and glutamate circulating pool may be indicative of skeletal muscle dysfunction, and are commonly encountered in age-related chronic conditions and models of muscle atrophy [54,55]. Notably, both alanine and glutamic acid levels were positively associated with insulin resistance and risk of T2DM in several independent study cohorts [56][57][58].…”

A Distinct Pattern of Circulating Amino Acids Characterizes Older Persons with Physical Frailty and Sarcopenia: Results from the BIOSPHERE Study

“…The higher levels of asparagine, aspartic acid, and glutamic acid observed in persons with PF&Ss may be suggestive of perturbations in muscle energy metabolism associated with muscle wasting. Interestingly, a pattern of metabolic changes accompany muscle remodeling after disuse, including energy substrate accumulation (e.g., asparagine) in atrophied muscles [ 71 , 72 ].…”