Much is known about the positive effects of branched‐chain amino acids (BCAA) in regulating muscle protein metabolism. Comparatively much less is known about the effects of these amino acids and their metabolites in regulating myotube formation. Using cultured myoblasts, we showed that although leucine is required for myotube formation, this requirement is easily met by α‐ketoisocaproic acid, the ketoacid of leucine. We then demonstrated increases in the expression of the first two enzymes in the catabolism of the three BCAA, branched‐chain amino transferase (BCAT2) and branched‐chain α‐ketoacid dehydrogenase (BCKD), with ~3× increase in BCKD protein expression (p < .05) during differentiation. Furthermore, depletion of BCAT2 abolished myoblast differentiation, as indicated by reduction in the levels of myosin heavy chain‐1, troponin and myogenin. Supplementation of incubation medium with branched‐chain α‐ketoacids or related metabolites derivable from BCAT2 functions did not rescue the defects. However, co‐depletion of BCKD kinase partially rescued the defects. Collectively, our data indicate a requirement for BCAA catabolism during myotube formation and that this requirement for BCAT2 likely goes beyond the need for this enzyme to generate the α‐ketoacids of the BCAA.
The mechanistic (mammalian) target of rapamycin complex 1 (mTORC1) signaling is vital for optimal muscle mass and function. Although the significance of mTORC1 in stimulating muscle growth is unequivocal, evidence in support of its role during muscle regeneration is less clear. Here, we showed that the abundance (protein and mRNA) of the mTORC1/S6K1 substrate, programmed cell death protein 4 (PDCD4), is upregulated at the onset of differentiation of L6 and C2C12 cells. The increase in PDCD4 was not associated with any changes in S6K1 activation, but the abundance of beta transducing repeat‐containing protein (β‐TrCP), the ubiquitin ligase that targets PDCD4 for degradation, increased. Myoblasts lacking PDCD4 showed impaired myotube formation and had markedly low levels of MHC‐1. Analysis of poly (ADP‐ribose) Polymerase (PARP), caspase 7 and caspase 3 indicated reduced apoptosis in PDCD4‐deficient cells. Our data demonstrate a role for PDCD4 in muscle cell formation and suggest that interventions that target this protein may hold promise for managing conditions associated with impaired myotube formation.
The importance of branched‐chain amino acids (BCAAs) in promoting skeletal muscle anabolism has been well studied. BCAAs isoleucine, leucine, and valine have been shown to have a profound effect on activating anabolic signaling pathways in skeletal muscle. This occurs in part by the upregulation in activity of the mammalian target of rapamycin complex‐1 (mTORC1), resulting in increased protein synthesis. However, the regulation of branched‐chain amino acids during the development of muscle remains yet to be elucidated. Here, we studied BCAA metabolism during a 5‐day differentiation of L6 myoblasts. Although no change in intracellular BCAA concentrations was observed during differentiation, L6 cells cultured in the absence of leucine were severely impaired in their ability to differentiate as expression of myosin heavy chain (MHC) was completed abrogated at day 5. Two enzymes which are critical for BCAA metabolism are the branched‐chain amino transferase‐2 enzyme (BCAT2) and the branched chain α‐keto acid dehydrogenase complex (BCKD). BCAT2 catabolizes BCAAs to their corresponding alpha‐keto acids, which are then irreversibly decarboxylated by the BCKD complex. The abundance of BCAT2 did not change during differentiation, whereas levels of the E1α subunit of BCKD was increased 7x on day 5 compared to day 1 (p<0.05). Finally, when either BCAT2 or BCKDE1α was knocked down in the presence of leucine, myoblast differentiation was abrogated (BCKDE1α RNAi: MHC decreased 4.5x from day 3 to day 5 (p<0.05); BCAT2 RNAi: MHC levels completely abolished). Our findings suggest that BCAA catabolism may be a critical process in facilitating muscle differentiation.
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