Abstract
Muscle protein synthesis and proteolysis are tightly coupled processes. Given that muscle growth is promoted by increases in net protein balance, it stands to reason that bolstering protein synthesis through amino acids while reducing or inhibiting proteolysis could be a synergistic strategy in enhancing anabolism. However, there is contradictory evidence suggesting that the proper functioning of proteolytic systems in muscle is required for homeostasis. To add clarity to this issue, we sought to determine if inhibiting different proteolytic systems in C2C12 myotubes in conjunction with acute and chronic leucine treatments affected markers of anabolism. In Experiment 1, myotubes underwent 1-h, 6-h, and 24-h treatments with serum and leucine-free DMEM containing the following compounds (n = 6 wells per treatment): (i) DMSO vehicle (CTL), (ii) 2 mM leucine + vehicle (Leu-only), (iii) 2 mM leucine + 40 μM MG132 (20S proteasome inhibitor) (Leu + MG132), (iv) 2 mM leucine + 50 μM calpeptin (calpain inhibitor) (Leu + CALP), and (v) 2 mM leucine + 1 μM 3-methyladenine (autophagy inhibitor) (Leu + 3MA). Protein synthesis levels significantly increased (p < 0.05) in the Leu-only and Leu + 3MA 6-h treatments compared to CTL, and levels were significantly lower in Leu + MG132 and Leu + CALP versus Leu-only and CTL. With 24-h treatments, total protein yield was significantly lower in Leu + MG132 cells versus other treatments. Additionally, the intracellular essential amino acid (EAA) pool was significantly greater in 24-h Leu + MG132 treatments versus other treatments. In a follow-up experiment, myotubes were treated for 48 h with CTL, Leu-only, and Leu + MG132 for morphological assessments. Results indicated Leu + MG132 yielded significantly smaller myotubes compared to CTL and Leu-only. Our data are limited in scope due to the utilization of select proteolysis inhibitors. However, this is the first evidence to suggest proteasome and calpain inhibition with MG132 and CALP, respectively, abrogate leucine-induced protein synthesis in myotubes. Additionally, longer-term Leu + MG132 treatments translated to an atrophy phenotype. Whether or not proteasome inhibition in vivo reduces leucine- or EAA-induced anabolism remains to be determined.
Highlights
The maintenance of skeletal muscle mass relies upon the achievement of a net neutral protein balance, or the balance of protein synthesis and protein breakdown rates [1]
Protein synthesis is catalyzed in skeletal muscle by ribosomes that exist in the sarcoplasm and between myofibrils, and the mechanistic target of rapamycin complex 1 is the signaling hub that leads to the formation of translation-competent ribosomes [2,3,4]
Effects of 1-h Treatment on mechanistic target of rapamycin complex 1 (mTORC1) Signaling mTOR phosphorylation was significantly higher in all treatments compared to CTL
Summary
The maintenance of skeletal muscle mass relies upon the achievement of a net neutral protein balance, or the balance of protein synthesis and protein breakdown rates [1]. Protein synthesis is catalyzed in skeletal muscle by ribosomes that exist in the sarcoplasm and between myofibrils, and the mechanistic target of rapamycin complex 1 (mTORC1) is the signaling hub that leads to the formation of translation-competent ribosomes [2,3,4]. There is evidence suggesting proteolysis is required for skeletal muscle homeostasis. ATG7 is involved in autophagosome formation, and reports from Masiero and colleagues show robust muscle atrophy occurs in Atg7-null mice [10,11]. While heightened and prolonged proteolysis lead to muscle atrophy, there is evidence to suggest a certain degree of proteolysis is required for muscle mass maintenance
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