Proteasome- and Calpain-Mediated Proteolysis, but Not Autophagy, Is Required for Leucine-Induced Protein Synthesis in C2C12 Myotubes
Abstract
<jats:p>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.</jats:p>
Type
Department
Description
Provenance
Subjects
Citation
Permalink
Published Version (Please cite this version)
Publication Info
Osburn, Shelby C, Christopher G Vann, David D Church, Arny A Ferrando and Michael D Roberts (n.d.). Proteasome- and Calpain-Mediated Proteolysis, but Not Autophagy, Is Required for Leucine-Induced Protein Synthesis in C2C12 Myotubes. Physiologia, 1(1). pp. 22–33. 10.3390/physiologia1010005 Retrieved from https://hdl.handle.net/10161/29756.
This is constructed from limited available data and may be imprecise. To cite this article, please review & use the official citation provided by the journal.
Collections
Scholars@Duke
Chris Vann
Dr. Vann is an exercise physiologist with a research focus centered in skeletal muscle physiology. His research focuses on elucidating mechanisms of tissue-to-tissue crosstalk and understanding how exercise-induced changes in epigenetic, genetic, and protein-level factors relate to health and performance outcomes across the age span. As rates of obesity, cardiometabolic disease, and sarcopenia increase in the U.S., Dr. Vann's research is centered on understanding the role of exercise in improved health outcomes at the molecular level and applying this knowledge to develop precise evidence based exercise interventions.
Unless otherwise indicated, scholarly articles published by Duke faculty members are made available here with a CC-BY-NC (Creative Commons Attribution Non-Commercial) license, as enabled by the Duke Open Access Policy. If you wish to use the materials in ways not already permitted under CC-BY-NC, please consult the copyright owner. Other materials are made available here through the author’s grant of a non-exclusive license to make their work openly accessible.