Proteasome- and Calpain-Mediated Proteolysis, but Not Autophagy, Is Required for Leucine-Induced Protein Synthesis in C2C12 Myotubes.
Proteasome- and Calpain-Mediated Proteolysis, but Not Autophagy, Is Required for Leucine-Induced Protein Synthesis in C2C12 Myotubes.
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DOI：
10.3390/physiologia1010005
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发表时间：
2021-12
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影响因子：
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通讯作者：
Roberts MD
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作者：
Osburn SC;Vann CG;Church DD;Ferrando AA;Roberts MD
肌肉蛋白质合成和蛋白质降解是紧密耦合的过程。鉴于肌肉生长是通过增加净蛋白质平衡来促进的，因此通过氨基酸促进蛋白质合成，同时减少或抑制蛋白质分解可能是促进合成代谢的一种协同策略。然而，有相互矛盾的证据表明，肌肉中的蛋白分解系统的正常功能是动态平衡所必需的。为了澄清这个问题，我们试图确定抑制C2C12肌管中不同的蛋白分解系统与急性和慢性亮氨酸治疗一起是否影响合成代谢的标志物。在实验1中，肌管用含血清和无亮氨酸的DMEM进行1h、6h和24h的处理(每个处理6孔)：(I)二甲基亚砜载体(CTL)，(Ii)2 mM亮氨酸+赋形剂(仅亮氨酸)，(Iii)2 mM亮氨酸+40μM MG132(20S蛋白酶体抑制剂)(亮氨酸+MG132)，(Iv)2 mM亮氨酸+50μM钙蛋白(钙蛋白酶抑制剂)(亮氨酸+钙蛋白)，(V)2 mM亮氨酸+1μM3-甲基腺嘌呤(自噬抑制剂)(Leu+3MA)。与CTL相比，Leu+MG132和Leu+CALP组蛋白质合成水平显著降低(p<；0.05)。在处理24小时时，Leu+MG132细胞的总蛋白产量显著低于其他处理。此外，24小时Leu+MG132处理的细胞内必需氨基酸(EAA)池显著大于其他处理。在后续实验中，肌管分别用CTL、Leu-Only和Leu+MG132处理48h以进行形态学评估。结果表明，与CTL和单纯Leu相比，Leu+MG132产生的肌管明显更小。由于精选蛋白分解抑制剂的使用，我们的数据在范围上是有限的。然而，这是首次有证据表明，MG132和CALP分别抑制了蛋白酶体和钙蛋白酶，阻断了亮氨酸诱导的肌管蛋白合成。此外，长期的Leu+MG132治疗转化为萎缩表型。在体内抑制蛋白酶体是否会减少亮氨酸或EAA诱导的合成代谢仍有待确定。
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.