Cruz André, Ferian Andrea, Alves Paula K N, Silva William Jose, Bento Mirella Ribeiro, Gasch Alexander, Labeit Siegfried, Moriscot Anselmo Sigari
Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Institute for Integrative Pathophysiology, Faculty for Clinical Medicine Mannheim of the University of Heidelberg, Mannheim, Germany.
DNA Cell Biol. 2020 Nov 2. doi: 10.1089/dna.2020.5423.
Lack of mechanical load leads to skeletal muscle atrophy, and one major underlying mechanism involves the myostatin pathway that negatively regulates protein synthesis and also activates Atrogin-1/MAFbx and MuRF1 genes. In hindlimb immobilization, leucine was observed to attenuate the upregulation of the referred atrogenes, thereby shortening the impact on fiber cross-sectional area, nonetheless, the possible connection with myostatin is still elusive. This study sought to verify the impact of leucine supplementation on myostatin expression. Male Wistar rats were supplemented with leucine and hindlimb immobilized for 3 and 7 days, after which soleus muscles were removed for morphometric measurements and analyzed for gene and protein expression by real-time PCR and Western blotting, respectively. Muscle wasting was prominent 7 days after immobilization, as expected, leucine feeding mitigated this effect. Atrogin-1/MAFbx gene expression was upregulated only after 3 days of immobilization, and this effect was attenuated by leucine supplementation. Atrogin-1/MAFbx protein levels were elevated after 7 days of immobilization, which leucine supplementation was not able to lessen. On the other hand, myostatin gene expression was upregulated in immobilization for 3 and 7 days, which returned to normal levels after leucine supplementation. Myostatin protein levels followed gene expression at a 3-day time point only. Follistatin gene expression was upregulated during immobilization and accentuated by leucine after 3 days of supplementation. Concerning protein expression, follistatin was not altered neither by immobilization nor in immobilized animals treated with leucine. In conclusion, leucine protects against skeletal muscle mass loss during disuse, and the underlying molecular mechanisms appear to involve myostatin inhibition and Atrogin-1 normalization independently of follistatin signaling.
缺乏机械负荷会导致骨骼肌萎缩，一个主要的潜在机制涉及肌生成抑制素途径，该途径会负向调节蛋白质合成，还会激活Atrogin-1/MAFbx和MuRF1基因。在后肢固定模型中，观察到亮氨酸可减弱上述萎缩相关基因的上调，从而缩短对肌纤维横截面积的影响，尽管如此，其与肌生成抑制素之间的可能联系仍不清楚。本研究旨在验证补充亮氨酸对肌生成抑制素表达的影响。给雄性Wistar大鼠补充亮氨酸，并后肢固定3天和7天，之后取出比目鱼肌进行形态测量，并分别通过实时PCR和蛋白质免疫印迹法分析基因和蛋白质表达。正如预期的那样，固定7天后肌肉萎缩明显，亮氨酸喂养减轻了这种效应。Atrogin-1/MAFbx基因表达仅在固定3天后上调，亮氨酸补充可减弱这种效应。固定7天后Atrogin-1/MAFbx蛋白水平升高，亮氨酸补充无法减轻这种升高。另一方面肌生成抑制素基因表达在固定3天和7天时上调，亮氨酸补充后恢复到正常水平肌生成抑制素蛋白水平仅在3天时间点与基因表达一致。卵泡抑素基因表达在固定期间上调，补充亮氨酸3天后加剧。关于蛋白质表达，卵泡抑素在固定组和亮氨酸处理的固定动物组中均未改变。总之亮氨酸可防止废用期间骨骼肌质量丢失，其潜在分子机制似乎涉及独立于卵泡抑素信号传导的肌生成抑制素抑制和Atrogin-1正常化。
