Effects of myostatin, a negative regulator of muscle mass, that modulates and improves beef production

本研究旨在分析日本短刺牛（自发性肌生成抑制素缺乏牛：DM牛）双肌表型诱导肌肉质量增加的整个机制。从2005年到2007年为期三年的研究得出了以下几点新信息。1)在DM牛胸最长肌成肌细胞培养物中，肌生长抑制素抑制IGF-2的表达，但不抑制IGF-1的表达，而刺激成肌细胞增殖的HGF和aFGF增强了肌生长抑制素的表达。而肌生长抑制素对HGF的表达无影响。这些发现表明，DM牛肌肉质量的增加在很大程度上取决于成肌细胞中产生的HGF产生的肌肉生长抑制素不足。2)通过微阵列分析，确定与肌生长抑制素细胞内信号通路相关的TGF-β诱导早期基因家族2 （TIEG-2）在DM牛中的表达量是对照牛的4.5倍。siRNA敲低TIEG-1导致细胞生长和肌管形成增加，MyoD家族、MRF4和IGF-2的表达增加。这些发现表明TIEG-1与肌生长抑制素细胞内信号传导密切相关，是一个特定的因素。3)首先从DM成肌细胞中建立了9种克隆的DM成肌细胞系（DMc），然后从DMc中获得了长生克隆的DM成肌细胞系（DMc-t）。这些细胞系非常便于精确地了解肌肉生长抑制素对肌肉分化和生长的作用。4)在IGF-1敲除小鼠和野生型小鼠的肌肉中检测到肌生长抑制素相似。IGF-2在DM牛胸最长肌的表达强于对照牛。在DMc培养中，肌生长抑制素抑制IGF-2表达，对IGF-1表达无影响。而激活素受体IIB （ActRIIB）作为肌肉生长抑制素受体和IGF-1受体的表达不受肌肉生长抑制素的影响。结果表明，肌肉生长抑制素和IGF-2相互作用，肌肉生长抑制素和IGF-1单独作用于肌肉的分化和再生。5)免疫组化结果显示，牛和猪腺垂体的促甲状腺激素中主要含有肌生长抑制素，促皮质激素中检测到ActRIIB。许多肌生长抑制素阳性的促甲状腺激素与ActRIIB阳性的促皮质激素相邻。结果表明，促甲状腺激素释放的肌生长抑制素通过旁分泌途径作用于促肾上腺皮质激素。6)小鼠ACTH腺瘤细胞系，at -20细胞表达ActRIIB。在AtT-20细胞培养中加入肌生长抑制素，可显著抑制细胞中POMC的表达和ACTH的免疫反应性。结果首次表明MSTN直接抑制ACTH细胞中ACTH的产生。目前的研究是按照时间表进行的，所获得的结果可以充分了解肌抑制素对肌肉分化和生长的作用。少

The present study was conducted to analyze the entire mechanisms that double muscled phenotype of Japanese shorthorn cattle (spontaneous myostatin-deficient cattle: DM cattle) induces the increased muscle mass. The research for three years form 2005 to 2007 resulted in several new information as follows.1) In the myoblast (DM myoblast) cultures derived from the M. longissimus thoracis of DM cattle, myostatin suppressed the expression of IGF-2 but not IGF-1, and HGF and aFGF that stimulate myoblast proliferation augmented the myostatin expression. But myostatin did not influence the HGF expression. These findings indicate that the increased muscle mass in DM cattle largely depends on deficient myostatin production by HGF produced in myoblasts.2) By microarray analysis, it was defined that the expression of TGF-β inducible early gene family 2 (TIEG-2) that is associated with myostatin intracellular signal pathway was 4.5 times more in DM cattle than control cattle. Knockdown of TIEG-1 by … More siRNA led the increased cell growth and myotube formation, and further the enhanced expression of number of MyoD family, MRF4 and IGF-2. These findings indicate that TIEG-1 is closely associated with myostatin intracellular signaling as one of specific factors.3) Nine kinds of cloned DM myoblast cell line (DMc) were first established from DM myoblasts and then the inmortal cloned DM myoblast cell line (DMc-t) was produced from DMc. These cell lines are very convenient to precisely understand myostatin action on muscle differentiation and growth.4) Myostatin was similarly detected in the muscle of IGF-1 knockout and wild type mice. IGF-2 expression was stronger in the M. longissimus thoracis of DM cattle than control cattle than control cattle. Myostatin suppressed IGF-2 expession and was no effect on IGF-1 expression in DMc cultures. But the expression of activin receptor type IIB (ActRIIB) to be myostatin receptor and IGF-1 receptor was not influenced by myostatin The results show that myostatin and IGF-2 reciprocally act and myostatin and IGF-1 independently act on muscle differentiation and regeneration of muscle.5) Immunohistochemistry revealed that myostatin was mainly contained in thyrotrophs and ActRIIB was detected in corticotrophs in the adenohypophysis of cattle and pigs. A lot of thyrotrophs positive for myostatin was located adjacently the corticotrophs positive for ActRIIB. The findings show that myostatin released from thyrotrophs acts on corticotrophs by paracrine pathway.6) Mouse ACTH adenoma cell line, AtT-20 cells were expressed ActRIIB. By myostatin addition to the AtT-20 cell cultures, the POMC expression and ACTH immunoreactivity in the cells were substantially suppressed. The results first show that MSTN directly suppresses the ACTH production in ACTH cells.The present study was proceeded according to schedules and obtained the results available to fully understand myostain action on muscle differentiation and growth. Less