Crown A L, He X L, Holly J M, Lightman S L, Stewart C E
Department of Medicine, University of Bristol, Bristol Royal Infirmary, Marlborough Street, Bristol BS2 8HW, UK.
J Endocrinol. 2000 Dec;167(3):403-15. doi: 10.1677/joe.0.1670403.
In an attempt to address the complex and clinically challenging question of the causes of muscle wasting in patients with cachexia, we have developed a primary adult human skeletal muscle cell model. The cultured cells were characterised by immunocytochemistry using antibodies to the myofibrillar protein constituents desmin and titin. Myotube formation was confirmed biochemically by a fourfold increase in the activity of the muscle-specific enzyme creatinine kinase, and myoblast withdrawal from the cell cycle, which is essential for terminal differentiation, was associated with progressive retinoblastoma protein dephosphorylation. Having successfully confirmed the phenotype of these adult human muscle cells, we assessed their interaction with the insulin-like growth factor (IGF) system. IGF-I is known to stimulate myoblast survival, proliferation and differentiation in cell lines, and, like insulin, is a potent anabolic agent in the regulation of protein metabolism. We have shown that IGF-I stimulated both replication and differentiation of myoblasts, whilst fibroblast growth factor-2 stimulated replication but inhibited differentiation. Examining the IGF system during the process of terminal differentiation, we found that both myoblasts and myotubes expressed insulin, IGF-I and insulin-IGF-I hybrid receptors, with the levels of all three receptor types increasing on differentiation. The cells also produced a wide range of IGF binding proteins (IGFBPs) including IGFBP-2, IGFBP-4 and abundant IGFBP-3, which has not been shown to be produced by any other skeletal muscle cell line examined to date. Both insulin and IGF-I had anabolic effects on myotube protein metabolism at physiological concentrations. Insulin was more potent than IGF-I: use of the IGF analogue long R(3)IGF-I demonstrated that the effects of exogenous IGF-I on protein metabolism were not affected by the high levels of endogenous IGFBP production. In summary, we have developed and characterised a clinically relevant in vitro model with which to address the aetiology of muscle wasting associated with chronic catabolic conditions, and we anticipate that future work will enable the development of novel, effective therapeutic interventions.
为了解决恶病质患者肌肉萎缩原因这一复杂且具有临床挑战性的问题，我们建立了一个原代成人人类骨骼肌细胞模型。使用针对肌原纤维蛋白成分结蛋白和肌联蛋白的抗体，通过免疫细胞化学对培养的细胞进行了表征。通过肌肉特异性酶肌酸激酶活性增加四倍，从生化角度证实了肌管形成，而成肌细胞退出细胞周期（这对终末分化至关重要）与视网膜母细胞瘤蛋白的逐步去磷酸化相关。在成功确认这些成人人类肌肉细胞的表型后，我们评估了它们与胰岛素样生长因子（IGF）系统的相互作用。已知IGF-I可刺激细胞系中成肌细胞的存活、增殖和分化，并且与胰岛素一样，是蛋白质代谢调节中一种强大的合成代谢剂。我们已经表明，IGF-I刺激了成肌细胞的复制和分化，而成纤维细胞生长因子-2刺激了复制但抑制了分化。在终末分化过程中检查IGF系统时，我们发现成肌细胞和肌管均表达胰岛素、IGF-I和胰岛素-IGF-I杂交受体，所有这三种受体类型的水平在分化时均增加。这些细胞还产生了多种IGF结合蛋白（IGFBPs），包括IGFBP-2、IGFBP-4和大量的IGFBP-3，迄今为止，尚未发现其他任何所检测的骨骼肌细胞系能产生这种蛋白。胰岛素和IGF-I在生理浓度下对肌管蛋白代谢均有合成代谢作用。胰岛素比IGF-I更有效：使用IGF类似物长效R(3)IGF-I表明，外源性IGF-I对蛋白质代谢的作用不受内源性IGFBP高水平产生的影响。总之，我们已经建立并表征了一个与临床相关的体外模型，用以研究与慢性分解代谢状况相关的肌肉萎缩的病因，并且我们预计未来的工作将能够开发出新颖、有效的治疗干预措施。
