Characterisation of the IGF system in a primary adult human skeletal muscle cell model, and comparison of the effects of insulin and IGF-I on proteinmetabolism

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 c haracterised by immunocytochemistry using antibodies to the myofibrillar pr otein constituents desmin and titin. Myotube formation was confirmed bioche mically by a fourfold increase in the activity of the muscle-specific enzym e creatinine kinase, and myoblast withdrawal from the cell cycle, which is essential for terminal differentiation, was associated with progressive ret inoblastoma protein dephosphorylation. Having successfully confirmed the ph enotype of these adult human muscle cells, we assessed their interaction wi th the insulin-like growth factor (IGF) system. IGF-I is known to stimulate myoblast survival, proliferation and differentiation in cell lines, and, l ike insulin, is a potent anabolic agent in the regulation of protein metabo lism. We have shown that IGF-I stimulated both replication and differentiat ion of myoblasts, whilst fibroblast growth factor-2 stimulated replication but inhibited differentiation. Examining the ICF system during the process of terminal differentiation, we found that both myoblasts and myotubes expr essed insulin, IGF-I and insulin-IGF-I hybrid receptors, with the levels of all three receptor types increasing on differentiation. The cells also pro duced a wide range of IGF binding proteins (IGFBPs) including IGFBP-2, IGFB P-4 and abundant IGFBP-3, which has not been shown to be produced by any ot her skeletal muscle cell line examined to date. Both insulin and IGF-I had anabolic effects on myotube protein metabolism at physiological concentrati ons. Insulin was more potent than IGF-I: use of the IGF analogue long R-3 I GF-I demonstrated that the effects of exogenous IGF-I on protein metabolism were not affected by the high levels of endogenous IGFBP production. III s ummary, we have developed and characterised a clinically relevant in vitro model with which to address the: aetiology of muscle wasting associated wit h chronic catabolic conditions, and we anticipate that future work will ena ble the development of novel, effective therapeutic interventions.