Isolation and validation of human prepubertal skeletal muscle cells: maturation and metabolic effects of IGF-I IGFBP-3 and TNFα

摘要

We have developed a primary skeletal muscle cell culture model derived from normal prepubertal children to investigate the effects of insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-3 (IGFBP-3) and tumour necrosis factor α (TNFα) on growth, differentiation and metabolism. Cells of myoblast lineage were characterized morphologically by desmin staining and differentiated successfully into multinucleated myotubes. Differentiation was confirmed biochemically by an increase in creatine kinase (CK) activity and IGFBP-3 secretion over time. IGF-I promoted whilst TNFα inhibited myoblast proliferation, differentiation and IGFBP-3 secretion. IGF-I partially rescued the cells from the inhibiting effects of TNFα. Compared to adult myoblast cultures, children's skeletal muscle cells demonstrated higher basal and day 7 CK activities, increased levels of IGFBP-3 secretion, diminished IGF-I/TNFα action and absence of the inhibitory effect of exogenous IGFBP-3 on differentiation. Additional studies demonstrated that TNFα increased basal glucose transport via GLUT1, nitric oxide synthase and p38MAPK-dependent mechanisms. These studies provide baseline data to study the interactivity effects of growth factors and cytokines on differentiation and metabolism in muscle in relation to important metabolic disorders such as obesity, type II diabetes or chronic wasting diseases.