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J Physiol Volume 568, Number 1, 229-242, October 1, 2005 DOI: 10.1113/jphysiol.2005.093906
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Isolation and validation of human prepubertal skeletal muscle cells: maturation and metabolic effects of IGF-I, IGFBP-3 and TNF{alpha}

Malcolm Grohmann1, Emily Foulstone1, Gavin Welsh3, Jeff Holly1, Julian Shield2, Elizabeth Crowne2 and Claire Stewart4

1 Department of Surgery, University of Bristol, Bristol Royal Infirmary, Upper Maudlin Street, Bristol, UK
2 Department of Paediatric Endocrinology, Institute of Child Health, Royal Hospital for Children, Upper Maudlin Street, Bristol, UK
3 Department of Biochemistry, University of Bristol, University Walk, Bristol, UK
4 Department of Exercise and Sport Science, Manchester Metropolitan University, Hassall Road, Alsager, UK

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 {alpha} (TNF{alpha}) 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 TNF{alpha} inhibited myoblast proliferation, differentiation and IGFBP-3 secretion. IGF-I partially rescued the cells from the inhibiting effects of TNF{alpha}. 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/TNF{alpha} action and absence of the inhibitory effect of exogenous IGFBP-3 on differentiation. Additional studies demonstrated that TNF{alpha} 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.

(Received 30 June 2005; accepted after revision 27 July 2005; first published online 4 August 2005)
Corresponding author C. Stewart: Department of Exercise and Sport Science, Manchester Metropolitan University, Hassall Road, Alsager, UK. Email: castewart{at}mmu.ac.uk

E. Crowne and C. Stewart contributed equally to this work.




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