ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2006) 12 P35

Effects of androgens on differentiation of human skeletal muscle-derived cells in vitro

AM Solomon1, ACM Sinanan2, PMG Bouloux1 & MP Lewis2


1Department of Endocrinology, Royal Free and University College Medical School, London, United Kingdom; 2Department of Tissue Engineering, Eastman Dental Insitute, UCL, London, United Kingdom.


Introduction

The mechanism by which hypertrophy of skeletal muscle occurs in response to anabolic androgens is incompletely understood. This study used a cell culture model of human skeletal muscle-derived cells, with samples obtained from consenting subjects and local ethics committee approval. The objective was to determine changes in myogenicity upon exposure to exogenous androgen +/− IGF-1 and to see whether an additive effect would be observed. IGF-1 has been shown to accelerate differentiation in human primary cultures and thus acted as a positive control.

Method

Upon growth to confluence, differentiation of muscle-derived cells was induced using 2% fetal calf serum +/− androgen (testosterone 0–500 nM or DHT 0–30 nM) and/or IGF-1 0–50 ng/ml. Specificity of IGF-1 mediated effects were assessed using a specific IGF-1 receptor antagonist (0–10 ug/ml). All cultures were performed in triplicate.

After 3 (early) or 7 (late) days’ differentiation, immunostaining was performed using primary antibodies directed towards the muscle-specific proteins desmin, and α-sarcomeric actin. The DNA-binding probe DAPI was used for nuclear staining. Using fluorescence microscopy, the relative numbers of myogenic (desmin +) vs non-myogenic (desmin −) cells were counted and % desmin+ve myogenicity calculated. Fusion index was obtained by calculating percentages of nuclei incorporated into myotubes.

Results

Increased myogenicity occurred in cells exposed to IGF-1 (P<0.02) in early differentiation. After 7 days’ differentiation supraphysiological doses of testosterone (+) and IGF-1(+++) both increased fusion index with no observable synergistic effect (P<0.05).

Conclusions

Although androgens may increase muscle hypertrophy by increasing myogenicity of pluripotent muscle-derived cells, this does not appear to occur in the earliest stage of differentiation; mechanisms for increased later fusion need to be further explored. Moreover, this system has potential for investigating the signalling mechanisms associated with the effects of anabolic steroids on skeletal muscle.

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