Insulin-like growth factor-I protects myoblasts from apoptosis but requires other factors to stimulate proliferation

Insulin-like growth factor-I (IGF-I) has been shown to stimulate myoblast p roliferation for a Limited time after which serum is required to reactivate IGF-I-stimulated myoblast proliferation. The aim of these studies was to d etermine whether IGF-I can stimulate myoblast proliferation and/or inhibit apoptosis alone or whether co-factors are necessary. This was achieved by i nvestigating the proliferative response of L6 myoblasts to IGF-I and horse serum (HS) and by examining the status of cells in terms of cell number, su bstrate adherence, cell viability and DNA laddering following incubation wi th IGF-I and HS. L6 myoblasts proliferate in response to IGF-I for 36 h; failure to respond to IGF-I after 36 h is not due to accumulation of waste products or lack of IGF-I. The addition of a low level (1% v/v) of HS restores the ability of myoblasts to proliferate in response to IGF-I and this supports the existen ce of a mitogenic competence factor. Furthermore, myoblasts failing to prol iferate in response to IGF-I after 36 h regain the capacity to respond to I GF-I for a further period of 36 h when exposed to fetal bovine serum. Following the initial (36 h) phase of IGF-I-stimulated proliferation, remov al of both IGF-I and HS led to a dramatic (60%) reduction in the number of cells fully attached to the culture vessel, with 60% of the completely deta ched cells dead. Agarose gel electrophoresis of extracts from these detache d cells revealed higher levels of DNA laddering than extracts prepared from attached cells with IGF-I present. This suggests that IGF-I acts as a surv ival factor by protecting cells from apoptosis. In conclusion these experim ents support the presence of a mitogenic competence factor in horse serum, which restores the ability of cells to proliferate in response to IGF-I. Un like proliferation protection against apoptosis is achieved by IGF-I or HS independently of each other.