Endocrine Abstracts

Introduction: Prolonged elevations of proinflammatory cytokines are associated with sarcopenia muscle wasting (ageing) and in cancer cachexia. Increased activation of the IGF/insulin pathway is an attractive target for combating such wasting.

Aims: Using rodent skeletal muscle cell lines we have investigated TNF-α/IGF-I interactions, in an attempt to mimic and understand mechanisms underlying skeletal muscle loss.

Methods and results: We report a novel role of IGF-I whereby co-incubation of C2 myoblasts with IGF-I (1.5 ng/ml) and a non-apoptotic dose of TNF-α (1.25 ng/ml; sufficient to block differentiation) unexpectedly facilitate a significant four-fold increase in myoblast death (P<0.05). Using RT-PCR we showed that levels of IGFBP-5 and IGF-II, produced by C2 myoblasts as potent regulators of differentiation and survival, are significantly downregulated (∼60%±3 and 50%±5 respectively, P<0.05) under these conditions. Using array technology we further established that 11 potential apoptotic genes were upregulated >2-fold, including: Caspases 2, 6 and 14, Trafs 2 and 3, Birc 2 and 3, bad, fadd, fas and faim. To address the potential signalling roles in TNF/IGF-induced apoptosis we co-administered the NF-κB inhibitor (10 nM) and observed only a small, non-significant decrease in TNF/IGF-induced death. Similarly Z-VAD-FMK (1 and 5 μM), a pan-caspase specific inhibitor induced a small, non-significant decrease in death. Interestingly, pre-administration of PD98059 (20 μM), a MAPK inhibitor followed by 1.25 ng/ml TNF-α and 1.5 ng/ml IGF-I, reduced death to baseline levels (P<0.05). PI(3)K inhibition, using LY294002 (5 μM), was also shown to reduce myoblast death although levels remained (~2-fold) above baseline. In future we aim to discover the mechanisms by which PD98059 and to a lesser extent LY294002 prevent the death inducing potential of 1.25 ng/ml TNF-α and 1.5 ng/ml IGF-I and promote survival.

Conclusion: These experiments demonstrate that IGF-I (1.5 ng/ml) can facilitate apoptosis in the presence of non-apoptotic doses of TNF-α (1.25 ng/ml), which appears to depend not only on the upregulation of specific apoptosis genes (potentially downstream of MAPK/PI(3)K), but also on the suppression of survival factors IGF-II and IGFBP-5.