Burn injury-induced IRS-1 degradation in mouse skeletal muscle.

Insulin resistance is a major effect of burn injury and insulin receptor substrate-1 (IRS-1) plays an important role in signal transduction. Here, we explored the integrity of IRS-1 in muscle after burn injury. A murine model of severe burn injury was used to explore IRS-1 integrity/degradation in muscle and to map Ser/Thr phosphorylations which represent the trigger sites for degradation. The findings are: three C-terminal IRS-1 cleavage fragments were confirmed with tandem mass spectrometry; MWs 95, 44 and 42 kD. In sham burn animals the level of intact IRS-1 was 51.9 ng/g, whereas, total IRS-1 which includes degradation fragments and post-translationally modified protein was 196.7 ng/g. After burn, intact and total IRS-1 were reduced to 47.8 (92.1 % sham, p<0.05) and 86.9 ng/g (44.2 % sham, p<0.005). In contrast, ubiquitinated IRS-1 increased from 24.5 to 28.4 ng/g (15.9% increment, p< 0.05) in the burned mice. In cytosol, membrane and nuclear fractions, total IRS-1 was reduced by 89.8% (p<0.005), 25.8% (p<0.05) and 87.3% (p<0.005). To further evaluate the IRS-1 degradation pathway, SOCS-3 mRNA levels after burn injury were found to be increased by 35% (p<0.05), 110% (p<0.05) and 140% (p<0.005). However, phosphorylation of Ser473 and Thr308 of Akt1 were reduced to 26.2% (p<0.05) and 49.8% (p<0.005). We conclude: burn injury is associated with IRS-1 degradation via SOCS-3 and ubiquitin-mediated pathways and reduced subcellular levels of IRS-1, serve as molecular basis for burn injury induced alteration in insulin function.