Two studies have demonstrated that Glu is elevated in the cerebrospinal fluid of RTT patients

h was 16785615 in accordance with higher level of 2583244 phospho-Akt in KD-C2C12 myoblasts. Conversely, KD-NIH3T3 and KD-MC3T3-E1 cells exhibited greater cleavage of Caspase-3 as compared to their respective si-Con cells. In NIH3T3 and MC3T3-E1 cells, knocking down of IGF-1R reduced total PARP expression . Hence, interpretation of PARP cleavage in these cell lines was difficult. Moreover, pretreatment with wortmannin resulted in increased cleavage of H2O2-induced caspase 3 and PARP levels in both si-Con and KD-C2C12 myoblasts, albeit the cleavage was more pronounced in the si-ConC2C12 myoblasts. To further validate our findings in C2C12 myoblasts, we performed DAPI staining. We found that treating cells with H2O2 for 10 h resulted in a significant reduction in si-Con-C2C12 nuclei number by,59% and KD-C2C12 nuclei number by,41%. However, the reduction in total nuclei numbers was significantly less for KD-C2C12 myoblasts when compared to si-Con-C2C12 myoblasts. In addition, the apoptotic nuclei counted in si-Con-C2C12 myoblasts were,15% of total nuclei, which was significantly higher than the KDC2C12 myoblasts, which showed,7% apoptotic nuclei. These results suggest that deficiency of IGF-1R in C2C12 myoblasts confers resistance to oxidative stress by enhancing Akt phosphorylation. Effect of Akt Activation on its Downstream Substrate Bad Bad promotes apoptosis through the mitochondrial intrinsic death pathway. Akt phosphorylates Bad at serine 136, resulting in dissociation of Bad from Bcl-2/Bcl-XL and increased Bad XAV-939 biological activity association with cytosolic 14-3-3, thereby suppressing pro-death signals. Our result demonstrate that pBad was significantly higher in basal as well as H2O2 or UV treated KD-C2C12 myoblasts. Our data with p-Bad is consistent with increased Akt activation in oxidatively stressed C2C12 cells deficient in IGF-1R, suggesting that Akt exerts its anti apoptotic effect through its downstream substrate Bad. Discussion IGF-1R is a membrane-spanning tyrosine kinase receptor which plays an important role in survival and antiapoptotic pathways through its downstream regulator Akt. On activation, Akt inactivates various pro-apoptotic proteins like, Ask1, BAD, Bax, FoxO transcription factors and caspase-9 by phosphorylating them on specific sites. In the present study we IGF-1R Deficiency Protects C2C12 Myoblasts from H2O2induced Apoptosis The role of Akt in driving anti-apoptotic signaling pathways in cells is well documented. To determine whether significantly higher activation of Akt on H2O2 treatment, protects KD-C2C12 Deficiency of IGF-1 Receptor and Oxidative Stress found that deficiency of IGF-1R in C2C12 myoblasts paradoxically confers resistance to oxidative stress in association with significantly enhanced Akt phosphorylation as compared to control C2C12 myoblasts. The results of Caspase-3 and PARP cleavage assays were in accordance with Akt phosphorylation; more phosphorylation of Akt was associated with reduced cleavage of caspase-3 and PARP, reflecting decreased apoptosis. Moreover, phosphorylation of Bad at the Ser136 Akt phosphorylation site was enhanced in KD-C2C12 cells treated with H2O2. Our findings in C2C12 myoblasts deficient in IGF-1R are similar to Holzenberger et al., where they showed that MEFs isolated from Igf1r+/2 mice were more resistant to oxidative stress induced by hydrogen peroxide. Resistance to oxidative stress was explained by hypophosphorylation of p66 Shc in IGF-1R deficient MEFs. However, the role of Akt in mediating r

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