In support of the hypothesis that protein prenylation could be involved in impaired activation of mTORC2, the addition of mevalonate has been shown to partially prevent particular aspects of the toxicity of statins about skeletal muscle cells36. Based on the effects of the current study, the promotion of apoptosis by simvastatin can be explained by three mechanisms. simvastatin caused accumulation of the insulin receptor -chain in the endoplasmic reticulum (ER) and improved cleavage of procaspase-12, indicating ER stress. Insulin reduced the manifestation of the insulin receptor -chain but improved procaspase-12 activation in the presence of simvastatin. In conclusion, simvastatin impaired activation of Akt Ser473 most likely as a consequence of reduced activity of mTORC2. Insulin could prevent the effects of simvastatin within the insulin signaling pathway and on apoptosis, but not within the endoplasmic reticulum (ER) stress induction. 0.1% DMSO; +P?0.05 10?M simvastatin. SMV: simvastatin, INS: insulin. Simvastatin treatment improved the synthesis and processing of the insulin receptor and Ziprasidone hydrochloride triggered procaspase-12 in the endoplasmic reticulum In order to explore the toxicity of simvastatin on C2C12 myotubes and the prevention by insulin, we 1st focused on the insulin receptor. In cell lysates, simvastatin significantly decreased the phosphorylation of the insulin receptor (Fig.?3A), while insulin alone or in co-treatment stimulated and prevented the phosphorylation (Fig.?3A). In addition, protein manifestation of the -subunit of the insulin receptor was improved by 10?M simvastatin (Fig.?3A). In contrast, 100?ng/mL insulin decreased the expression of the insulin receptor and prevented the increase in the presence of simvastatin. Subsequently, Ziprasidone hydrochloride we separated the endoplasmic reticulum (ER) and analyzed the manifestation of the insulin receptor. Again, simvastatin alone improved the manifestation of the insulin receptor and insulin prevented this increase (Fig.?3B). Build up of proteins in the ER due to various insults is known to induce ER stress25. We evaluated the induction of ER stress in cells by Western blot analysis using an anti-caspase-12 antibody that recognizes the cleaved and GADD45B the pro forms of this caspase (Fig.?3C). After 24?hours of exposure, simvastatin alone increased cleavage of procaspase-12, indicating ER stress (Fig.?3D). An increase in procaspase-12 was also observed in the presence of insulin and was accentuated for the combination of simvastatin and insulin. After 48?h of exposure, we found that the manifestation of the cleaved form of caspase-12 was stable in the control (DMSO) and insulin samples compared to exposure for 24?hours. However, in the presence of simvastatin or the combination of simvastatin and insulin, cleavage of procaspase-12 was significantly improved compared to incubation for 24?hours (Fig.?3C,D). These findings suggested that simvastatin induced ER stress by retaining proteins such as the insulin receptor in the ER and that insulin improved the ER stress in the presence of simvastatin despite suppressing the synthesis of the insulin receptor -chain. Open in a separate window Number 3 Simvastatin improved protein manifestation of the insulin receptor -chain, but impaired its phosphorylation, and induced endoplasmic reticulum stress in C2C12 myotubes. (A) Quantification of the phosphorylation and total protein manifestation of the Ziprasidone hydrochloride insulin receptor -chain in the whole cells and corresponding Western blots. -actin manifestation was utilized for standardization. (B) Quantification of the insulin receptor -chain manifestation in the rough endoplasmic reticulum and corresponding Western blots. Calreticulin manifestation was utilized for standardization and organelle specificity. (C) Immunoblots showing the full and cleaved forms of the caspase-12 in myotubes treated for 24 and 48?hours. (D) Quantification of the caspase-12 activation. The groups of images were cropped from different blots. Full-length blots are offered in Supplementary Fig.?1. Data symbolize the imply??SEM of three indie experiments. *P?0.05 versus 0.1%DMSO; +P?0.05 versus 10?M simvastatin; $P?0.05 48?hours versus 24?hours. SMV: simvastatin, INS: insulin. Insulin prevented the impairment of protein synthesis and atrogin-1 manifestation in C2C12 myotubes treated with simvastatin Next, we investigated the effects of simvastatin and insulin within the insulin receptor signaling pathway (Fig.?1). As previously reported16, 10?M simvastatin was associated with a significant decrease in Akt phosphorylation in the Ser473, whereas the Thr308 phosphorylation was decreased by pattern only (Fig.?4A). Co-treatment with insulin restored the phosphorylation of Akt at both phosphorylation sites at both concentrations used (Fig.?4A). Activation of Akt indirectly activates mTORC1, which phosphorylates S6K. S6K phosphorylates and therefore activates the ribosomal protein S6 (rpS6), which promotes mRNA translation and thus protein synthesis (Fig.?1). In order to investigate the activity of Akt, we 1st evaluated the Ser9 phosphorylation of GSK3. As demonstrated in Fig.?4B, simvastatin decreased the phosphorylation of GSK3 in the Ser9, which is associated with activation of caspases and apoptosis26. Importantly, insulin partially restored the phosphorylation of GSK3 at Ser9. Ziprasidone hydrochloride Next, we investigated the mRNA manifestation of MAFbx, which encodes for atrogin-1, an ubiquitin.
In support of the hypothesis that protein prenylation could be involved in impaired activation of mTORC2, the addition of mevalonate has been shown to partially prevent particular aspects of the toxicity of statins about skeletal muscle cells36