20-22 Wildtype mice treated with APAP for 6 hours exhibited increased p-JNK that was not found with Wy-14,643-pretreatment, whereas Ppara-null mice have increased p-JNK following Wy-14,643-pretreatment (Fig. 3C). To ensure that p-JNK was associated with increased activity, kinase assays were performed and increased p-JNK levels were indeed consistent with elevated p-c-jun levels (Fig. 3C, bottom panel). APAP treatment results in a decrease in hepatic levels of GSH at 2 hours and 6 hours, due in learn more part to the production of the quinone NAPQI from APAP that is rapidly neutralized by GSH conjugation by glutathione S-transferase.

This decrease was partially restored by Wy-14,643-pretreatment. However, the maintenance of GSH levels was even more pronounced in isolated mitochondria (Fig. 4A). H2O2 levels are inversely correlated with GSH levels and reflect increase oxidative stress. Indeed, Wy-14,643-pretreatment decreased H2O2 levels elevated by APAP treatment, and this was most pronounced in isolated mitochondria (Fig. 4B). APAP toxicity is also associated with increased levels of long-chain acylcarnitines in serum that are likely due to mitochondrial damage.15 Metabolomics comparison of serum revealed marked differences in serum metabolites between APAP-treated and Wy-14,643-pretreatment/APAP as indicated by the scores plot

separation of the two groups (Supporting Fig. 4A). This difference was driven, among others, by differences in levels of palmitoylcarnitine that were elevated in APAP-treated mouse serum and normal in Wy-14,643-pretreatment/APAP (Supporting Fig. 4B). Pretreatment with click here Wy-14,643 prior to APAP administration blocks the increase in palmitoylcarnitines, as indicated by direct quantification of palmitoylcarnitine MCE (Fig. 4C). At 2 hours post-APAP treatment, both APAP- and Wy-14,643/APAP-treated mice exhibited

extensive GSH depletion in both the liver and mitochondria (Fig. 4D). Because APAP toxicity results in elevated mitochondrial oxidative stress and mitochondrial damage, a role for UCP2 in Wy-14,643 protection against APAP-induced hepatic damage was investigated. UCPs are located in the mitochondrial inner membrane and are associated with decreased hepatic ROS.23, 24 Wy-14,643 treatment induced UCP2 mRNA in the absence and presence of APAP (Fig. 5A, left panel); similar induction was not observed in Ppara-null mice. Protein levels of UCP2 were also measured in mitochondrial extracts from control and mice treated with Wy-14,643 for 24 hours (Supporting Fig. 5). To determine whether UCP2 has a role in Wy-14,643 protection against APAP hepatotoxicity, Ucp2-null mice were subjected to Wy-14,643 and APAP treatment. Mice lacking expression of UCP2 were not protected against APAP-induced toxicity following Wy-14,643, as revealed by serum ALT and AST enzyme levels (Fig. 5B) and liver histology (Fig. 5C).