rease. APN restored loss of mTOR phosphorylation induced by H2O2: Numerous autophagic pathways converge at the mammalian target of rapamycin, which when phosphorylated becomes a potent inhibitor of autophagy. Thus loss of mTOR phosphorylation activates autophagy. In order to determine whether H2O2-mediated autophagy is occurring via an mTOR mediated pathway in our system, phospho-mTOR protein expression was measured. H2O2 decreased phospho-mTOR expression in ARVMs by 3613%. Conversely, APN pretreatment restored phospho-mTOR protein expression back to baseline levels. Beclin-1 protein expression, an upstream promoter of autophagic induced cell death pathways, was also measured. H2O2 decreased beclin-1 protein expression by 352%. However, pretreatment with APN had no significant effect on beclin-1 expression. Finally, neither H2O2 nor APN significantly affected ATG5 or ATG7 expression. 4 Adiponectin Modulates Cardiac Myocyte Autophagy doi: 10.1371/journal.pone.0068697.g002 17266205 H2O2-induced autophagy involves AMPK-mTOR phosphorylation AMPK activation phosphorylates tuberous sclerosis complex 2, which inhibits mTOR and induces autophagy. The role of the AMPK-mTOR dependent pathway in H2O2induced autophagy was therefore investigated. In ARVMs treated with H2O2, p-AMPK protein expression was increased 564%. In some experiments, ARVM were also treated with both compound C and H2O2 and caused complete inhibition of p-AMPK protein expression. APN pretreatment significantly decreased H2O2-induced phospho-mTOR expression almost back to baseline. As shown in either via AMPK activation or inhibiting mTOR; APN modulates H2O2- induced autophagy only by restoring mTOR activation. H2O2 decreased phospho-Akt gene expression which does not involve mTOR Since Akt mediates several processes important to cardiac adaptation including cell death inhibition and metabolism, we sought to determine if Akt was also involved in H2O2-induced autophagy. There was a 212% decrease in phospho-Akt gene expression in ARVMs stimulated with H2O2. Interestingly in ARVM treated with APN alone or H2O2simulated ARVM pretreated with APN, phospho-Akt expression was increased by comparable amounts. In order to examine the role of Akt in the regulation of mTOR by H2O2, ARVM were 487-52-5 biological activity infected with a HA-tagged dominantnegative Akt or -gal. Transduction with dn-Akt had no effect on the decreased H2O2-induced mTOR gene expression 5 Adiponectin Modulates Cardiac Myocyte Autophagy doi: 10.1371/journal.pone.0068697.g003 . These data indicate that H2O2 decreased phospho-Akt expression but is not involved in H2O2induced mTOR phosphorylation and signaling. ERK is required for mTOR phosphorylation in H2O2induced autophagy In general ERK is believed to activate mTOR; however mTOR inhibition has been associated with increased ERK activity in response to non-starvation stress. Since H2O2 increases ERK expression in cardiomyocytes, we sought to investigated the role of the ERK pathway in H2O2induced autophagy using the MEK1/2 inhibitor U0126. H2O2 reduced phospho-mTOR protein expression by 684%. However pretreatment with U0126 only partially restored phospho-mTOR protein expression by 5722%, suggesting incomplete ERK involvement. Additionally, H2O2 increased LC3-II/LC3-I protein expression ratio by a factor of 1.680.36, while ERK inhibition significantly attenuated this increase by 348%. These results indicate that an ERK-mTOR pathway is also at least partially involved in 9521749 H2O2mediated autophagy. APN atte

These experiments demonstrate that activation of the GCGR receptor increases TCF promoter activity

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