Abstract 17681: A Redox Dependent Mechanism for Regulation of AMPK Activation by Thioredoxin1 During Energy Starvation
5’-AMP-activated protein kinase (AMPK) regulates metabolism and survival in cardiomyocytes (CMs). Although energy starvation, known to activate AMPK, increases reactive oxygen species levels, whether or not AMPK is directly regulated by oxidative modification is unknown. We here demonstrate that AMPK is negatively regulated by cysteine oxidation. Glucose deprivation (GD) increased expression of thioredoxin1 (Trx1), a reducing enzyme that cleaves disulfides, in CMs. Downregulation of Trx1 enhanced cysteine oxidation of AMPK and attenuated GD-induced phosphorylation of AMPK at Thr172 (0.6 fold, p<0.05). In vitro kinase assays revealed that AMPK oxidation inhibits its phosphorylation by LKB1, an AMPK kinase, indicating that oxidation suppress AMPK activation. Downregulation of Trx1 exacerbated GD-induced cell death (60% vs. 37% p<0.05). Dominant negative Trx1 transgenic mice (Tg-DN-Trx1) exhibited greater myocardial infarction (MI, 62% vs. 39% p<0.05) than WT mice in response to prolong ischemia. These data suggest that Trx1 plays an essential role in mediating energy-starvation-induced AMPK activation by preventing oxidation. Proteomic analyses showed that knockdown of Trx1 enhances oxidation of AMPKα at Cys130 (16.2% vs. 3.8%, p<0.05) and Cys174 (20.4% vs. 2.3%, p<0.05) during GD. Mutations of either Cys130 or Cys174 to serine abolished Thr172 phosphorylation of AMPK, indicating that maintaining Cys130 and Cys174 in reduced form is required for AMPK activation. In a mouse model of obesity, high-fat diet (HFD) decreased Trx1 expression (-55%, p<0.05) compared to normal diet, and enhanced cysteine oxidation of AMPK, which was accompanied by decreases in Thr172 phosphorylation of AMPK (-45%, p<0.05). However, increased expression of Trx1 in Trx1 transgenic mice (Tg-Trx1) prevented AMPK oxidation and maintained its activity even after HFD treatment. Tg-Trx1 was protected against the HFD-induced exacerbation of MI compared with WT mice (41% vs 65%, p<0.05), suggesting that restoration of Trx1 expression rescues HFD-induced myocardial injury by preventing AMPK oxidation. In conclusion, cysteine oxidation negatively regulates AMPK, and Trx1 acts as an essential co-factor to prevent oxidation and activate AMPK during metabolic stress.
- © 2013 by American Heart Association, Inc.