Methylglyoxal-induced AMPK activation leads to autophagic degradation of thioredoxin 1 and glyoxalase 2 in HT22 nerve cells
Methylglyoxal (MGO) is a major glycating agent that reacts with basic residues of proteins and promotes the formation of advanced glycation end products which are believed to play key roles in a number of pathologies, such as diabetes, Alzheimer's disease, and inflammation. We previously showed that MGO treatment targets the thioredoxin and the glyoxalase systems, leading to a decrease in Trx1 and Glo2 proteins in immortalized mouse hippocampal HT22 nerve cells. Here, we propose that autophagy is the underlying mechanism leading to Glo2 and Trx1 loss induced by MGO. The autophagic markers p62, and the lipidated and active form of LC3, were increased by MGO (0.5 mM). Autophagy inhibition with bafilomycin or chloroquine prevented the decrease in Trx1 and Glo2 at 6 and 18 h after MGO treatment. Proteasome inhibition by MG132 exacerbated the effect of MGO on Trx1 and Glo2 degradation (18 h), further suggesting a role for autophagy. ATG5 small interfering RNA protected Trx1 and Glo2 from MGO-induced degradation, confirming Trx1 and Glo2 loss is mediated by autophagy. In the search for the signals that control autophagy, we found that AMPK activation, a known autophagy inducer, was markedly increased by MGO treatment. AMPK activation was confirmed by increased acetyl coenzyme A carboxylase phosphorylation, a direct AMPK substrate and by decreased mTOR phosphorylation, an indirect marker of AMPK activation. To confirm that MGO-mediated Trx1 and Glo2 degradation was AMPK-dependent, AMPK-deficient mouse embryonic fibroblasts (MEFs) were treated with MGO. Wildtype MEFs presented the expected decrease in Trx1 and Glo2, while MGO was ineffective in decreasing these proteins in AMPK-deficient cells. Overall, the data indicate that MGO activates autophagy in an AMPK-dependent manner, and that autophagy was responsible for Trx1 and Glo2 degradation, confirming that Trx1 and Glo2 are molecular targets of MGO.