? Activated AMPK is crucial to achieving resilient hypometabolic state during dormancy in the Echinococcus granulosus larval stages.

LOOS JULIA A; CUMINO ANDREA C

Mar del Plata

Congreso; LVIII Reunión Anual de la Sociedad Argentina de Investigación Clínica, Reunión Sociedad Argentina de Fisiología y XLV Reunión Sociedad Argentina de Farmacología Experimental; 2013

Sociedad Argentina de Investigaciones Clínicas

ACTIVATED AMPK IS CRUCIAL TO ACHIEVING RESILIENT HYPOMETABOLIC STATE DURING DORMANCY IN THE ECHINOCOCCUS GRANULOSUS LARVAL STAGES. Julia A Loos,1,2Andrea C Cumino.1,2 1 CONICET, 2Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata Email: julialoos@hotmail.com AMPK, hypometabolism, Echinococcus granulosus, LKB1, FoxO Molecular pathways which are activated to induce the hypometabolism include stimulation of the AMP-activated protein kinase (AMPK) in nematodes and insects. The inverse relationship between TOR and AMPK activity is critical for long term survival when nutrients are limiting. It is unknown in Echinococcus granulosus larval stages, causative agent of the human hydatidosis. Previously, we identified and analyzed encoding genes for α, β and γ subunits of Eg-AMPK and carried out transcriptional expression studies of these prototypical subunits in the parasite. Furthermore, we demonstrated that an AMPK agonist, the metformin, reduced the vitality of protoscoleces and metacestodes in a dose-dependent manner; decreased the transcriptional expression level of pepck, g6p and mdh, increased the type II fermentation and induced autophagy, indicating that a prolonged exposure to the drug impairs the energy asset of larval stages. In this work, we showed that metformin effects resulted in a mitochondrial membrane depolarization as shown by JC-1 stain, indicating a raise in the AMP levels and the allosteric activation of AMPK. Also, AMPK was highly activated in control protoscoleces and was confirmed its sensibility to metformin by two-fold increases in the amount of active phosphorylated AMPK (Eg-AMPKα-PThr176) assessed by immunoblotting. The phosphorylation of Eg-AMPKα implicates upstream kinases, LKB1 and CaMKK, both identified by their conserved structures in the cestode. Additionally, we identified highly conserved C-terminal hydrophobic amino acids that function as a nuclear export sequence (NES) in the AMPKα and its nuclear expression in control protoscoleces by confocal microscopy was verified. Due to the recent description of FoxO proteins as transcriptional regulators targeted by AMPK linked with signaling during hypometabolism states, we identified the only one FoxO ortholog in E. granulosus genome in accordance to the occurrence in others invertebrates.