Oxidative stress in the induction of apoptosis mediated by protein-kinase a in glucose-deprived hepatocytes.

FERRETI AC; MATTALONI SM; OCHOA EJ; LAROCCA MC; FAVRE C

Orlando Fl

Congreso; 17th Annual Meeting de la Society for Free Radical Biology and Medicine.; 2010

Society for Free Radical Biology and Medicine y Society for Free Radical Research International

Nutritional stress regulates survival in eukaryotic cells and thekinase pathways involved are conserved as part of an ancestralresponse to regulate cell viability during substrate scarceness.We have previously demonstrated that 6-h-glucose deprivationin normal cultured hepatocytes induces some of the hallmarks ofapoptosis (caspase 3 activation, nuclear fragmentation) in aPKA-dependent fashion. In this study we first characterized theevents that underlain such apoptotic activation. We found thatglucose withdrawal lead to significant depolarization of themitochondria, which was accompanied by Bax translocation andcytochrome c release. These effects were completely preventedby inhibiting either PKA activity or anchoring. Glucose absencepromotes oxidative stress in different cell types. On this regard,it has been reported that PKA may modulate either theproduction or the scavenging of reactive oxygen species (ROS),by regulating the respiratory chain activity or the transcriptionalactivity of stress-defense genes. Thus, we further analyzedwhether glucose deprivation induces oxidative stress inhepatocytes, PKA role in the oxidative stress generation, andthe relationship of these effects with apoptotic activation. Thecells were cultured for 1-6 h in the presence (C) or absence (G0)of glucose, PKA inhibitor H89, or anti-oxidant vitamin C (vit c)and ROS levels and caspase 3 activity were determined. 2-hglucosedeprivation induced a significant ROS increase whichwas prevented by the presence of H89 or vit c (C: 100 %, G0:149.5 ± 22.9; G0 + H89: 87.4 ± 23.5; G0 + vit c: 109.7 ± 26.2%). The increase in caspase 3 was a later event, not significantuntil 6 h and prevented by vit c (C: 5.1 ± 0.5; G0: 7.1 ± 0.5; G0 +vit c: 4.1 ± 0.7 pU/mg). These results point to a PKA-regulatedoxidative component in the apoptotic activation in glucosedeprivedhepatocytes. In this connection, qPCR assays in thesegroups indicated that PKA inhibition during glucose deprivationleads to an increase in Sod1 and Cat mRNA levels, which partlyexplain the prevention in ROS imbalance with H89. A putativerole of PKA in ROS production during glucose deprivationthrough the phosphorylation of cytochrome c oxidase should notbe disregarded.