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

Congreso; 17 th Annual Meeting of the Society for Free Radicals Biology and Medicine (SFRBM); 2010

Society for Free Radicals Biology and Medicine

Nutritional stress regulates survival in eukaryotic cells and the kinase pathways involved are conserved as part of an ancestral response to regulate cell viability during substrate scarceness. We have previously demonstrated that 6-h-glucose deprivation in normal cultured hepatocytes induces some of the hallmarks of apoptosis (caspase 3 activation, nuclear fragmentation) in a PKA-dependent fashion. In this study we first characterized the events that underlain such apoptotic activation. We found that glucose withdrawal lead to significant depolarization of the mitochondria, which was accompanied by Bax translocation and cytochrome c release. These effects were completely prevented by inhibiting either PKA activity or anchoring. Glucose absence promotes oxidative stress in different cell types. On this regard, it has been reported that PKA may modulate either the production or the scavenging of reactive oxygen species (ROS), by regulating the respiratory chain activity or the transcriptional activity of stress-defense genes. Thus, we further analyzed whether glucose deprivation induces oxidative stress in hepatocytes, PKA role in the oxidative stress generation, and the relationship of these effects with apoptotic activation. The cells 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-hglucose deprivation induced a significant ROS increase which was 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 significant until 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-regulated oxidative component in the apoptotic activation in glucosedeprived hepatocytes. In this connection, qPCR assays in these groups indicated that PKA inhibition during glucose deprivation leads to an increase in Sod1 and Cat mRNA levels, which partly explain the prevention in ROS imbalance with H89. A putative role of PKA in ROS production during glucose deprivation through the phosphorylation of cytochrome c oxidase should not be disregarded