| Abstract |
Autophagy is a defence mechanism of the cell that is activated under conditions of stress or due to environmental or nutrition changes of the cell. During this process, the cell gets rid of damaged or dysfunctional organelles or invading pathogens that are engulfed in a double-membrane vesicle which is known as autophagosome. Autophagosome fuses with the lysosome and as a result its entire content is degraded and recycled. In this way, autophagy promotes cell survival. Autophagy is one of the many cell functions that are controlled by the PI3K-Akt-mTOR intracellular signaling pathway whose key components are Akt kinases. Akt kinases through mTORC1 complex inhibit autophagy, therefore we presume that their absence should lead to autophagy induction. The purpose of this study was to identify the effect of Akt1 and Akt2 loss on macrophage autophagy on cells treated with different stimuli including LPS, IL4, IFNγ and insulin. The results showed that LPS treatment of both Akt1 and Akt2 deficient macrophages did not induce the expression of LC3 autophagy marker. Serum starvation did not affect LC3-II levels in Raw cells and there was a small induction of autophagy after 6h and 24h of IFNγ or IL4 treatment in both peritoneal macrophages and Raw264.7. Moreover, treatment with rapamycin, an inhibitor of mTOR, did not increase LC3 above basal levels. Quantitative real-time PCR was used to evaluate the expression levels of two important autophagy genes, Atg5 and Atg7. The results indicated an increase in both Atg5 and Atg7 after 24h of LPS treatment on Raw264.7 under starvation conditions. In the presence of serum, Atg7 expression was decreased after 6h and 24h of LPS treatment of both WT and Akt1-/- macrophages and Atg5 expression was increased 24h following LPS treatment of both WT and Akt1-/- cells. Finally, both genes were increased after 6h and 24h of IFNγ treatment in Akt1-/- cells compared to WT controls and only Atg7 levels were higher after simultaneous treatment with LPS and IFNγ on Raw macrophages. As for the mitophagy marker Pink-1, its expression levels varied in response to different stimuli. Specifically, absence of Akt1 resulted in increased basal mitophagy. While LPS suppressed mitophagy in WT macrophages this suppression was not
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observed in Akt1-/- macrophages 24 hours following stimulation. At 48 hours of treatment or at serum starvation conditions these differences were not observed. We can therefore conclude that Akt1 differentially controls Atg5 and Atg7 expression in macrophages and that absence of either Akt1 or Akt2 does not affect LC3-dependent autophagy, suggesting a potential redundancy between the two Akt isoforms.
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Contribution of AKT kinases in macrophage autophagy
