Talk:Zujovic 2019 MitoFit Preprint Arch EA
In Fig1B palmitate group do not seem to be permeabilized even after 6 titrations. There is no significant increase in respiration as it is visible for controls. In Fig2 palmitate group permeabilization is also questionable, because you see an effect on respiration already after 4 ug of digitonin (which is less than in Fig1B) but then there is almost no ADP stimulation: OXPHOS coupling efficiency seems to be less than 50%! And after Rot and Ama the ROX is significantly lower suggesting that endogenous substrates may be also present. Another point in Fig 1A and 2 respiration in OXPHOS for S pathway is smaller than LEAK respiration for N-pathway.
Reply from Nina Krako (2019-07-04)
Palmitate treated cells are not expected to have the same respiration and OXPHOS coupling efficiency as control cells. Considering the fact that palmitate here decreases the respirometric viability index to ~ 50%, as well as literature data that demonstrate the negative effect of palmitate on mitochondria and bioenergetic of cells. The ROX is not lower, but maybe higher (what cannot be seen on Fig2, but we noticed it in different repeats of this protocol), and that could be due to the presence of endogenous substrates but probably in non-viable cells (consequence of palmitate treatment), and not in non efficiently permeabilized after 6ug of Dig. Correct? How one can be sure that PA treated cells are optimally permeabilized, if not to do a digitonin optimization simultaneously with control cells (1B)? Application of higher than 6ug of digitonin in palmitate treated cells causes a cyt c increase (not shown). What is your suggestion about how to improve this study and to evaluate the respiration of PA treated Huh7 cells?
Reply from Ondrej Sobotka (2019-08-09) Dear Nina, I would suggest more digitonin. According to our recent data we applied up to 10ug/mL/mio. cells with our HuH-7 controls without any release of cytochrome c. The question remains if the positive cyt c test in your results could have been caused by palmitate treatment or digitonin titrations, but considering that respiration in ROUTINE is higher than S pathway in OXPHOS (in all your figures) seems to me that Huh-7 cells are not well permeabilized. The figure where you observed this cytochrome c release would be interesting to see. To improve your study you can find digitonin titration protocol here: https://www.bioblast.at/index.php/SUIT-010_O2_ce-pce_D008 or you can combine digitonin titrations with other protocols here: https://suitbrowser.oroboros.at/
- Good luck!
What is the reason behind your substrate concentrations (P 8.8 mM, M 4.4mM and S 10 mM)? According to recent work of Sumbalova et. al 2016 it was shown that Malate significantly inhibits S pathway which can be partially overcome by 50 mM succinate. (ref: http://www.bioblast.at/index.php/Sumbalova_2016a_Abstract_MitoFit_Science_Camp_2016) For next experiments you can find SOPs on titration concentrations here:
Reply from Nina Krako (2019-07-04)
The reason for this is that we followed one of our old protocol in the lab (described in Krako et al, JAD 2013, adapted from Kuznetsov et al, Nature Protocols 2008) for concentration and stock preparation, that does not differ a lot from Oroboros recommendations from O2K manual titrations. However this is a good point, we will try to increase S to 50mM and decrease M to 2mM, to see how it affects the S pathway in controls and in PA treated cells. We will start to use P at 5mM according to [Krako 2013 J Alzheimers Dis] ([O2k manual titrations]) to stay coherent with other SUIT protocols and thus facilitate the harmonization and interpretation of results in line with MITOEAGLE objectives.