I performed a preliminary mitochondrial assay using Wild-type and ApoE KO Sca1+ cells, fed with glucose or galactose. Under both conditions, the ApoE KO cells showed a significant increase in ATP production in the 2-hour assay (especially when glucose was given), in comparison to Wild-type, with the latter producing similar ATP levels in both conditions.
My hypothesis is that ApoE KO cells have dysfunctional mitochondria. Could this be in line with increased ATP production?
Many thanks in advance
Hi there. I am not familiar with this method, so excuse me if I get something wrong.
Quite an obvious idea: maybe wild type "eat" more ATP, while others cell has dysfunction not in mytochondria, but in some system which consumes ATP?
In you situation I would firstly try some mito check, like TMRE/DCF dye for potential/ros. If both dyes are equal in both groups, it would rather say, that mitochondria are OK. Though not garantee in, of course.
By the way, if you don't have seehorse, you can check mito respiration in isolated mitochondria. It requires more skill and is not so "native". I won't recommend to do it by yourself if you dont know how to, but chances to have instrument in facility is bigger, since its cheaper and older. Thought you'd rather stop on fluorescent dyes - less problems :)
Dear Vasily, interesting idea! You mean more ATP detected means less ATP being used?
Has this been reported in any cell type? Can ATP overload cause higher ROS?
Hi Daniel, I thought that increased mito membrane potential is indicative of high ATP and low ROS, while a lower one, has lower ATP and higher ROS values.
Another aspect to consider is that ATP levels are affected by the cell cycle. For example, highly proliferative cells have higher ATP levels than less proliferative cells (Sweet & Singh 1999, J Cell Physiol, 180, pp. 91-6). So it could be that you observed higher ATP levels in your ApoE KO cells because these cells are proliferating faster than your WT cells.
Dear Alexandra, that could be the case, since, indeed I have observed that the ApoE KO cells do reach confluence faster than the WT. Thank you for the input.
Since you had glucose in the system, how could you come to the conclusion that the increased ATP came from oxidative phosphorylation but not from increased glycolytic activity? Typically if the mitochondria are defective, cells would increase the rate of glycolysis to meet the cellular need of ATP. One additional experiment that you could do would be to measure the ATP coming specifically from glycolysis or from mitochondria. For glycolytic ATP determination, you could fed the cells with glucose and Na-azide (to block mitochonndrial activity). For mitochondrial ATP production you treat cells with Na-Pyruvate and deoxy d-glucose (hexokinase ingibitor).
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