I can see several reasons: 1) not enough substrate ( you need to check if you add enough substrates - Pyruvate, or Glutamate and Malate calculated per the number of cells in culture); 2) you did not write how you measured the rate of oxidative phosphorylation/or measured the decrease of oxygen in culture; the problem might be in the assay approach; 3) It does not look like the extracted cells are stressed and enter the very early stage of apoptosis – as the level of oxygen consumption would be most likely increased; thus, it is possible that the cells are at the stage of energy conservation and for some reason started to “hibernate”, maybe the cells miss blood hormones for the normal respiratory level ( insulin, thyroxin).4) did you compare the 4h rate with the 0h rate? It is possible that 0h (right after cell extraction) is the increased respiratory rate as cells are stressed, phosphorylation is uncoupled and the oxygen consuption is increased.

 Thanks for the quick answer and the suggestions.

1) The culture medium is RPMI so it could be good to add some substrates, at least to exclude them as a limiting factor. 

2) To asses mitochondrial oxygen consumption, we resuspend cells in a common respiration medium and observe oxygen flux in respiration chambers. Cells are permebealized by the addition of digitonin inside the chamber at the beginning of the experiment.

3) The absence of hormones could be an issue. The hibernation theory seems plausible too. Our next step would be to check for enzyme activity and expression (enzymes of energy metabolism).

4) Yes we did compare 4h and 0h. The intensity of the decrease in oxygen consumption depends of the respiratory state. The decrease is more intense for respiratory state 3 (Pyruvate, Malate, Glutamate, Succinate and Malate) and uncoupled respiration than for  State 3 respiration without Succinate and complex IV activity.

We compare our control measures to some treatments were the decrease is alleviated, so thats's good but we still want to know where the decrease originates from.

Thanks again

the Enzymes half life were differ in vitro and in vivo, and the deterioration of these enzymes mainly related with metabolites accumulation.