We evaluated here how oxidative energetic metabolism is affected by CR during chronological aging, based on the fact that increases in respiratory rates have been pinpointed as essential for the lifespan-expanding effects of CR 
. Interestingly, we found that, while cellular respiration is slightly higher in CR cells during the early hours in culture - as had been previously observed 
- the effect of CR on respiration is much more marked between 24 and 74 h (). This finding is remarkable, since it demonstrates that the most notable effects of CR on oxidative metabolism occur long after glucose is totally exhausted from the media, characterizing a hysteretic effect.
Hysteresis, or the dependence of a biological response not only on the current environment but also on the past environment, is involved in the control of many cellular functions including the modulation of the cell cycle 
. Therefore it seems consistent to find that it is also implicated in the control of respiration during chronological lifespan. Indeed, we found that CR hysteretically primes cells to respond earlier and more intensely with an increase in respiratory rates during aging (). Although we do not yet know what signaling mechanisms mediate this hysteretic effect of CR, it is central toward the lifespan expanding effects, since multiple different interventions generating respiratory-incompetent cells result in a lack of response to CR (, 
A result of enhanced respiratory metabolism in S. cerevisiae
is the change in production of acidic products associated with alterations in media pH. This is especially relevant in yeast cultures, which are generally poorly buffered. Indeed, it has been proposed that the shift in pH and, specifically, prevention of acetate formation, is the mechanism through which CR increases chronological lifespan 
. Although we see large changes in pH over time and under CR or control culture conditions (), our results suggest this is not the sole direct mechanism responsible for limited cell survival under control culture conditions, for a series of reasons: (i) CR is ineffective as a lifespan extension intervention in ρ0
cells (, 
), yet pH in the culture media is significantly different under these conditions (); (ii) buffering pH does not eliminate lifespan extension promoted by CR () and (iii) differences in pH between CR and control WT cultures peak around 200 h (), however, most significant differences in lifespan occur much later (). Conversely to our data, other groups have found that buffering media pH to 6.0 in control cells increases chronological lifespan 
. One important difference in these studies is the use of synthetic complete medium, which is more poorly buffered (cultures reach pH as low as 2.5 
) and supports more limited survival than YPD. Furthermore, the effects of buffering pH in 2% glucose cultures were not compared to those of CR in these studies, and it is therefore not possible to conclude if pH buffering was sufficient to promote the full effects of CR under those conditions. While we certainly believe that acidic pH is toxic to cells, our results indicate that buffering extracellular pH is not sufficient to induce the fully extended lifespans observed in CR, a concept in line with previous data showing that acidification of CR cells is not sufficient to decrease lifespan 
. For a two-sided review on the ongoing debate regarding the effects of pH in yeast lifespan and CR, we recommend reference 
Our results also suggest that direct acetate toxicity is not responsible for the differences in survival under CR and control conditions 
since: (i) it was undetectable in WT cells under both culture conditions after 72 h; (ii) the levels of this acid in the culture media did not surpass ~0.6 g/L (10 mM), while levels of 200 mM are necessary to promote a decrease in S. cerevisiae
; (iii) acetate levels differ significantly between CR and control ρ0
cell cultures (), but CR does not extend lifespan in these cells (, 
). It should again be noted that previous experiments that suggest the central role of acetate in CR lifespan extension 
were conducted in synthetic media, while our results are in YPD, which is less prone to dramatic pH changes. Again, we recommend reference 
for further insight into the ongoing debate on the role of acetate in chronological lifespan extension by CR.
Another often-proposed lifespan-extending effect discussed for the yeast CR model has been the shift toward oxidative metabolism of glucose 
. Very surprisingly, our results show this is not the case. Maximum specific growth rates on glucose (), glucose specific consumption rates (), glucose-to-cell conversion () and ethanol production from glucose () were unaffected by CR. Taken together, these data demonstrate that the ability of cells to metabolize and grow on glucose, as well as the proportion of ethanol produced per glucose molecule metabolized, is identical under both growth conditions. Thus, our results clearly indicate, through different but highly consistent findings, that glucose is predominantly fermented under both CR and control conditions, and that CR does not stimulate the respiratory metabolism of glucose in the early hours in culture.
A higher biomass production observed under control conditions versus CR () was observed, but it is explained by the total glucose available in the culture media, not a higher efficiency in use of energy (which is in fact lower, ). Likewise, the increased total amount of ethanol generated by cells in control media () is also explained by the higher initial concentration of glucose. Again, these results show that CR does not alter glucose metabolism, which is predominantly fermentative (as indicated by similar quantitative results obtained with ρ0 cultures).
Although glucose-to-cell conversion () and glucose-to-ethanol production () were unaffected by CR, WT cell growth in ethanol/glycerol (), ethanol/glycerol consumption () and conversion to biomass () are all significantly enhanced under CR conditions. This indicates that, while CR does not enhance the respiratory metabolism of glucose, it increases the speed and efficiency of use of exclusively respiratory substrates. The effect is seen for both ethanol and glycerol, although experiments using the substrates separately () indicate that the change in specific growth in ethanol is far more substantial. These results again demonstrate a hysteretic effect of CR, which primes cells to utilize respiratory substrates faster and more efficiently, after the complete elimination of glucose from the culture media.
Interestingly, this hysteretic effect is intrinsic of the cells, since it persists in substituted media (). Furthermore, since CR is ineffective in cells that are not capable of respiring (, 
), we propose that the hysteretic preparedness for earlier, faster and more efficient oxidative metabolism of ethanol is central toward the lifespan-enhancing effects of CR.