On 2014 Aug 06, Xudong Fu commented:

Thanks for your comments. It is really interesting. I hope I could explain why I don't think alpha-ketoglutarate works as an antioxidant to extend lifespan.

It has been shown that α-ketoglutarate is an antioxidant. However, antioxidants are not necessarily able to extend lifespan. Actually, the relationship between ROS and lifespan has not been established. Increase of ROS may contribute to lifespan extention potenially through downstream defensim activation and overexpression of antioxidant enzyme is not able to extend lifespan in mice (http://www.sciencedirect.com/science/article/pii/S0092867413006454, http://onlinelibrary.wiley.com/doi/10.1111/j.1474-9726.2008.00449.x/full).

Our paper discovers that α-ketoglutarate can bind and inhibit ATP synthase and extend lifespan in C. elegans. The inhibition of ATP synthase is actually likely to generate more ROS. It has been shown that oligomycin, a known ATP synthase inhibitor, could induce ROS in vivo. In our hand, we also find that α-ketoglutarate could induce ROS (by DCFDA dye measurement) both in C. elegans and cells. That suggest α-ketoglutarate is unlikely to increase lifespan simply through antioxidant function. It's more likely α-KG regulates lifespan through induction of ROS instead. We actually have explained why we don't think ROS plays a major role in the life extension effect for α-ketoglutarate; see Supplementary Information (http://www.nature.com/nature/journal/v510/n7505/extref/nature13264-s1.pdf). Briefly speaking, although α-ketoglutarate could induce ROS, additional antioxidants does not decrease the lifespan extension effect of α-KG in our hands. In addition, ROS has been suggested to activate TOR but we observed α-ketoglutarate decrease TOR.

In terms of the dosage we used, it has been known that α-KG is not highly membrane permeable. To solve this issue, we made octyl α-KG, an ester form of α-KG which is more membrane permeable. After octyl α-KG goes into the membrane, it gets hydrolyzed and releases free α-KG. Most of the assays in Fig. 2 were done with octyl α-ketoglutarate instead of original α-KG. That's the main reason we don't need to use 8mM dose for Fig. 2 assays. We also tried octyl α-KG in lifespan assays (see Extended Data Table 2). It only required micromolar scale of octyl α-KG instead of millimolar for us to observe the lifespan extension. In addition, after treatment of 400 uM of octyl α-KG on cells or after treatment of 8mM α-KG on C. elegans, the in vivo concentration of α-kG increased comparably around 50-100%. That suggest 50-100% increase of endogenous α-KG could inhibit ATP synthase and extend lifespan, nonetheless the different forms or concentration of α-KG we used, consolidating our hypothesis that α-KG extend lifespan through direct ATP synthase inhibition. Also, because the endogenous increase of α-KG is only 50-100%, this concentration does not seem to be in line with the concentration suitable for non-enzymatic reaction.

On 2014 Aug 04, Paul Brookes commented:

This is an interesting result, but I believe there's a fundamental mechanism that may have been overlooked, and is especially prescient given the high concentration of alpha-ketoglutarate that was required for this effect (8mM).

Like all alpha-keto acids, a-KG is an antioxidant. It can react freely with H2O2 (the products being succinate and CO2). Such oxidative decarboxylation has been widely studied (e.g. http://www.ncbi.nlm.nih.gov/pubmed/16781453), and essentially amounts to a short-cut in the TCA cycle (see our editorial on Fedotcheva's paper - http://www.ncbi.nlm.nih.gov/pubmed/16781451). This is why such acids exhibit short half-lives in vitro. For example if you keep a solution of pyruvate on ice it'll all be decarboxylated to acetate within a few hours.

Bottom line, letting worms swim around in an 8mM solution of an antioxidant MIGHT give rise to some effects on lifespan that are completely independent of any of the claimed mechanisms herein.

On 2014 Aug 04, Paul Brookes commented:

This is an interesting result, but I believe there's a fundamental mechanism that may have been overlooked, and is especially prescient given the high concentration of alpha-ketoglutarate that was required for this effect (8mM).

Like all alpha-keto acids, a-KG is an antioxidant. It can react freely with H2O2 (the products being succinate and CO2). Such oxidative decarboxylation has been widely studied (e.g. http://www.ncbi.nlm.nih.gov/pubmed/16781453), and essentially amounts to a short-cut in the TCA cycle (see our editorial on Fedotcheva's paper - http://www.ncbi.nlm.nih.gov/pubmed/16781451). This is why such acids exhibit short half-lives in vitro. For example if you keep a solution of pyruvate on ice it'll all be decarboxylated to acetate within a few hours.

Bottom line, letting worms swim around in an 8mM solution of an antioxidant MIGHT give rise to some effects on lifespan that are completely independent of any of the claimed mechanisms herein.

On 2014 Aug 06, Xudong Fu commented:

Thanks for your comments. It is really interesting. I hope I could explain why I don't think alpha-ketoglutarate works as an antioxidant to extend lifespan.

It has been shown that α-ketoglutarate is an antioxidant. However, antioxidants are not necessarily able to extend lifespan. Actually, the relationship between ROS and lifespan has not been established. Increase of ROS may contribute to lifespan extention potenially through downstream defensim activation and overexpression of antioxidant enzyme is not able to extend lifespan in mice (http://www.sciencedirect.com/science/article/pii/S0092867413006454, http://onlinelibrary.wiley.com/doi/10.1111/j.1474-9726.2008.00449.x/full).

Our paper discovers that α-ketoglutarate can bind and inhibit ATP synthase and extend lifespan in C. elegans. The inhibition of ATP synthase is actually likely to generate more ROS. It has been shown that oligomycin, a known ATP synthase inhibitor, could induce ROS in vivo. In our hand, we also find that α-ketoglutarate could induce ROS (by DCFDA dye measurement) both in C. elegans and cells. That suggest α-ketoglutarate is unlikely to increase lifespan simply through antioxidant function. It's more likely α-KG regulates lifespan through induction of ROS instead. We actually have explained why we don't think ROS plays a major role in the life extension effect for α-ketoglutarate; see Supplementary Information (http://www.nature.com/nature/journal/v510/n7505/extref/nature13264-s1.pdf). Briefly speaking, although α-ketoglutarate could induce ROS, additional antioxidants does not decrease the lifespan extension effect of α-KG in our hands. In addition, ROS has been suggested to activate TOR but we observed α-ketoglutarate decrease TOR.

In terms of the dosage we used, it has been known that α-KG is not highly membrane permeable. To solve this issue, we made octyl α-KG, an ester form of α-KG which is more membrane permeable. After octyl α-KG goes into the membrane, it gets hydrolyzed and releases free α-KG. Most of the assays in Fig. 2 were done with octyl α-ketoglutarate instead of original α-KG. That's the main reason we don't need to use 8mM dose for Fig. 2 assays. We also tried octyl α-KG in lifespan assays (see Extended Data Table 2). It only required micromolar scale of octyl α-KG instead of millimolar for us to observe the lifespan extension. In addition, after treatment of 400 uM of octyl α-KG on cells or after treatment of 8mM α-KG on C. elegans, the in vivo concentration of α-kG increased comparably around 50-100%. That suggest 50-100% increase of endogenous α-KG could inhibit ATP synthase and extend lifespan, nonetheless the different forms or concentration of α-KG we used, consolidating our hypothesis that α-KG extend lifespan through direct ATP synthase inhibition. Also, because the endogenous increase of α-KG is only 50-100%, this concentration does not seem to be in line with the concentration suitable for non-enzymatic reaction.