Research shows sirtuins regulated by NAD+ concentration, no direct NAD/NADH ratio influence

Sirtuins are NAD-dependent deacetylases that are highly conserved from bacteria to human. SIR2 was originally shown to extend lifespan in budding yeast. Since then, sirtuins have been shown to also regulate longevity in other lower organisms. In mammals, there are seven sirtuins (SIRT1-7). They have different specific substrates and biological functions, and are found in various cell compartments. Sirtuins require NAD+ for their enzymatic activity which connects metabolism to aging and aging-related diseases.

Multiple studies have described the sirtuins as sensors of the NAD / NADH ratio, with some suggesting this implies that the NAD / NADH ratio is a contributing factor in driving sirtuin activity. It is known that the NAD/NADH ratio can be affected in vitro by hypoxia or by substrate metabolism, where low glucose leads to an increased NAD/NADH ratio, and high glucose, lactate, or ethanol leads to a decrease in the NAD/NADH ratio. Similarly, changes in the ratio can be accomplished in vivo by exercise, by calorie restriction, or by allowing high fat or high ethanol intake. Because changing the NAD/NADH ratio has been shown to impact protein acetylation levels this has led to the aforementioned suggestion that sirtuins could be affected by this ratio.

Upto now however no clear test results exist to support the “sensor” hypothesis. The researchers in this study set up an experiment to validate this hypothesis with mammalian sirtuins (SIRT1-7). In order to do this they developed a novel set of peptide based probes which were used to identify the range of hydrolytic activities of human sirtuins. Upon establishing and validating the probes they then tested the ability of NADH, ADP-ribose, and nicotinamide to inhibit the NAD -dependent deacylase activities of the sirtuins.

The experiments demonstrated that NADH inhibits the long chain deacylase activities of human SIRT1–3 and SIRT6 as well as the dicarboxyl-derived deacylase activities of SIRT5. The corresponding IC50 concentration values necessary to inhibit any of the tested deacylase activities of human sirtuins were in the low millimolar range. The researchers compared that to previous studies of estimates of in-vivo cytosolic and mitochondrial free NADH. The estimates for free NADH are 110nM and 30uM, respectively. Thus, the current knowledge of physiological levels of free NADH in the different organelles combined with the observed much higher IC50 values strongly suggests an insignificant direct inhibitory effect of NADH for all sirtuins even if there would be strong relative fluctuations in NADH levels. This also corroborates the previous conclusions by Guarente for SIRT2-mediated deacetylation.

Therefore the study concluded that the observed data shows that sirtuins are unlikely to be sensors of the NAD/NADH ratio in vivo as previously assumed. But given that the sirtuins require NAD as a co-substrate they are still metabolic sensors. The researchers added the caveat that as one of the most important redox couples in the cell, changes in NAD and NADH levels (e.g. as a result of calorie restriction) are very complex and hence there is a possibility that ratio changes may affect sirtuin activity and protein acylation levels indirectly.

While not mentioned in the study but as described in various posts on this blog there are supplements available that likely increase the NAD+ concentration in the body for example with nicotinamide riboside or possibly indirectly with quercetin. The study is, as such, another indication that an absolute rise in the NAD+ concentration as seen with supplementation of nicotinamide riboside may indeed activate sirtuins and hence is beneficial for health and anti-aging.

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