NAD+ has become one of the most discussed molecules in ageing research, which has not helped the quality of information available about it. The underlying biochemistry is well established and considerably more interesting than the marketing version.
What NAD+ is
Nicotinamide adenine dinucleotide is a dinucleotide built from an adenine nucleotide and a nicotinamide nucleotide joined through their phosphate groups. It is present in every living cell and has been understood as a redox cofactor since the early twentieth century.
What changed in the last two decades is the recognition that this is only half of what it does.
Two jobs, not one
NAD+ operates in two fundamentally different modes, and conflating them is the most common error in discussions of the molecule.
As a redox carrier, NAD+ accepts a hydride ion to become NADH, then donates it elsewhere, cycling between the two forms. In this role it is catalytic and is not consumed. Glycolysis, the citric acid cycle and oxidative phosphorylation all depend on it.
As an enzyme substrate, NAD+ is cleaved and genuinely consumed. Sirtuins, PARPs and CD38 all degrade NAD+ as part of their catalytic cycle, releasing nicotinamide. This consumptive use is what creates demand for resynthesis, and it is the reason cellular NAD+ levels can fall at all.
Covarrubias and colleagues set out this architecture and its relationship to ageing in Nature Reviews Molecular Cell Biology.[1]
The sirtuin connection
Sirtuins are NAD+-dependent deacetylases that remove acetyl groups from protein lysine residues, including histones. Because their activity depends on NAD+ availability, they couple the metabolic state of the cell to gene expression. Imai and Guarente reviewed this relationship in Trends in Cell Biology.[2]
This dependency is the mechanistic basis for most interest in NAD+ in ageing research. Verdin's review in Science extended the picture across metabolism and neurodegeneration.[3] Lautrup and colleagues examined the neural dimension specifically in Cell Metabolism.[4]
Compartmentalisation
A point frequently missed: NAD+ is not distributed uniformly through the cell. Distinct pools exist in the cytosol, the mitochondria and the nucleus, and these are not freely interchangeable. The inner mitochondrial membrane is impermeable to NAD+ itself.
This has direct experimental consequences. A measurement of whole-cell NAD+ averages across compartments that may be changing in opposite directions, and an intervention that raises cytosolic NAD+ does not necessarily raise the mitochondrial pool.
Handling and stability
NAD+ is chemically awkward to work with. The oxidised form is unstable in alkaline conditions; the reduced form NADH is unstable in acid. There is no pH at which both are comfortably stable, which complicates any protocol measuring both.
Practical consequences:
- Solutions are prepared fresh rather than stored, and kept cold and shielded from light during use.
- The lyophilised solid is hygroscopic and should be returned to −20°C storage promptly, protected from moisture.
- Measured NAD+/NADH ratios are highly sensitive to extraction method, and results from different protocols often cannot be compared directly.
What the literature does not establish
Most of what is confidently asserted about raising NAD+ levels rests on animal and cell culture work. Amjad and colleagues reviewed the signalling roles in Molecular Metabolism, and the picture that emerges is of a regulatory network rather than a simple quantity to be increased.
NAD+ itself is poorly taken up by cells intact, which is why so much research attention has gone to precursors instead. Any experimental design assuming applied NAD+ translates to intracellular NAD+ is making an assumption the literature does not support.
References
- Covarrubias AJ. NAD+ metabolism and its roles in cellular processes during ageing. Nature Reviews Molecular Cell Biology. 2021. PMID 33353981
- Imai S. NAD+ and sirtuins in aging and disease. Trends in Cell Biology. 2014. PMID 24786309
- Verdin E. NAD+ in aging, metabolism, and neurodegeneration. Science. 2015. PMID 26785480
- Lautrup S. NAD+ in Brain Aging and Neurodegenerative Disorders. Cell Metabolism. 2019. PMID 31577933
Research use only
NAD+ is supplied by MMpeptides strictly for laboratory and in vitro research. It is not approved for human or veterinary use, is not a cosmetic or medicinal product, and nothing in this article should be read as describing use in people. Third-party studies are cited for scientific context only. Researchers are responsible for observing all applicable regulations in their jurisdiction.