NAD+ Homeostasis: Redox Metabolism, Salvage Pathways and NAD-Dependent Signalling

NAD+ Is a Metabolic Cofactor, Not a Peptide

NAD+ is a dinucleotide based on pyridine and consists of two nucleotide units joined by means of their phosphate groups; it has an oxidized form, NAD+, and a reduced form, NADH.

The NAD+/NADH pair takes part in reversible electron-transfer reactions at various stages of cellular metabolism, and the enzymes taking place in glycolysis, the tricarboxylic acid cycle and oxidative metabolism make use of this redox couple to transfer reducing equivalents between metabolic reactions.

The fact that this chemistry is the reason why the NAD+/NADH ratio is an important experimental parameter means that it is not possible to fully describe the cellular redox state by measuring NAD+ alone since NADH stands for the corresponding reduced pool.

NAD+ also has a second function which is mechanistically different from its redox function since several enzymes use NAD+ as a substrate when modifying proteins or producing signalling metabolites.

The NAD+ Salvage Pathway

Mammalian cells are able to maintain their levels of NAD+ by means of a number of biosynthetic pathways, such as de novo synthesis, the Preiss-Handler pathway and the salvage pathways.

The salvage pathway is so important because it recycles the nicotinamide that is produced when NAD+-dependent enzymes use up NAD+.

Nicotinamide phosphoribosyltransferase is a key enzyme in this pathway and is usually referred to as NAMPT. It converts nicotinamide into nicotinamide mononucleotide, this being then transformed into NAD+ by enzymes known as nicotinamide mononucleotide adenylyltransferase.

A recycling system is thus established in which the consumption of NAD+ leads to the production of nicotinamide, which in turn can be used in the synthesis of further NAD+.

The salvage pathway is therefore more than just a pathway for production; it is part of the mechanism by which cells keep NAD+ homeostasis.

De Novo NAD+ Biosynthesis

NAD+ can also be synthesised by pathways which start with tryptophan.

The de novo pathway consists of a number of enzymatic steps which finally lead to the formation of quinolinic acid before it is converted into intermediates in the biosynthesis of NAD+. It is especially important when looking at how the metabolism of amino acids is linked to the production of nucleotide cofactors.

The Preiss-Handler pathway offers yet another route, starting out with nicotinic acid and then going through nicotinic acid mononucleotide and other intermediates before reaching NAD+.

These pathways show that the concentration of NAD+ in the cells cannot be attributed to any one metabolic reaction, since the intracellular pool is the result of a balance between synthesis, recycling, transport and enzymatic consumption.

NAD+ Consumption by Sirtuins

Sirtuins are NAD+-dependent enzymes that remove acyl modifications from proteins.

During a sirtuin-catalysed reaction, NAD+ is consumed and converted into products including nicotinamide. Because NAD+ is required as a substrate, changes in NAD+ availability can influence sirtuin activity under experimental conditions.

Seven mammalian sirtuins, SIRT1 through SIRT7, occupy different cellular compartments and have different substrate preferences. This compartmentalization means that total cellular NAD+ concentration does not necessarily provide a complete picture of NAD availability at every site within the cell.

The research is therefore beginning to take into account both the concentration of NAD+ and its distribution within the cells when studying sirtuin biology.

NAD+ and PARP Signalling

Poly(ADP-ribose) polymerases, which are generally referred to as PARPs, are also a major class of enzymes that consume NAD+.

PARP enzymes use NAD+ to transfer ADP-ribose units to target proteins. Poly(ADP-ribosyl)ation and mono(ADP-ribosyl)ation can alter protein interactions, localization and activity.

PARP activity is of particular importance in research into DNA damage responses since some PARP enzymes react to DNA lesions and take part in repair-associated signalling.

This sets up a significant metabolic link since higher PARP activity can lead to increased consumption of NAD+, and alterations in NAD+ availability can in turn affect the substrate conditions for NAD-dependent signalling enzymes.

CD38 and NAD+ Consumption

CD38 also denotes a major system for consuming NAD+.

In addition to its role in intracellular NAD metabolism, CD38 has important extracellular and membrane-associated functions unlike those of sirtuins and PARPs. The enzyme activity of CD38 is capable of converting NAD+ into various metabolites such as cyclic ADP-ribose and ADP-ribose.

These products are concerned with calcium signalling and thus provide CD38 with a functional link between NAD+ metabolism and the regulation of intracellular calcium.

Consequently, research on NAD+ homeostasis looks at CD38 together with sirtuins and PARPs rather than considering the concentration of NAD+ in isolation.

NAD+ Is Compartmentalized Within Cells

One of the more important developments in NAD+ research is the recognition that NAD+ is distributed across different cellular compartments.

NAD+ pools exist in the cytosol, nucleus and mitochondria, and their regulation is not necessarily identical.

The NAD+-using enzymes found in the nucleus include various sirtuins and PARPs. The NAD+ in the mitochondria is closely associated with oxidative metabolism and redox reactions. NAD+ in the cytosol is involved in a number of metabolic pathways and is able to interact with signalling processes.

As a result, the amount of total NAD+ obtained from a whole-cell extract may mask the differences between the various compartments.

Recent research has stressed the importance of measuring NAD levels in a compartment-specific manner when investigating NAD homeostasis.

NAD+ and Redox Biology

The pair consisting of NAD+ and NADH is essential to cellular redox chemistry.

In oxidation reactions NAD+ takes in electrons and is thus reduced to NADH; NADH can then donate its reducing equivalents by means of the metabolic pathways linked with energy production.

This redox function differs from the signalling role of NAD+ as an enzymatic substrate.

It is important from an experimental point of view. A change in the concentration of NAD+ might affect the redox balance, the availability of substrates for NAD-dependent enzymes, or both. For this reason researchers must specify which aspect of NAD biology the experiment is intended to measure.

Measurements of NAD+/NADH, enzyme activity assays and metabolic flux measurements can together offer complementary information.

NAD+ and Mitochondrial Research

The mitochondria have a large amount of NAD which supports oxidative metabolism.

Mitochondrial NAD+ participates in reactions associated with the tricarboxylic acid cycle and other pathways that generate reducing equivalents for the respiratory chain.

The maintenance of NAD levels in different parts of the cell is not entirely independent of the rest of the cell since transport, biosynthesis and metabolic demand all play a role in it.

The research in this field therefore looks at NAD availability together with mitochondrial metabolism rather than considering the cofactor on its own.

NAD+ and Cellular Signalling Are Mechanistically Connected

The link between NAD+ metabolism and signalling exists since NAD+ functions as both a metabolic cofactor and a substrate.

Sirtuins use up NAD+ when they modify proteins, PARPs use NAD+ during ADP-ribosylation reactions and CD38 converts NAD+ into metabolites which have a signalling function.

A network results in which the metabolic demand has an effect on signalling capacity and signalling activity in turn changes the available pool of NAD+.

The relationship is therefore dynamic not linear. The effects of increasing or decreasing NAD+ at one point in the network will differ depending on which enzymes are active, where the NAD+ pool is located and how quickly it is being consumed.

NAMPT as a Key Experimental Target

NAMPT is widely studied due to its role in the NAD+ salvage pathway.

The enzyme causes nicotinamide to be converted into nicotinamide mononucleotide, thus providing an important pathway for the regeneration of NAD+.

It is therefore possible for experimental studies to alter NAMPT activity in order to examine the effects of changes in NAD+ availability on subsequent processes.

NAMPT should not, however, be looked at by itself. Changing one enzyme in the NAD network can affect substrate availability, metabolite concentrations and other compensatory pathways elsewhere in the system.

This is one reason why NAD research increasingly uses pathway-level measurements rather than a single endpoint.

Experimental Models Used in NAD+ Research

NAD+ biology can be investigated using biochemical assays, cultured cells, isolated organelles, animal models and human research.

Biochemical systems can measure enzyme kinetics and NAD-dependent reactions directly. Cell culture experiments can examine changes in NAD concentration, NAD+/NADH ratios, gene expression and enzyme activity.

Organelle-specific studies can investigate mitochondrial or nuclear NAD pools. Animal research allows investigators to examine tissue-specific NAD metabolism in a more complex biological environment.

Other studies involving humans offer a further kind of evidence, but the results vary according to the population, the tissue examined, the analytical method and the experimental intervention.

The models should not be regarded as being interchangeable; the fact that the concentration of NAD has changed in cultured cells does not mean that the same change occurs in a particular human tissue.

Measuring NAD+ Requires Analytical Control

NAD+ and NADH are molecules that are chemically related but can be distinguished analytically. It is therefore necessary to use an analytical method which can differentiate between the relevant metabolites.

Measurements can also be affected by sample preparation since the NAD metabolites may change during handling.

Researchers can employ chromatographic and mass-spectrometric methods when determining the amounts of NAD-related metabolites. The extraction procedure, the choice of internal standards, the calibration strategy and the conditions for storing the samples all have an effect on the measurements obtained.

It is especially important to carry out consistent sample preparation and use a consistent analytical methodology when conducting comparative research.

The method employed to measure NAD+ should therefore be taken into account when assessing any reported change in it.

NAD+ Research and Aging Biology

NAD+ research is frequently discussed in connection with aging, but the scientific question is more specific than simply asking whether NAD+ increases longevity.

Studies have reported age-associated changes in NAD metabolism and have investigated mechanisms involving NAD biosynthesis, consumption, mitochondrial function, sirtuin activity and CD38.

The subject is still one that is the focus of ongoing research. Recent reviews have still looked at how NAD+ metabolism changes with age and at how compartment-specific NAD regulation might affect cellular physiology.

For the sake of research it is better to look at the NAD-dependent pathways rather than regard "longevity" as a single biological endpoint.

Current Limitations in NAD+ Research

NAD+ biology is complex because its concentration reflects several competing processes.

Synthesis, salvage, enzymatic consumption, transport and compartmentalization can all influence the measured NAD pool.

A further limitation is that bulk measurements of NAD may fail to show changes within particular organelles or cell types.

Experimental models also differ greatly. Results from isolated cells might indicate metabolic conditions which are not duplicated in whole tissues. In the same way, findings from animals might not translate directly into human biology.

In the past, research into human intervention has been more restricted than the large amount of mechanistic literature, and the reviews have highlighted the importance of gaining a better understanding of NAD dynamics that are specific to tissues and to compartments.

NAD+ as a Research Model for Metabolism and Signalling

NAD+ is a useful example of a metabolic cofactor being able to function as well within cellular signalling networks.

Its redox role connects NAD+ with energy metabolism. Its substrate role connects it with sirtuins, PARPs, CD38 and other NAD-consuming enzymes.

The salvage pathway connects NAD+ with nicotinamide metabolism, while compartmentalization determines where specific NAD-dependent reactions occur.

The fact that NAD+ homeostasis is therefore a useful research framework for the study of metabolic regulation, enzyme activity, redox biology and intracellular signalling without reducing the subject to a single outcome oriented towards consumers.

Research Use Only

It is intended for use in scientific and educational research only.

The subject of NAD+ is treated as one of biochemical research rather than as a medicine, a treatment, a supplement recommendation or a product for human consumption and no dosing, administration protocol or therapeutic recommendation is given.

When interpreting published research, one should take into account the experimental model, the analytical method, the biological compartment, and the limitations of each particular study.

In the case of laboratory research, the investigators ought to look over the precise identity, the analytical documentation and the information specific to each batch relating to any material being investigated.

FAQs

References

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