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NAD+ Integration in Cellular Pathways

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The importance of NAD+ and ROS

The coenzyme nicotinamide adenine dinucleotide (NAD+) is the molecular link between cellular metabolism, signaling, and transcription. Its synthesis, availability, and degradation are therefore central to the function of the cell and in healthy human physiology.

Cellular metabolism is the beginning of all other activities in living cells, providing energy for the millions of biochemical reactions that take place in any given moment. As such, any dysfunction in this process has immediate effects both on an intracellular scale and for overall human health. One of the most heavily studied aspects of this dysfunction is the changing levels and ratios of NAD+ and its related forms and enzymatic cofactors and byproducts.

Reactive Oxygen Species, or ROS, are a potentially damaging biochemical byproduct of multiple cellular pathways that involve redox reactions, with the most prominent contributors being the energy pathways facilitated by the coenzymes NAD+ and NADP. The reduced forms of these coenzymes are used for detoxification of ROS, and the various ROS themselves are used in a temporary fashion for intra- and inter-cellular signaling. There are currently ~400 known redox reactions in the cell that involve NAD+/NADH, and another ~30 for the phosphorylated variation NADP/NADPH. The far-reaching influence of these molecules makes their measurement and visualization useful even in seemingly unrelated experiments.

A phenotype of age is decreased resistance to cellular stress, which can be treated by increased NAD+ levels. The full influence of NAD+ is likely largely unknown and should be explored in multiple experimental fields and contexts. By measuring NAD+ both specifically and in ratio of its oxidized and reduced forms, researchers can discern the many factors at play within both healthy and dysfunctional cell behavior.

Healthy versus cancerous cell regulation

NAD+ levels and associated enzymes are essential for genomic maintenance. Since genomic stress and damage is the root source of all cancers, NAD+ regulation is of interest in fields that study cancer susceptibility and prevention. Cancer can be simplified as being the condition in which cells forget how to die.

There has been a huge amount of progress and discoveries made in recent years about the protein links between multiple cellular pathways and physiological conditions such as metabolic dysfunction and aging. Better methods of detection and visualization of NAD+ and associated biomolecules are pivotal in gaining further understanding of these interconnected aspects of human health and function.

There is constant crosstalk between the activity of the mitochondria, peroxisomes, and nucleus, which not only is representative of the typical cooperation between the organelles within the cell, but also that of the communication between cell metabolism, regulation, and gene transcription.

There are separate pools of NAD+ used by the cytosol, nucleus, and mitochondria, interconnected by multiple redox processes. Affected cell pathways include glycolysis (occurring within the cytoplasm) and TCA cycle as well as oxidative phosphorylation (within the mitochondria). Although these pathways are location-specific, the cell is able to move NAD+ to the appropriate organelles to a small extent. However, cytoplasmic NAD+ cannot directly cross the mitochondrial membrane, so biosynthesis of NAD+ is of high importance. Compartmentalized NAD+ biosynthesis is also studied in its relationship to cancer cell growth and the deregulation of normal cellular activity.

NAD+ biosynthesis is essential for the energy production of the cell, and the resultant coenzymes are kept in various locations within the cell. This compartmentalized synthesis is one aspect of the integration of cell transcription, signaling, and metabolism. Increases in cytoplasmic NAD+ reduce nuclear NAD+ levels. Cells require increased cytoplasmic NAD+ to regulate nuclear events during cell differentiation, an example of the close relationship between cell cycle regulation and metabolic activity.


Interconnected Cycles- Chronobiology, Metabolism and Aging Pathologies

Simultaneous measurement of related biomolecules

There are many molecular metabolic regulators, some of which have been studied intensely in recent years as their importance in cellular biology gains more validation. Some of the major molecular families include: peroxisome proliferator activated receptors (PPARs), sirtuins, and multiple kinases, such as AMP-activated protein kinase (AMPK), and protein kinase A (PKA). The interdependent molecular mechanisms that govern cellular metabolism are still being explored, and improved understanding of the overall process requires exploration of their synergistic and additive/subtractive effects of these molecules and their involved pathways on each other.

Consider the well-known sensation of sleepiness after a large meal. This innocuous physiological response is the end result of an intricate biochemical interplay that relates directly to these dinucleotides. Namely, they are the communication between the sleep/wake cycle (circadian clock) and metabolism.

Nocturnin (NOCT) is an enzyme expressed as a factor of the circadian clock. NOCT, potentially along with other proteins, target the mitochondria and dephosphorylate NADP+ and NADPH into their NAD+ and NADH forms, which are immediately accessible to multiple energy-producing pathways. This enzymatic link is one of the most obvious biochemical ties between mammalian circadian rhythm and metabolic activity.

Focusing on a single target or metabolic mechanism can lead to a lack of understanding of the underlying biochemistry and interplay between various pathways that regulate cell behavior and survival. NAD+ has a role in physiological conditions far more dire than after-dinner naps, and measuring its fluctuation is a worthwhile effort regardless of currently-known relations to a particular experiment.

ROS, enzymatic activity, and health

Mitochondrial respiratory chain deficiency has multiple physiological effects, and improving the NAD+/NADH ratio is a promising treatment for at least some of the symptoms.

Intracellular NAD+ levels are significantly lower in diseases associated with aging and metabolic disorders, including mitochondrial dysfunction. NAD+ is critical as an electron carrier for energy production in the cell, as the reversible redox reaction that converts NAD+ to NADH is central for mitochondrial metabolic function and has no NAD+ net loss. There is loss, however, for many cell signaling processes, which use NAD+ as an enzymatic substrate. These include the well-known sirtuin family, which is implicated in metabolic and age-related conditions.

The NAD+/NADH ratio effects SIRT1 enzymatic activity and the metabolism of adipogenic transcriptional factors. Adipogenesis is regulated by oxidative signaling, so the presence of various ROS within organelles such as peroxisomes or more generally within the cytosol has long-reaching effects. Some types of ROS dysregulation have been shown to have physiological responses with adipose tissue differentiation, leading to dramatic obesity and diabetic symptoms.


Growing Understanding-Methods of Measurement in Living Cells

Intracellular visualization of NAD(P)+/NAD(P)H

Mitochondrial NAD+ transporters have not yet been identified and isolated in mammals, although they have been in both yeast and bacteria. This is just one of the many aspects of NAD(P)+ movement in the cell that is being explored. Intracellular assays are necessary to measure the changes in nicotinamide levels that indicate the intricate biochemical ballet of the living cell. Outside of the more common cell cycle analyses and similar experiments, there are multiple applications that employ intracellular probes (Figure 1 and 2). NADP+/NADPH ratio can be used to evaluate the redox status of cellular peroxisomes, for example.

Fluorescence images of NADH/NADPH in HeLa cells using Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit (Cat No. 15290). HeLa cells were incubated with 100 µM NADH or 100 µM NADPH in serum-free medium for 30 minutes and then co-incubated with JZL1707 NAD(P)H sensor working solution for another 30 minutes. The fluorescence signal was measured using fluorescence microscope with a Cy3® filter.

Flow cytometric analysis of NADH/NADPH measurement in Jurkat cells using Cell Meter™ Intracellular NADH/NADPH Flow Cytometric Analysis Kit (Cat No. 15296). Cells were incubated with or without 100 µM NADH in serum-free medium for 30 minutes and then co-incubated with JJ1902 NAD(P)H sensor working solution for another 30 minutes. Fluorescence intensity was measured using ACEA NovoCyte flow cytometer in APC channel.
The prominence of crosstalk between multiple cellular regulatory pathways has become more apparent in recent years, commensurate with the importance of measuring multiple regulatory factors in bioenergetics experiments.

As diminishing levels of NAD+ are associated with both general age and its attendant pathologies, or in more acute or genetically-caused diseases, there has been increasing interest in therapies that treat these conditions by increasing NAD+ levels within the cell. This intracellular increase is typically attempted by either NAD+ precursor supplements to aid in natural intracellular synthesis, or by inhibiting the enzymes that destructively use NAD+ as a substrate. The benefits observed so far have been in mouse models, and are likely accomplished by the improved accessibility of NAD+ for general cellular metabolic use, for genetic disorders, or alternatively by providing NAD+ sources for the sirtuin enzyme family, of which SIRT1 is one of the most prominently studied.


Living versus fixed cell protocols and comparisons

Fixed cells, which have been treated with formaldehyde or similar fixative, are effectively 'stopped' at the moment of fixation, allowing exhaustive examination. This allows exact measurements and detailed answers, but limits the questions that can be answered, given that the cells are dead. Cell lysate is the cytosolic component of lysed cells, separated via centrifuging. The destructive nature of lysate preparation gives limited information about the normal behavior of the cell. In order to improve comprehension of the constantly-changing cellular landscape, living cells are the most difficult and potentially most rewarding subject of study.


Pushing the Envelope-Cell Lysate NAD+/NADH Visualization Techniques

Lysate isolation protocols and uses

Results of Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit with Jurkat cell lysate.
In order to produce cell lysate, appropriately chosen cells will be incubated in lysis buffer, which breaks down the outer cell membrane. This controlled destruction is necessary to allow the interior of the cell to be accessed by antibodies (such as in IHC procedures) and gives consistent results. After the cells have been lysed, they will be centrifuged and the supernatant (containing the collected cytosol of the entire population of treated cells) will typically be selected.

Some procedures make use of the organelle pellet instead, which is usually composed of the cell nuclei or mitochondria, for studies targeting cell metabolism.

Using cell lysates provides several advantages for experiments. Multiple sample types (such as tissue or cultures) can be used, including those that have been previously frozen, and they are easy to reproduce. Unfortunately, it is difficult to completely replicate physiological conditions, and pathways such as cell respiration are uncoupled by the lysing procedure. Despite these issues, for experimental questions about enzymatic or catalytic behavior, lysate procedures are often the simplest method to obtain answers.

Western blot, immunoassays and other techniques

For immunoprecipitation assays, western blots, and similar procedures, use of cell lysates is standard protocol and gives dependable results. Choosing to employ either colorimetric or fluorimetric detection techniques typically depends on the degree of sensitivity required, the experimental design, and the equipment available. In general, colorimetric tests are simple to run and require more basic laboratory equipment, and fluorimetric assays give more sensitive results.

Table 2. Detection Sensitivity of Colorimetric Amplite® NAD(P) and NAD(P)H Assay Kits

Cat No.
Product Name
Assay Target
Detection Limit
Dynamic Range
15258Amplite® Colorimetric Total NAD and NADH Assay KitNAD+NADH0.3 µM0-10 µM
15260Amplite® Colorimetric Total NADP and NADPH Assay KitNADP+NADPH0.1 µM0-3 µM
15271Amplite® Colorimetric NADH Assay KitNADH3 µM1-200 µM
15272Amplite® Colorimetric NADPH Assay KitNADPH3 µM1-200 µM
15273Amplite® Colorimetric NAD/NADH Ratio Assay KitNAD/NADH0.1 µM0-3 µM
15274Amplite® Colorimetric NADP/NADPH Ratio Assay KitNADP/NADPH Ratio0.03 µM0.03-1 µM
15275Amplite® Colorimetric Total NAD and NADH Assay Kit *Enhanced Sensitivity*NAD+NADH0.1 µM0.01-10 µM
15276Amplite® Colorimetric Total NADP and NADPH Assay Kit *Enhanced Sensitivity*NADP+NADPH0.03 µM0.03-1 µM

Table 3. Detection Sensitivity of Fluorimetric Amplite® NAD(P) and NAD(P)H Assay Kits



The edge of science is informed by new information giving rise to new patterns of thought. The paired nicotinamide molecules within the cell still do much that researchers are on the edge of understanding. Employing and improving NAD+ detection in multiple applications will be central to interpreting the interrelated nature of cellular dynamics. NAD+ gains more biological importance with each new discovery and is likely to be a core component of future personalized medical therapies for currently incurable human conditions.


Additional Resources

Interactive Spectrum Viewer Online Tool
A common pitfall of multicolor flow cytometric analysis is fluorescence spillover caused by two or more fluorophores with overlapping emission spectra. In order to minimize this problem and improve confidence in the results, fluorophores should be chosen with an eye towards avoiding emission wavelength intersection. The interactive Spectrum Viewer tool consists of a large library of fluorophores to compare in order to assist researchers with experimental design.



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