NAD+ Metabolism – A Link to Age-Associated Pathologies
Novel Fluorescence Probes for Sensitive Detection of Exogenous and Endogenous Nitric Oxide in Live Cells

NAD+ Metabolism – A Link to Age-Associated Pathologies

Practical Guide for Live Cell Cycle Analysis in Flow Cytometry

Intracellular pH Measurement with Dual Excitation Fluorescence Sensor BCFL

Novel Esterase Substrate for the Quantification of Bacterial Viability

AssayWise Letters Vol. 7(2)

Cellular metabolism involves a host of complex biochemical reactions which are essential for cell survival and energy homeostasis. Research in metabolism has contributed to the understanding that perturbations in these metabolic pathways, particularly mitochondrial dysfunction, have major implications on the onset and progression of metabolic disorders, neurodegenerative diseases and other age-associated pathologies. This has sparked interest in the discovery and development of therapeutic solutions aimed at recovering metabolic functionality to treat such disorders.

The manipulation of intracellular NAD+ is emerging as a promising therapeutic focus. Studies in yeast, worms and mice have shown that an increase in intracellular nicotinamide adenine dinucleotide (NAD+) by dietary supplementation with NAD+ precursors boosted sirtuin activity and subsequently improved metabolic efficiency by enhancing mitochondrial functionality and biogenesis [1,3,5]. Picciotto et al. 2016 discovered that supplementation with nicotinamide mononucleotide (NMN), a key NAD+ intermediate, restored arterial SIRT-1 activity and ameliorated endothelial dysfunction and arterial stiffening in aged mice. This provides evidence that increasing intracellular NAD+ levels can reverse vascular dysfunction and oxidative stress in aging mice, advocating the therapeutic potential of NAD+ supplementation.


What is NAD+ and why is it important?

NAD+ is a key coenzyme found in all living cells. NAD+ was initially discovered over a century ago as a cofactor in yeast fermentation (Harden et al., 1906) and since then, the roles of NAD+ and its metabolite have been expanded. Research has shown NAD+ is a critical coenzyme for oxidoreductases and dehydrogenases in cellular respiration pathways that generate adenosine triphosphate (ATP) from nutrient breakdown. NAD+ is a major degradation substrate, key signaling molecule and rate-limiting cofactor in enzymatic reactions involving sirtuins, poly-ADP ribose polymerases (PARPs) and cyclic ADP ribose synthases (CD38/CD157) [2,10]. These NAD+ dependent proteins consume NAD+ in order to execute posttranslational modifications, such as protein deacetylation, which influence many vital physiological processes including DNA repair, mitochondrial biogenesis and functionality, and calcium signaling [10].


NAD+ Bioavailability and Metabolic Homeostasis

Among the NAD+-consuming proteins, sirtuins play a fundamental role in the regulation of cellular metabolism and adaptation. Mammals have seven sirtuin homologs and each has distinct catalytic activities, biological functions and subcellular localizations [9].  Sirtuins utilize a collection of metabolic targets to translate stress induced changes, such as caloric restriction and oxidative stress, into metabolic adaptations. For example, a well-studied nuclear sirtuin, SIRT-1, targets various transcriptional co-activators (e.g. PGC-1α) and cofactors (e.g. FOXO1) during times of fasting and caloric restriction. PGC-1α acts as the primary regulator of mitochondrial biogenesis and functionality, while FOXO1 modulates mitochondrial fatty acid metabolism and protects against oxidative stress [5].

SIRT-1 is involved in promoting fat mobilization, fatty acid oxidation, glucose production, and insulin secretion in response to nutrient availability in mammals. Moynihan et al., 2005 demonstrated BESTO mice at 3 and 8 months of age have enhanced glucose-stimulated insulin secretion (GSIS) and improved glucose tolerance. This phenotype was found to be due to SIRT-1 mediated repression of UCP2 which causes increased ATP levels in response to glucose stimulation thus resulting in GSIS. Further research by Ramsey et al., 2008, demonstrated that aged mice lose their glucose-responsive phenotypes with age (18-24 months) even though SIRT-1 expression remained high. Isolation of islets of aged BESTO mice notably, no longer showed SIRT-1-mediated repression of UCP2 expression or increased ATP levels. Further investigation showed the depletion in NAD+ levels, suggesting that SIRT-1 activity is contingent upon NAD+ bioavailability. NAD+ levels were replenished using dietary supplementation of a NAD+ precursor, nicotinamide mononucleotide (NMN). The supplementation was followed by a restoration of GSIS and an improved resistance to diabetes in aged mice. This suggests that sirtuin activation can be therapeutically stimulated to ameliorate age-associated disorders and improve health through NAD+ replenishment. This paves new therapeutic avenues for increasing subcellular NAD+ levels through NAD+-precursor supplementation, increased activation of de novo or salvages NAD+ synthetic pathways, or reduced competition for NAD+ via pharmacological inhibition of PARPs and cADPR synthases.


Amplite™ Quantitative Tools for NAD+ and NADH Determination

Quantification of the generation or consumption of NAD+ and its reduced form, NADH, is important for monitoring NAD+/NADH enzyme-mediated reactions or for screening for modulators and substrates of these reactions. Unfortunately, traditional NAD+ and NADH determination assays are not without their caveats. Many of the existing methods monitor changes in NADH using absorption at an ultraviolet wavelength of 340 nm. At this wavelength, biological samples and plastics generate high levels of interference due to light scattering and autofluorescence. These interfering artifacts significantly decrease assay sensitivity and may require the use of counter-assays to identify potential compounds that interfere with the detection method. In addition, assays done in the UV range require the use of expensive quartz cuvettes and microplates. To remedy this, AAT Bioquest® has developed an array of colorimetric and fluorimetric assays for the determination of NAD+ and NADH in the visible spectrum (400 nm – 700 nm).  Our fluorimetric assays offer improved sensitivity and require the use of a fluorescence microplate reader or analogous setup. While colorimetric assays are simple to handle and can be read with standard absorbance microplate readers. 


NAD+ Determination

While several assays are commercially available for quantifying NADH as well as the total or ratio of NAD+/NADH within a biological sample, very little exists for the quantification of NAD+. This is due to the intrinsic properties of NAD+. Upon peak absorption of ultraviolet light at 259 nm, NAD+ is non-fluorescent, making its measurement impractical. However, AAT Bioquest® has developed a fluorimetric NAD+ determination assay that utilizes a proprietary fluorimetric sensor, Quest Fluor™ NAD, which specifically measures NAD+. Our assay can detect as little as 30 nM NAD+ in a 100 μL assay volume. The Quest Fluor™ NAD probe has little to no response towards NADH and upon association with NAD+ generates a fluorescence signal proportional to the concentration of NAD+. The simple add-mix-read protocol makes this assay amenable for high-throughput screening in 96-well or 384-well microtiter plate formats.


Figure 1. NAD+ dose response was measured with Amplite™ Fluorimetric NAD+ Assay Kit (Cat# 15280) in a 96-well black/solid bottom plate using a Germini microplate reader (Molecular devices). A: NAD+ standard curve, as low as 30 nM of NAD+ can be detected with 20 min incubation (n=3). B: Comparison of NAD+ and NADH response. C: NAD+ standard curve with 100 µM NADH in presence in the solution. As low as 0.3% of NAD+ (~300 nM) converted from NADH can be detected with 20 min incubation (n=3).


NADH Determination

The determination of NADH, the reduced form of NAD+, can be measured colorimetrically or fluorimetrically. Amplite™ Colorimetric NADH Assay Kit utilizes a novel chromogenic sensor that is superior to traditional NADH probes. Compared to traditional probes, which maximally absorb at 340 nm, our chromogenic sensor has a maximum absorbance at 460 nm upon NADH reduction. From absorbance increase at 460 nm, a concentration of as little as 3 µM of NADH in a 100 µL assay volume can be easily calculated, as these two values are proportional. Amplite™ Fluorimetric NADH Assay Kit is a homogenous single-reagent-addition assay that specifically recognizes NADH in an enzyme cycling reaction. This enzyme cycling reaction significantly enhances assay sensitivity detecting as little as 1 µM of NADH in a 100 µL assay volume.


Figure 2. NADH dose response was measured with Amplite™ Fluorimetric NADH Assay Kit (Cat# 15261) in a 96-well black plate using a NOVOStar microplate reader (BMG Labtech). As low as 1 μM (100 pmols/well) of NADH can be detected with 1 hour incubation (n=3) while there is no response from NAD+.


Total NAD+/NADH and NAD+/NADH Ratio Determination

The NAD+/NADH ratio (balance between the oxidized and reduced forms) is an important indicator of a cell’s redox state and when measured provides insight to the metabolic activities and health of a cell. The Amplite™ colorimetric and fluorimetric total NAD+/NADH and NAD+/NADH ratio assays are a series of homogenous assays for the sensitive detection of total oxidized and reduced NAD+ as well as their ratio in biological samples. These assays rely on a system of enzyme cycling reactions that specifically recognize NAD+/NADH with superior sensitivity. In addition, the fluorimetric assays have been shown to have reduced interference from biological samples due to its spectral properties, which lie in the red visible range. For researchers concerned about usability, these kits are simple and convenient. Purification of NAD+/NADH from the sample prior to use is not necessary and each assay can easily be adapted for automation without a separation step. Depending on detection format, simply mix-and-read using either an absorbance microplate reader at 460 nm or a fluorescence microplate reader at Ex/Em = 540/590 nm.



Figure 3. Amplite™ Colorimetric NAD+/NADH Ratio Assay Kit (Cat# 15273) was used to measure NAD+/NADH ratio in a 96-well white wall/clear bottom microplate using a SpectraMax® microplate reader (Molecular Devices). Equal amount of NAD+ and NADH mixtures were treated with or without NAD+ extraction solution for 15 minutes, and then neutralized with extraction solution at room temperature. The signal was read at 460 nm. NAD+/NADH ratio was calculated based on the absorbance shown in the figure.


Figure 4. Total NADH/NAD+ and their extract dose responses were measured with Amplite™ Fluorimetric NAD+/NADH Ratio Assay Kit (Cat# 15263) in a 96-well black plate using a Gemini microplate reader (Molecular Devices). 25 µL of equal amount of NAD+ and NADH was treated with or without NADH or NAD+ extraction solution for 15 min, and then neutralized with extraction solutions at room temperature. The signal was read at Ex/Em = 540/590 nm 30 min after adding 75 µL of NADH reaction mixture. The blank signal was subtracted from the values for those wells with the NADH reactions.


Table 1. Product Ordering Information

Cat. #Product NameAssay TargetDetection Mode
15280Amplite™ Fluorimetric NAD Assay Kit *Blue Fluorescence*NADFluorescence
15271Amplite™ Colorimetric NADH Assay KitNADHAbsorption
15261Amplite™ Fluorimetric NADH Assay Kit *Red Fluorescence*NADHFluorescence
15258Amplite™ Colorimetric Total NAD and NADH Assay KitNAD+NADHAbsorption
15275Amplite™ Colorimetric Total NAD and NADH Assay Kit *Enhanced Sensitivity*NAD+NADHAbsorption
15257Amplite™ Fluorimetric Total NAD and NADH Assay Kit
*Red Fluorescence*
NAD+NADHFluorescence
15273Amplite™ Colorimetric NAD/NADH Ratio Assay KitNAD/NADH RatioAbsorption
15263Amplite™ Fluorimetric NAD/NADH Ratio Assay KitNAD/NADH RatioFluorescence


References

  1. Belenky, P., Racette, F.G., Bogan, K.L., McClure, J.M., Smith, J.S., and Brenner, C. (2007). Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+. Cell 129, 473–484.
  2. Cantó, Carles, et al. “NAD Metabolism and the Control of Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus.” Cell Metabolism, vol. 22, no. 1, 2015, pp. 31–53., doi:10.1016/j.cmet.2015.05.023.
  3. Cerutti, R., Pirinen, E., Lamperti, C., Marchet, S., Sauve, A.A., Li, W., Leoni, V., Schon, E.A., Dantzer, F., Auwerx, J., et al. (2014). NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease. Cell Metab. 19, 1042–1049.
  4. De Picciotto, N. E., Gano, L. B., Johnson, L. C., Martens, C. R., Sindler, A. L., Mills, K. F., … Seals, D. R. (2016). Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice. Aging Cell, 15(3), 522–530. http://doi.org/10.1111/acel.12461.
  5. Harden A, Young WJ. The Alcoholic Ferment of Yeast-Juice. Part II.--The Conferment of Yeast-Juice. Proceedings of the Royal Society of London Series B, Containing Papers of a Biological Character. 1906;78:369–3
  6. Mouchiroud, L., Houtkooper, R.H., Moullan, N., Katsyuba, E., Ryu, D., Canto´ , C., Mottis, A., Jo, Y.-S., Viswanathan, M., Schoonjans, K., et al. (2013). The NAD(+)/Sirtuin Pathway Modulates Longevity through Activation of Mitochondrial UPR and FOXO Signaling. Cell 154, 430–441.
  7. Moynihan, Kathryn A., et al. “Increased Dosage of Mammalian Sir2 in Pancreatic β Cells Enhances Glucose-Stimulated Insulin Secretion in Mice.” Cell Metabolism, vol. 2, no. 2, 2005, pp. 105–117., doi:10.1016/j.cmet.2005.07.001.
  8. Ramsey, Kathryn Moynihan, et al. “Age-Associated Loss of Sirt1-Mediated Enhancement of Glucose-Stimulated Insulin Secretion in Beta Cell-Specific Sirt1-Overexpressing (BESTO) Mice.” Aging Cell, vol. 7, no. 1, 2008, pp. 78–88., doi:10.1111/j.1474-9726.2007.00355.x.
  9. Schwer, Bjoern, and Eric Verdin. “Conserved Metabolic Regulatory Functions of Sirtuins.” Cell Metabolism, vol. 7, no. 2, 2008, pp. 104–112., doi:10.1016/j.cmet.2007.11.006.
  10. Srivastava, Sarika. “Emerging Therapeutic Roles for NAD Metabolism in Mitochondrial and Age-Related Disorders.” Clinical and Translational Medicine, vol. 5, no. 1, 2016, doi:10.1186/s40169-016-0104-7.