A Look at NAD-Dependent SIRT-1

It has long been suspected that SIRT1 plays a role in modulating cellular metabolism. For example, early studies showed that SIRT1 is critical to the regulation of glucose homeostasis through regulation of gluconeogenic and glycolyic pathways. Specifically, a study by Rodgers et al. found that SIRT1 interacts with a transcriptional cofactor called PGC-1α. Through the deacetylation of PGC-1α at lysine residues, SIRT1 is able to induce gluconeogenic genes, which leads to increases in glucose output in hepatic cells. Additional studies have supported these initial findings, with research showing that SIRT1 acts as a metabolic switch in cardiac and skeletal myocytes amongst other cell lines.

While it seems fairly evident that SIRT1 plays a role in modulating cellular metabolism, what is much more controversial is the idea that SIRT1 plays a role in cellular longevity and aging. In particular, a wealth of research seems to suggest that caloric restriction, or fasting, will activate SIRT1 pathways and lead to lifespan extension. Such findings have spanned different kingdoms, with the effects of caloric restriction present in organisms like yeast, flies and rats. Such findings have also prompted a frenzy of research into the potential links between caloric restriction, SIRT1 and neurodegenerative diseases like Alzheimer's dementia. The increased interest in caloric restriction has led many researchers to investigate the possible mechanisms in which SIRT1 may be involved.

Research by Brunet et al. in 2004 seems to suggest that SIRT1 may play a role in the regulation of FOX (Forkhead box) transcription factors, particularly one called FOXO3. The FOX proteins are well-known to have important influences on genes regulating cell growth, proliferation and apoptosis, so it is certainly a possible pathway by which SIRT1 can modulate cell longevity. Brunet et al. argue that SIRT1 acts to deacetylate FOXO3, which in experiments resulted in increased cellular resistance to oxidative stress while simultaneously inhibiting induced cell death. The overall effect, they posit, is an increase in cell longevity.

While FOXO3 may be the mechanism by which SIRT1 leads to lifespan extension, it does not explain why caloric restriction specifically will lead to activation of FOX pathways. For this, many researchers have pointed to studies in S. cerevisiae. In particular, researchers have argued that SIRT1's NAD-dependent activation is the link between caloric restriction and lifespan extension. In S. cerevisiae, caloric restriction causes a metabolic shift in which the more efficient respiration pathway becomes preferred over the less efficient fermentation pathway. This change in metabolic pathway causes an alteration in the NAD+/NADH ratio, leading to an increase in cellular NAD+, an increase in SIRT1 activity and, as researchers have argued, an increase in cell longevity. More recently, however, some contradictory evidence has arisen which seems to at least cast doubt onto this explanation. For example, in S. cerevisiae, which are unable to undergo respiration, the same correlation between caloric restriction and longevity has been found. Studies in worms have also found that reduced mitochondrial function is correlated with increases in lifespan. This has motivated many researchers to seek alternative explanations for the link between caloric restriction and SIRT1 up-regulation, with many suggesting that NAD+ regeneration from nicotinamide (NAM) should receive closer investigation.

Our Tools

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We offer an extensive catalog of kits for life science researchers interested in studying NAD and NADH. These kits come in two varieties: colorimetric and fluorimetric. In general, we recommend fluorimetric kits as these tend to be more sensitive. However, many researchers may prefer colorimetric kits due to compatibility with existing equipments. Fluorimetric kits will require a fluorescence microplate reader or analogous setup. Colorimetric kits can be read with standard absorbance microplate readers.

Our colorimetric assay kits utilize a novel chromogenic sensor that is superior to traditional NAD/NADH probes. Unlike traditional probes which maximally absorb at 340 nm, our probe has a maximum absorbance at 460 nm upon NADH reduction. This means that it does not require the use of quartz microplates, which are not only expensive but tend to result in low sensitivity and high interference. The concentration of NADH in a sample solution can easily be calculated from the absorbance at 460 nm as these two values are proportional. Our probe is sensitive enough that it can detect as little as 3 µM NADH in a 100 µL assay volume and is excellent for studies involving oxidative stress in which the NAD/NADH system is involved. For fluorimetric detection, we have our Amplite^® Fluorimetric NAD/ NADH Ratio Assay Kit *Red Fluorescence*. This kit consists of a system of enzymes that specifically recognize NAD/NADH through use of an enzyme cycling reaction. Compared with other detection methods, our enzyme cycling reaction significantly increases detection sensitivity. In addition, this assay has been shown to have very low background. This is due to its spectral properties, which lie in the red visible range, significantly reducing interference from biological samples. For researchers concerned about usability, our kit is very convenient in its application. There is no need to purify NAD/NADH from the sample prior to use; simply mix-and-read using a fluorescence microplate reader (Ex/Em = 540/590 nm).