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`Author Manuscript
`Crit Rev Biochem Mol Biol. Author manuscript; available in PMC 2013 December 11.
`Published in final edited form as:
`Crit Rev Biochem Mol Biol. 2013 ; 48(4): . doi:10.3109/10409238.2013.789479.
`
`NAD+ metabolism, a therapeutic target for age-related metabolic
`disease
`
`Laurent Mouchiroud#1, Riekelt H. Houtkooper#2, and Johan Auwerx1,*
`
`1Laboratory for Integrative and Systems Physiology, Ecole Polytechnique Fédérale de Lausanne
`(EPFL), Lausanne, Switzerland 2Laboratory Genetic Metabolic Diseases, Academic Medical
`Center, Amsterdam, The Netherlands
`# These authors contributed equally to this work.
`Abstract
`Nicotinamide adenine dinucleotide (NAD) is a central metabolic cofactor by virtue of its redox
`capacity, and as such regulates a wealth of metabolic transformations. However, the identification
`of the longevity protein Sir2, the founding member of the sirtuin protein family, as being NAD+-
`dependent reignited interest in this metabolite. The sirtuins (SIRT1-7 in mammals) utilize NAD+
`to deacetylate proteins in different subcellular compartments with a variety of functions, but with a
`strong convergence on optimizing mitochondrial function. Since cellular NAD+ levels are limiting
`for sirtuin activity, boosting its levels is a powerful means to activate sirtuins as a potential therapy
`for mitochondrial, often age-related, diseases. Indeed, supplying excess precursors, or blocking its
`utilization by PARP enzymes or CD38/CD157, boosts NAD+ levels, activates sirtuins and
`promotes healthy aging. Here, we discuss the current state of knowledge of NAD+ metabolism,
`primarily in relation to sirtuin function. We highlight how NAD+ levels change in diverse
`physiological conditions, and how this can be employed as a pharmacological strategy.
`
`Keywords
`Aging; Metabolism; Mitochondria; PARPs; Sirtuins
`
`1. Introduction
`Nicotinamide adenine dinucleotide (NAD) is a metabolic cofactor that is present in cells
`either in its oxidized (NAD+) or reduced (NADH) form. Its function as a cofactor for a
`multitude of enzymatic reactions has been appreciated since the early 1900’s, when NAD+
`was described as a “cozymase” in fermentation and its characteristics were elucidated, not in
`the last place by several Nobel prize winners (Berger et al., 2004). In its function as an
`oxidoreductase cofactor, NAD+ is critical for a wide range of enzymatic reactions, including
`for instance GAPDH in glycolysis. NAD redox balance is tightly regulated (we refer the
`reader for more information of this aspect to (Houtkooper et al., 2010a)). After a period of
`relative anonymity, NAD+ became again in the spotlight because it was identified as a
`substrate for a major class of deacetylase proteins, the sirtuins, named after the founding
`member of the family yeast Sir2p (Ivy et al., 1986; Rine and Herskowitz, 1987). Sirtuins
`have pleiotropic metabolic effects, and since NAD+ levels reflect the energy state of the cell,
`
`*Correspondence: Phone, +41-21 693 9522; or admin.auwerx@epfl.ch.
`Declaration of interest The authors declare no conflict of interest with respect to this publication.
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`it was hypothesized that sirtuins could function as metabolic sensors that use NAD+ as a
`messenger and cosubstrate, translating this signal to a cellular adaptation (Canto and
`Auwerx, 2011). Due to this development, it has become apparent that pathways involved in
`synthesis or consumption of NAD+ are attractive targets for the management of conditions
`with dysfunctional metabolism, including not only obesity and diabetes, but also cancer and
`neurodegenerative diseases (Houtkooper and Auwerx, 2012).
`
`In this review, we will describe the pathways contributing to NAD+ homeostasis, and will
`discuss their potential benefits in the management of metabolic disease.
`2. NAD+ metabolism
`NAD+ metabolism is a careful balance between biosynthesis on one hand and its breakdown
`on the other. Importantly, both sides of the balance are composed of several pathways.
`
`NAD+ biosynthesis and salvage
`NAD+ can be synthesized from various precursors (figure 1). De novo biosynthesis, which
`starts from the amino acid tryptophan, occurs primarily in the liver and kidney but is
`considered a minor contributor to the total pool of NAD+ (reviewed in detail in (Houtkooper
`et al., 2010a)). On the other hand, biosynthesis from nicotinic acid (NA) or nicotinamide
`(NAM)—both present in our diet as vitamin B3—is the primary source of NAD+. These
`pathways, also known as the salvage or Preiss-Handler pathway, are important for NAD+
`homeostasis. This is illustrated by the human disease pellagra, which is caused by NAD+
`deficiency subsequent to poor dietary intake of precursors. Pellagra is clinically
`characterized by the 4 “D’s”, i.e. diarrhea, dermatitis, dementia, and if untreated ultimately
`death. Pellagra, in dogs prevalent as black tongue disease, is caused by deficiency of NAD+
`(precursors) and can be easily treated by providing the vitamin in the diet (Elvehjem et al.,
`1937). Synthesis of NAD+ from NA or NAM involves phosphoribosyl transfer followed by
`adenylyl transfer. In the case of NA, the resulting product requires a final ATP-dependent
`amidation step by NAD synthase to complete the synthesis of NAD+ (for more detail on
`NAD+ enzymology we refer the reader to (Houtkooper et al., 2010a; Magni et al., 2004).
`
`Recently, a “new” NAD+ precursor—NAM riboside (NR)—that also enhances NAD+ levels
`through the salvage pathways was described (Bieganowski and Brenner, 2004). Even though
`this pathway for NAD biosynthesis was already known in bacteria, it was only recently
`demonstrated that NR —which is found in milk and yeast—could also be used to synthesize
`NAD+ in eukaryotes (Bieganowski and Brenner, 2004). Indeed, supplementation of NR to
`cells or mice increases the levels of NAD+ and results in the activation of its downstream
`signaling cascades (Canto et al., 2012), as will be discussed in more detail below.
`
`Sirtuins
`
`Sirtuins are a class of metabolic regulators, of which seven orthologues exist in mammals
`(Blander and Guarente, 2004; Chalkiadaki and Guarente, 2012; Haigis and Sinclair, 2010;
`Houtkooper et al., 2012). The sirtuins differ in tissue expression, subcellular localization,
`enzymatic activity and targets. Sirtuins are named after their homology to yeast Sir2 (silent
`regulator 2) (Ivy et al., 1986; Rine and Herskowitz, 1987), which was originally described as
`a NAD+-dependent class III histone deacetylases (Imai et al., 2000). Sirtuins are categorized
`into four different classes according to the amino acid sequence-based phylogenetic analysis
`(Frye, 2000): Class I includes SIRT1, SIRT2, and SIRT3, Class II and Class III SIRT4 and
`SIRT5, respectively, and SIRT6 and SIRT7 come under Class IV. Mammalian sirtuins show
`a diverse subcellular localization. SIRT1, SIRT6 and SIRT7 are mainly found in the
`nucleus, SIRT2 is predominantly in the cytoplasm, while SIRT3, SIRT4 and SIRT5 are
`localized in mitochondria (Pirinen et al., 2012). It has become clear, however, that the
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`sirtuins not only deacetylate histones, but also a wide range of other proteins (figure 2).
`Most of the targets are involved in stress response pathways, whether metabolic in nature,
`genotoxic or otherwise. In addition, some of the sirtuins were reported to ADP-ribosylate
`proteins rather than deacetylate (Haigis et al., 2006), and SIRT5 was shown to act as a
`demalonylase and desuccinylase (Du et al., 2011; Peng et al., 2011; Wang et al., 2011).
`Future research will have to determine whether other sirtuins also possess such activity, but
`it seems likely that multiple family members function as deacylases.
`
`The nuclear sirtuin SIRT1 is the best-known member of the family especially after it was
`described as the target of the polyphenol, resveratrol (Howitz et al., 2003), which is found in
`low quantities in red wine (see section “Pharmacological control of NAD+ levels”). SIRT1
`deacetylates histones, but its key activity involves the regulation of mitochondrial biogenesis
`and stress response through the deacetylation of PGC-1α (Rodgers et al., 2005) and FOXO1
`(Brunet et al., 2004; van der Horst et al., 2004). Its role in stress response is further
`confirmed by the identification of p53, HIF-1α and NF-κB as SIRT1 targets (reviewed in
`(Canto and Auwerx, 2011; Houtkooper et al., 2012)). Less is known about the other sirtuins,
`but it is clear that they also impact on metabolism in various ways. The cytosolic SIRT2 was
`shown to deacetylate tubulin (North et al., 2003), as well as the sterol regulatory element
`binding protein-2 (SREBP-2) (Luthi-Carter et al., 2010), although genetic evidence for this
`latter association is so far lacking. Furthermore, SIRT2 deacetylates phosphoenolpyruvate
`carboxykinase (PEPCK) to control gluconeogenesis (Jiang et al., 2011) and was recently
`shown to deacetylate the receptor-interacting protein 1 (RIP1), and thereby serve as a critical
`component of the TNFα-mediated programmed necrosis pathway (Narayan et al., 2012).
`Finally, SIRT2 controls myelin formation in vivo through the atypical-PKC regulator PAR3
`(Beirowski et al., 2011). The mitochondrial sirtuins—SIRT3, SIRT4 and SIRT5—
`deacetylate protein targets involved in oxidative phosphorylation (Ahn et al., 2008), fatty
`acid oxidation (Hirschey et al., 2010), ketogenesis (Shimazu et al., 2010), oxidative stress
`(Someya et al., 2010), glutamate metabolism (Haigis et al., 2006), urea cycle (Nakagawa et
`al., 2009), as well as several other mitochondrial pathways (Hebert et al., 2013), and thereby
`regulate multiple facets of mitochondrial metabolism (reviewed in (Houtkooper et al., 2012;
`Pirinen et al., 2012; Verdin et al., 2010)). Surprisingly, mice deficient in either of the
`mitochondrial sirtuins do not develop an overt metabolic phenotype under basal non-
`challenged conditions (Fernandez-Marcos et al., 2012; Haigis et al., 2006; Haigis and
`Sinclair, 2010; Hirschey et al., 2010; Lombard et al., 2007; Nakagawa et al., 2009). SIRT6
`deacetylates both histones and DNA polymerase β, a DNA repair protein. As a result,
`deletion of SIRT6 in mice results in a severe premature aging phenotype associated with
`defects in DNA repair (Mostoslavsky et al., 2006). Additionally, Sirt6−/− mice have reduced
`IGF1 levels and are severely hypoglycemic (Mostoslavsky et al., 2006), possibly mediated
`by the HIF1α-dependent activation of glycolysis and subsequent decreases in glucose levels
`(Zhong et al., 2010). No in vivo molecular deacetylation targets have been described for the
`nucleolar SIRT7, but knockdown or overexpression of SIRT7 resulted in decreased or
`increased RNA polymerase I-mediated transcription, respectively (Ford et al., 2006). A
`thorough characterization of Sirt7−/− mice has not been performed but mice deficient for
`SIRT7 display hyperacetylation of p53, develop cardiomyopathy and die young
`(Vakhrusheva et al., 2008).
`
`Other NAD+ consumers: PARPs and CD38/CD157
`NAD+ is not only consumed by sirtuins, but also by the members of the poly(ADP-ribose)
`polymerase (PARPs) family and the NAD glycohydrolases CD38 and CD157 (figure 2). The
`nuclear PARP1 accounts for most of the PARP activity in vivo and is the best studied family
`member, but critical functions for other PARPs are emerging (Luo and Kraus, 2012;
`Schreiber et al., 2006). PARPs are best characterized for their role in DNA damage
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`pathways, but more generally PARPs regulate adaptive stress responses, including
`inflammatory, oxidative, proteotoxic, and genotoxic stresses (Luo and Kraus, 2012). For
`example, when protein translation is stalled, PARP13 localizes to cytosolic stress granules
`and regulates microRNA expression and activity, alleviating the protein stress (Leung et al.,
`2011). More recently, however, the role of the different PARPs role in metabolism has
`become more apparent, as Parp1 and Parp2 knockout mice are protected against high-fat
`diet induced obesity (Bai et al., 2011a; Bai et al., 2011b; Bai et al., 2007). Based on the
`functional links between PARPs and sirtuins, it was tempting to speculate that the levels of
`NAD+, the joint co-substrate, could in fact dictate these functions. The in vivo
`characterization of mutant mice for PARP1—the major PARP isoform—further
`corroborated this hypothesis (Bai et al., 2011b). Parp1−/− mice were protected from high-fat
`diet induced obesity and showed overall improved fitness compared to control littermates.
`The effects of Parp1 deletion were due to elevation of NAD+ levels and subsequent SIRT1-
`dependent activation of mitochondrial metabolism in brown adipose tissue and muscle (Bai
`et al., 2011b). Importantly, this genetic evidence was confirmed by pharmacological studies
`using PARP inhibitors (Bai et al., 2011b), as will be further discussed in section
`“Pharmacological control of NAD+ levels”. Interestingly, Parp2−/− mice were also
`protected against diet-induced obesity, but this effect was not mediated through changes in
`NAD+ levels and activation of SIRT1 as the case in Parp1−/− mice, but rather through the
`induction of muscle SIRT1 expression (Bai et al., 2011a). While PARPs are stress response
`proteins, the NAD+-consuming CD38 is an ubiquitous, but still quite enigmatic enzyme,
`involved in maintaining calcium homeostasis (Lee, 2012). Although CD38 is often referred
`to as an ectoenzyme, it may also have intracellular activity (Lee, 2012), although its full
`potential as an NAD+ consumer is yet to be discovered. Still, even if most CD38 activity
`occurs outside the cell, the resultant metabolites can be transported inside, most likely in the
`form of NR (Nikiforov et al., 2011). Similar to PARP deficient mice, CD38−/− mice display
`highly elevated NAD+ levels that are accompanied by SIRT1 activation and, at the
`organismal level, increased energy expenditure (Barbosa et al., 2007). The role of CD157,
`also known as Bst1, is not characterized in the context of metabolic disease. It is interesting
`to note, however, that a recent study demonstrated a role for CD157 in the response of
`intestinal Paneth cells to CR (Yilmaz et al., 2012). When Paneth cells are exposed to caloric
`restriction (CR), CD157 is activated to produce cyclic ADP-ribose, which in turn signals to
`intestinal stem cells to switch on maintenance programs rather than differentiation (Yilmaz
`et al., 2012). Whether or not CD157 is involved in metabolic regulation in other tissues as
`well remains to be investigated.
`3. Modulation of NAD levels by physiological processes
`Fasting and exercise
`SIRT1 activity is generally increased during restrictive metabolic conditions, and decreased
`in situations of caloric excess. Complying with the fact that SIRT1 activity is regulated by
`NAD+, these observations shed light on the potential role of NAD+ as a metabolic sensor in
`stress conditions, where the levels of NAD+ are generally affected. During fasting and
`exercise, the level of NAD+ increases (Canto et al., 2009; Canto et al., 2010). Interestingly,
`this increase in NAD+ levels is linked with sirtuin activation. In a similar way, CR in mouse
`models leads to an increase in the level of NAD+ in different tissues, such as muscle, liver
`and white adipose tissue (Canto et al., 2010; Chen et al., 2008). Conversely, caloric excess
`by means of a high-fat diet (Kim et al., 2011), but also aging (Braidy et al., 2011; Yoshino et
`al., 2011), lead to reduced NAD+ levels. Several studies have revealed through the prism of
`the sirtuin family the potential involvement NAD+ in longevity modulation during CR, and
`in a larger extent in the physiological aging mechanism.
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`Caloric restriction, NAD+ and aging
`Aging is characterized by a progressive accumulation of molecular, cellular and organ
`damage, leading to dys- or malfunction of many metabolic processes and a generalized
`physiological decline. If this decline is uncompensated, it can result in the development of
`age-associated diseases, like neurodegenerative diseases, such as Alzheimer’s and
`Parkinson’s disease, metabolic disorders, such as type 2 diabetes and atherosclerosis, or
`cancer. Despite the complexity of the aging process, many studies have demonstrated over
`the last two decades that aging is subject to regulation by common signaling pathways,
`transcription factors and their co-regulators. Among the different proposed mechanisms that
`impact on and modulate longevity, CR is by far the most consistent and reproducible
`intervention that increases lifespan and protects against the decline of biological functions
`with age in many different species.
`
`CR is defined as a moderate limitation of food intake below the ad libitum level, without
`malnutrition. It was already known for ages that moderation and composition of diet can
`influence the aging process (Schafer, 2005), but the modern day founder was Clive McCay,
`who put its benefits in the scientific spotlight in 1935 (McCay et al., 1989). CR remains the
`most effective and reproducible intervention to extend lifespan and delay the development
`of age-associated diseases in divergent species, from yeast to monkeys (Houtkooper et al.,
`2010b; Koubova and Guarente, 2003). The concept that enhanced mitochondrial function
`upon CR contributes to its beneficial effects on lifespan was extended to humans, in which a
`general improvement in metabolic health occurs during CR ((Civitarese et al., 2007) and
`reviewed in (Holloszy and Fontana, 2007)). The implication of NAD+ in aging is closely
`intertwined with the major role proposed for the NAD+-dependent sirtuin enzymes in CR.
`
`The role of NAD+ in CR emerged from studies in yeast, where pioneering work revealed
`that longevity mediated by CR requires the NAD+ biosynthesis pathway and the activity of
`the yeast sirtuin homolog Sir2 (Lin et al., 2000). It was proposed that increased Sir2 activity
`leads to the repression of recombination events at the homologous repeats present in the
`ribosomal DNA, preventing as such the formation of extra-chromosomal ribosomal DNA
`circles, which is one of the causes of replicative aging in yeast. Thus, the increased dosage
`of SIR2 in yeast prevents the formation of extra-chromosomal ribosomal DNA circles and
`prolongs lifespan, whereas its inhibition has the opposite effect, reducing the replicative life
`span by 50% (Kaeberlein et al., 1999). Interestingly, mimicking CR by reducing glucose
`concentration of the growth medium from 2% to 0.5% is sufficient to extend lifespan to a
`similar level as by overexpressing Sir2, and these effects were dependent on the Sir2 gene or
`the nicotinate phosphoribosyltransferase 1 (NPT1) gene, which is involved in the
`biosynthesis of NAD (Lin et al., 2000).
`
`The worm genome comprises four genes sharing homology with Sir2, with sir-2.1 being the
`closest homolog of Sir2 (Frye, 2000), and sir-2.1 is required for the lifespan extension in
`response to the CR-mutation eat-2 (Wang and Tissenbaum, 2006). In the fruitfly Drosophila
`melanogaster, which expresses five homologs of Sir2 (Frye, 2000), CR extends lifespan and
`increases dSir2 mRNA expression, but was unable to mediate lifespan extension in flies
`where dSir2 had been deleted (Rogina and Helfand, 2004). As discussed before, the
`mammalian genome encodes seven sirtuins and within this family of proteins, SIRT1 is the
`most extensively studied in the context of the lifespan regulation. Several in vivo studies
`assign a role for SIRT1 to explain the longer life in mice under CR. In fact, in Sirt1−/− mice
`the beneficial effects on metabolism and longevity induced by a CR diet are attenuated,
`although it should be noted that these mice are very sick to start with (Boily et al., 2008;
`Chen et al., 2005). Conversely, transgenic mice, constitutively overexpressing the Sirt1
`gene, exhibit a range of features that are reminiscent of the phenotypes seen in CR mice, as
`they are lighter and metabolically more active, show improved glucose homeostasis, and
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`develop less cancer (Banks et al., 2008; Bordone et al., 2007; Herranz et al., 2010; Pfluger et
`al., 2008).
`
`The beneficial effects mediated by SIRT1 under CR conditions have been proposed to be
`due to an improvement of the mitochondrial function and biogenesis (Guarente and Picard,
`2005), but also to a global increase in stress resistance and maintenance of the cellular and
`mitochondrial homeostasis (figure 3). Recent studies in worms added a new layer of
`complexity in this dynamic mechanism, by showing that an early burst of ROS is required
`for the induction of the ROS defense pathway and for the lifespan extension under CR
`conditions (Mouchiroud et al., 2011; Schulz et al., 2007). Moreover, compelling new
`evidence suggest that other pro-longevity pathways, induced by CR and/or stress conditions,
`could also potentially involve SIRT1, such as mitophagy, mitochondrial dynamics (fission/
`fusion) and mitochondrial unfolded response (Durieux et al., 2011; Egan et al., 2011; Yang
`et al., 2011). Further studies are needed to decipher the exact role of NAD+ and SIRT1 in
`lifespan regulation through these mechanisms.
`
`However, the requirement of sirtuin proteins in longevity modulation is not without
`controversy and still the object of an intense debate. Initial work reported that increased
`expression of the yeast protein Sir2 and of related sirtuin proteins in Caenorhabditis elegans
`and Drosophila melanogaster extends lifespan (Rogina and Helfand, 2004; Tissenbaum and
`Guarente, 2001). These observations were recently challenged by showing that the effect of
`overexpression of worm sir-2.1 and fly SIR2 on lifespan is, at best, limited or even absent
`(Burnett et al., 2011; Viswanathan and Guarente, 2011). In view of the predominant role of
`SIRT1 in metabolic homeostasis in mammals, we speculate that SIRT1 is rather a major
`keystone in health maintenance and stress response, instead of being crucial for the
`determination of lifespan per se. As such, NAD+ serves as a central metabolite that
`communicates the metabolic state under such stressful conditions and activates the sirtuins
`to trigger adaptive and protective responses. Further studies are needed to elucidate the role
`of the others sirtuin family members in longevity regulation.
`4. Pharmacological control of NAD+ levels
`Resveratrol and STACs
`As briefly mentioned above, various compounds can modulate the levels of NAD+ and
`thereby activate sirtuin enzymes. One of the best described is the polyphenol 3,5,4′-
`trihydroxystillbene, which was originally isolated in 1939 from the roots of the plant white
`hellebore (Veratrum grandiflorum O. Loes) (Takaoka, 1939). This fact is reflected in the
`common name, of the compound, i.e., resveratrol, a combination of res (from the fact that it
`is a resorcinol or dihydroxy benzene), veratr (Veratrum) and ol (for the alcoholic groups)
`(figure 4). A few decades later, in 1963, resveratrol was extracted from the roots of another
`plant, Japanese knotweed (Reynoutria japonica), commonly used in traditional Chinese and
`Japanese medicine (reviewed in (Baur and Sinclair, 2006)). In the wild, resveratrol is found
`in many edible fruits, such as grapes, blueberries, cranberries or peanuts (Baur and Sinclair,
`2006). It is also present in red wine at concentration ranging from 0.1 to 14.3 milligrams per
`liter (Soleas et al., 1997). Interestingly, higher levels of resveratrol are made by plants in
`response to infection or nutrient stress, thereby qualifying it as a phytoalexin. Resveratrol
`has attracted the attention of the scientific community when it was demonstrated that this
`molecule could be the origin cardioprotective effects specific to red wine which is
`commonly referred to as «the French paradox » (Kopp, 1998; Pace-Asciak et al., 1995).
`Since then resveratrol has been shown to be effective in preventing and delaying the
`progression of various diseases such as cancer (Jang et al., 1997), cardiovascular disease and
`glucose intolerance (Timmers et al., 2011) and ischemic stroke (Sinha et al., 2002; Wang et
`al., 2002). In 2003, an in vitro high throughput screening of small chemical compounds
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`identified resveratrol as the most potent activator of SIRT1, able to extend the yeast lifespan
`(Howitz et al., 2003). As in yeast, treatment with resveratrol increases lifespan of worms and
`fly in a SIRT1 (sir-2.1 or Sir2p, respectively) dependent manner (Howitz et al., 2003; Wood
`et al., 2004), although this is controversial (Bass et al., 2007; Kaeberlein et al., 2005). In
`mammals, resveratrol supplementation in mice fed with a high fat diet (HFD) improved
`physiological parameters, as these mice showed a decrease in HFD-induced weight gain, an
`improved glucose metabolism, and less damage to the pancreas and heart, all features
`associated with increased activity of AMPK and PGC-1α, culminating in increased
`mitochondrial number and function (Baur et al., 2006; Lagouge et al., 2006). Ultimately, this
`beneficial metabolic profile leads to a longer life expectancy (Pearson et al., 2008). This
`effect on longevity is, however, only observed when mice are fed with a HFD. Under chow
`diet, treatment with resveratrol does not extend mice lifespan, although it seems to improve
`their overall health (Barger et al., 2008; Pearson et al., 2008). In chow fed mice, resveratrol
`significantly attenuated several hallmarks of aging, such as reduced inflammatory and
`apoptotic events in vascular endothelium, limiting the formation of cataracts, preservation of
`bone density and conservation of motor activity with age (Barger et al., 2008; Baur et al.,
`2006; Lagouge et al., 2006; Pearson et al., 2008). These resveratrol treated mice also exhibit
`a transcriptional profile in heart, liver and muscle that is similar to that seen in animals under
`CR, supports the idea that resveratrol mimics the effects of food limitation in ad libitum fed
`individuals. The fact that the effects of the CR mimetic, resveratrol, also depend on the diet
`contributed to the controversy that CR could work only in animals maintained under
`“regular” laboratory conditions. Indeed, it has been questioned whether the beneficial effects
`observed under CR were not due to a simple “rescue” of the deleterious effects brought by
`the state of overnutrition specific of the artificial diets prepared in laboratories (Harper et al.,
`2006; Longo and Finch, 2003; Martin et al., 2010; Prentice, 2005). These observations could
`explain the conflicting results obtained with CR in non-human primates, where two
`independent studies—both of which using a different control diet and feeding regimen—
`showed a different extent of CR health benefits (Colman et al., 2009; Mattison et al., 2012).
`Since it was known that resveratrol acts as an inhibitor of the ATP synthase complex in the
`oxidative phosphorylation (Zheng and Ramirez, 2000), it was hypothesized that the effects
`of resveratrol could be mediated by activation of the AMP-activated protein kinase
`(AMPK), rather than direct SIRT1 activation (Beher et al., 2009; Canto et al., 2010;
`Pacholec et al., 2010; Um et al., 2010). Indeed, AMPK is activated upon resveratrol
`treatment and resveratrol’s effects are lost in cells or tissues devoid of AMPK (Canto et al.,
`2010; Um et al., 2010) (figure 5). Following AMPK activation, expression of NAMPT
`increased, leading to increased NAD+ levels and activation of SIRT1 (Canto et al., 2009;
`Canto et al., 2010; Fulco et al., 2008). It should be noted that the mode of action of
`resveratrol is still subject of debate. Recent reports suggest that resveratrol may act through
`phosphodiesterase 4 inhibition, thereby mobilizing calcium stores and activating AMPK
`(Park et al., 2012), or through allosteric activation of SIRT1 that is dependent on structural
`hydrophobic motifs in SIRT1 substrates (Hubbard et al., 2013), but further work is needed to
`clarify whether this also plays a physiological role.
`
`Regardless of these issues, resveratrol treatment in mice induces mitochondrial biogenesis
`and energy expenditure (Baur et al., 2006; Lagouge et al., 2006). The required dose
`(200-400 mg/kg/day), however, was in a range that is normally incompatible with human
`consumption (15-30 g per day for a 75 kg person). Importantly and reassuring from a
`clinical point of view, resveratrol supplementation in obese humans reached beneficial
`effects at a far lower dose (150 mg per day) (Timmers et al., 2011). It should be noted,
`however, that resveratrol failed to exert beneficial effects in non-obese female subjects
`(Yoshino et al., 2012), in line with mouse data where resveratrol is particularly effective in
`high-fat diet fed mice (Baur et al., 2006; Lagouge et al., 2006).
`
`Crit Rev Biochem Mol Biol. Author manuscript; available in PMC 2013 December 11.
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` Europe PMC Funders Author Manuscripts
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` Europe PMC Funders Author Manuscripts
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`Elysium Health Exhibit 1010
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`Increasing SIRT1 activity through the use of synthetic SIRT activating compounds or
`STACs, such as SRT1720, also prevents diet-induced obesity and delays the onset of
`associated metabolic abnormalities in mice models (Feige et al., 2008; Milne et al., 2007).
`Similar to resveratrol, the small molecule SIRT1 activator SRT1720, which is 1,000-fold
`more potent than resveratrol, also extends both mean and maximum mouse lifespan in mice
`fed with HFD (Minor et al., 2011). This effect is potentially explained by the improved
`metabolic homeostasis, as typified by insulin sensitization and increased mitochondrial and
`locomotor activity (Feige et al., 2008; Milne et al., 2007). It is important to mention that
`there is, however, still some debate whether SRT1720, as well as other SIRT1-activators, are
`targeting SIRT1 in a specific manner (Beher et al., 2009; Dai et al., 2010; Pacholec et al.,
`2010), although recent evidence suggests that this may be dictated by specific hydrophobic
`residues in SIRT1 substrates (Hubbard et al., 2013). Human efficacy studies with such
`synthetic SIRT1 activators should be reported in the near future.
`
`NAD+ boosters
`A more specific approach to modify NAD+ levels involves the supplementation of NAD+
`precursors or NAD+-consumption inhibitors (figure 3). The precursors NA, NMN, and NR,
`but also PARP or CD38 inhibitors increase NAD levels in various cell types and tissues of
`mice (Bai et al., 2011b; Barbosa et al., 2007; Canto et al., 2012; Yoshino et al., 2011).
`
`NA, also called niacin, has been used to treat dietary tryptophan deficits (pellagra) and
`hyperlipidemia (Elvehjem et al., 1937; Karpe and Frayn, 2004; Sauve, 2008). Along another
`line, reduced NAMPT expression in Nampt+/− mice, which decreased plasma NMN levels
`and lowered NAD+ levels in brown adipose tissue, at least in female mice, was shown to
`impair glucose-stimulated insulin secretion (Revollo et al., 2007). This effect can be rescued
`by NMN supplementation, which indicates that the maintenance of NAD+ levels is crucial
`for pancreatic function (Revollo et al., 2007). A recent study has confirmed this observation
`by demonstrating that NAMPT activity is compromised by HFD and aging, and could
`contribute to the pathogenesis of type 2 diabetes (Yoshino et al., 2011). Enhancing NAD+
`biosynthesis by intraperitoneal injection of NMN indeed improved glucose homeostasis in
`obese mice (Yoshino et al., 2011). NR is another potent naturally occurring NAD+
`precursor. Dietary NR supplementation increases NAD+ levels in brown adipose tissue,
`muscle and liver, but not in brain and white adipose tissue (Canto et al., 2012). In