The Lifespan Extension Ability of Nicotinic Acid Depends on Whether the Intracellular NAD+ Level Is Lower Than the Sirtuin-Saturating Concentrations

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International Journal of Molecular Sciences

Article The Lifespan Extension Ability of Nicotinic Acid Depends on Whether the Intracellular NAD+ Level Is Lower than the Sirtuin-Saturating Concentrations

Nae-Cherng Yang 1,2,* , Yu-Hung Cho 1 and Inn Lee 1

1 Department of Nutrition, Chung Shan Medical University, Taichung 402, Taiwan; [email protected] (Y.-H.C.); [email protected] (I.L.) 2 Department of Nutrition, Chung Shan Medical University Hospital, Taichung 402, Taiwan * Correspondence: [email protected]

Received: 8 November 2019; Accepted: 20 December 2019; Published: 24 December 2019

Abstract: Calorie restriction can extend lifespan by increasing intracellular nicotinamide adenine dinucleotide (NAD+), thereby upregulating the activity of sirtuins (Caenorhabditis elegans Sir-2.1; human SIRT1). Nicotinic acid (NA) can be metabolized to NAD+; however, the calorie restriction mimetic (CRM) potential of NA is unclear. This study explored the ability and mechanism of NA to extend the lifespan of human Hs68 cells and C. elegans. We found that NA can eﬃciently increase the intracellular NAD+ levels in Hs68 cells and C. elegans; however, NA was only able to extend the lifespan of C. elegans. The steady-state NAD+ level in C. elegans was approximately 55 µM. When intracellular NAD+ was increased by a mutation of pme-1 (poly (ADP-ribose) metabolism enzyme 1) or by pretreatment with NAD+ in the medium, the lifespan extension ability of NA disappeared. Additionally, the saturating concentration of NAD+ required by SIRT1 was approximately 200 µM; however, the steady-state concentration of NAD+ in Hs68 cells reached up to 460 µM. These results demonstrate that the lifespan extension ability of NA depends on whether the intracellular level of NAD+ is lower than the sirtuin-saturating concentration in Hs68 cells and in C. elegans. Thus, the CRM potential of NA should be limited to individuals with lower intracellular NAD+.

Keywords: nicotinic acid; calorie restriction mimetic; NAD+; lifespan; C. elegans; Hs68 cells

1. Introduction Calorie restriction mimetic (CRM) is a compound that mimics the metabolic, hormonal, and physiological effects of calorie restriction (CR) to produce CR-like effects on longevity and reduce age-related disease [1]. Nicotinamide adenine dinucleotide (NAD+) is a coenzyme involved in cellular energy metabolism, adaptive responses, neurodegenerative disorders, and aging [2–4]. One of the mechanisms of CR-mediated longevity of organisms is the NAD+–sirtuin pathway, which includes an increase of the intracellular NAD+ level by CR with subsequent activation of sirtuins (e.g., Sir-2.1 in nematodes and SIRT1 in humans) to extend the lifespan of the organisms [5]. Vitamin B3 (also known as niacin) can be metabolized to produce NAD+ and may activate sirtuins to promote longevity of organisms. However, the CRM potential of vitamin B3 remains unclear. It is known that nicotinic acid (NA) and nicotinamide (NAM) are two major forms of vitamin B3. Studies have found that NA has a good blood lipid-lowering effect and can reduce the progression of atherosclerosis and reduce the risk of cardiovascular events [6], while NAM has been reported to prevent the onset of type 1 diabetes. However, human clinical trials have failed to demonstrate these effects [7]. Our previous study has shown that using FK866, an Nampt (nicotinamide phosphoribosyltransferase; a rate-limiting enzyme in the NAD+ synthetic salvage pathway from NAM to NAD+) inhibitor, to mimic

Int. J. Mol. Sci. 2020, 21, 142; doi:10.3390/ijms21010142 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 142 2 of 22

vitamin B3 deficiency in human Hs68 cells can reduce the lifespan of Hs68 cells, suggesting that vitamin B3 deficiency can potentially induce aging [8]. On the other hand, it is reasonable to suggest that supplementation with vitamin B3 may potentially act as a CRM to extend the lifespan of organisms. Indeed, potential use of vitamin B3 as a CRM has been extensively discussed in the literature [9,10]. A major feature of CRM is induction of longevity; however, a previous study has shown that NAM cannot extend the lifespan of C57BL/6J mice [11]. Since NAM itself is an inhibitor of sirtuins [12] and can inhibit poly(ADP-ribose) polymerases (PARP) to induce apoptosis [13], the mechanism of action of NAM may be complex. Indeed, NAM actually increased the lifespan of Caenorhabditis elegans, and this effect is obvious even in the absence of sirtuins [14]. Therefore, this study selected NA as the vitamin B3 representative molecule to study the CRM potential. A previous study has shown that NA can extend the lifespan of C. elegans through Sir-2.1 [14], suggesting the CRM potential of NA. However, a previous study reported that NA does not to extend the lifespan of BJ cells [15]. In our previous paper, we found that calorie restriction can extend the replicative lifespan of Hs68 cells [5], suggesting that NA may be able to extend the lifespan of Hs68 cells if it has CRM potential. Thus, this study intended to explore the ability and mechanism of NA to extend the lifespan of Hs68 cells and C. elegans. We hypothesized that NA can extend the lifespan of Hs68 cells and C. elegans via the NAD+–sirtuin signaling mechanism. The growth curve of the cumulative population doubling level (CPD) was used to monitor the replicative lifespan of Hs68 cells; the senescence-associated β-galactosidase activity was detected in parallel as a cell senescence marker [16]. The effects of NA on the lifespan of C. elegans were assessed according to the lifespan assay and three physiological indexes—pharyngeal pumping, autofluorescence, and body bends, which were used as additional aging markers. The intracellular NAD+ levels of Hs68 cells and C. elegans were determined by acid extraction, followed by the enzymatic cycling method. Two mutants were used: sir-2.1 or pme-1. Pretreatment with NAD+ in the nematode growth medium (NGM) plate was used to reveal the role of Sir-2.1 and the effect of the steady-state level of intracellular NAD+ on the ability of NA to extend the lifespan of C. elegans. Additionally, a commercially available SIRT1 activity kit was used to determine the saturating concentration of NAD+ as a cosubstrate of SIRT1.

2. Results

2.1. Effects of NA on Intracellular NAD+ in Hs68 Cells and C. elegans After Hs68 cells were incubated with various concentrations of NA (0, 0.25, 0.5, 1, and 3 mM) for one day and nematodes were incubated with various dosages of NA (0, 100, 200, and 600 nmol/plate) for seven days, the intracellular NAD+ levels were determined by the enzymatic cycling method. It has been reported that adding 1 mM NA in NGM can extend the lifespan of C. elegans [14]. However, during the treatment with NA in NGM, only NA molecules on the surface of NGM can be taken up by C. elegans. In this study, we directly spiked NA in the LB medium and spread NA onto the center of the top surface of NGM with OP50 bacteria. According the concentration used in a previous report [14], we used 0.5, 1, and 3 mM NA in LB medium and spread 200 µL of the corresponding solutions onto the surface NGM with OP50; thus, the dosages of NA used for the C. elegans treatment were 100, 200, and 600 nmol/plate (note: The LB medium would dry out after spreading onto the surface of NGM, thus the dosage unit of nmol/plate was used rather than the molar concentration of NA). The results showed that NA can significantly increase the intracellular NAD+ levels in Hs68 cells and C. elegans. As shown, NA significantly increased (p < 0.05) intracellular NAD+ at concentrations from 0.25 to 3 mM in Hs68 cells (Figure1a) and dosages of 200 and 600 nmol /plate for nematodes (Figure1b). Int. J. Mol. Sci. 2020, 21, 142 3 of 22 Int. J. Mol. Sci. 2020, 21, 142 3 of 22

(a)

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+ Figure 1.FigureEffects 1. of Effects nicotinic of nicotinic acid (NA) acid on (NA) the intracellular on the intracellular nicotinamide nicotinamide adenine dinucleotideadenine dinucleotide (NAD ) (NAD+) levels inlevels Hs68 cellsin Hs68 and cellsCaenorhabditis and Caenorhabditis elegans.( elegansa) Hs68. ( cellsa) Hs68 were cells cultured were cultured in medium in medium containing containing 0, 0, b C. elegans 0.25, 0.5,0.25, 1, and 0.5, 3 mM1, and NA 3 mM for 24 NA h. for ( ) 24 h. (b) C.were elegans grown were on grown nematode on nematode growth mediumgrowth medium (NGM) (NGM) plates containing 0, 100, 200, and 600 nmol NA/plate in the center of the top surface of NGM for seven plates containing 0, 100, 200, and 600 nmol NA/plate in the center of the top surface of NGM for seven days. The intracellular NAD+ levels in Hs68 cells and C. elegans were measured. Values (mean SD, days. The intracellular NAD+ levels in Hs68 cells and C. elegans were measured. Values± (mean ± SD, n = 3 independent experiments) not sharing a common letter are significantly different (p < 0.05). n = 3 independent experiments) not sharing a common letter are significantly different (p < 0.05). 2.2. Effects of NA on the Replicative Lifespan and Senescence-Associated β-Galactosidase (SA-βG) Activity of Hs68 Cells2.2. Effects of NA on the Replicative Lifespan and Senescence-Associated β-Galactosidase (SA-βG) Activity of Hs68 Cells To estimate the lifespan of Hs68 cells, cells were incubated with various concentrations of NA (0, 0.25, 0.5,To 1, andestimate 3 mM) the and lifespan the cumulative of Hs68 cells, growth cells we curvesre incubated were obtained with various (Figure concentrations2a). During of NA cumulative(0, 0.25, growth 0.5, at 1, day and 91, 3 the mM) SA- andβG activitythe cumulative in Hs68 cellsgrowth was curves determined were by obtained the double-substrate (Figure 2a). During methodcumulative [8], i.e., qualitatively growth at byday X-Gal 91, the staining SA-β (FigureG activity S1; in see Hs68 the Supplementarycells was determined Materials) by the and double- quantitativelysubstrate by relativemethod fluorescein[8], i.e., qualitatively fluorescence by X-Gal (Figure st2ainingb). The (Figure results S1; showed see the that supplementary the replicative materials) and quantitatively by relative fluorescein fluorescence (Figure 2b). The results showed that the

Int. J. Mol. Sci. 2020, 21, 142 4 of 22 Int. J. Mol. Sci. 2020, 21, 142 4 of 22 lifespanreplicative or the SA- lifespanβG activity or the was SA- notβ signiﬁcantlyG activity was (p > not0.05) significantly inﬂuenced by (p NA, > 0.05) illustrating influenced that NAby NA, cannotillustrating extend the that lifespan NA cannot of Hs68 extend cells. the lifespan of Hs68 cells.

(a)

22 Control 20 NA 0.25 mM 18 NA 0.5 mM 16 NA 1 mM 14 NA 3 mM 12 10 8 6 Additional CPDs 4 2 0

0 7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 Incubation time (days) (b)

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80 cein fluorescence cein 60

20 Relative fluores Relative (% of control) 0 l tro mM mM mM mM on 25 .5 1 3 C 0. A 0 NA NA NA N FigureFigure 2. E ff2.ects Effects of nicotinic of nicotinic acid acid (NA) (NA) on theon the replicative replicative lifespan lifespan and and senescence-associated senescence-associated β- β-galactosidasegalactosidase (SA- (SA-βG)β activity.G) activity. Hs68 Hs68 cells cells were were serially serially cultured cultur ined medium in medium containing containing 0, 0.25, 0, 0.25, 0.5, 0.5, 1, 1, andand 3 mM 3 mM NA. NA. (a) Cumulative(a) Cumulative growth growth curves curves were were obtained obtained to determineto determine the the replicative replicative lifespan. lifespan. (b) (b) OnOn day day 91 91 of theof the serial serial culture, culture, SA- SA-βGβG activities activities were were measured measured by by the the double-substrate double-substrate method, method, and and thethe fluorescein fluorescein fluorescence fluorescence is is shown shown as as a %a % of of the the control. control. Values Values are are mean mean SD,± SD,n =n =3 3 plates. plates. ±

2.3. Effects of NA on the Lifespan and Physiological Indexes of C. elegans Different dosages of NA (0, 100, 200, and 600 nmol/plate) were spread onto the central surface of NGM to test the effects of NA on the lifespan of nematodes. The results showed that NA at a dosage of 600 nmol/plate could significantly (p < 0.05) extend the lifespan of nematodes; the average lifespan was increased by 17%, and the median lifespan was increased by four days (Figure 3a; Table

Int. J. Mol. Sci. 2020, 21, 142 5 of 22

2.3. Effects of NA on the Lifespan and Physiological Indexes of C. elegans Different dosages of NA (0, 100, 200, and 600 nmol/plate) were spread onto the central surface of NGM to test the effects of NA on the lifespan of nematodes. The results showed that NA at a dosage ofInt.600 J. Mol. nmol Sci. /2020plate, 21 could, 142 significantly (p < 0.05) extend the lifespan of nematodes; the average 5 of 22 lifespan was increased by 17%, and the median lifespan was increased by four days (Figure3a; Table S1).S1). Additionally, Additionally, NA NA could could significantly significantly (p < 0.05)(p < 0.05) increase increase the pharyngeal the pharyngeal pumping pumping of C. elegans of C. elegans (Figure3(Figureb), reduce 3b), the reduce autofluorescence the autofluorescence of C. elegans of C. (Figureelegans 3(Figurec) and significantly3c) and significantly increase increase the body the body bends ofbendsC. elegans of C. inelegans the swimming in the swimming test (Figure test3 (Figured). 3d).

(a)

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Survival (%) Survival 40

Control 20 NA 100 nmol/plate NA 200 nmol/plate NA 600 nmol/plate 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Days (b)

160 Control NA 100 nmol/plate 140 NA 200 nmol/plate NA 600 nmol/plate 120 a a a 100 a b d c a.bb 80 a b a 60

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Figure 3. Cont.

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Figure 3. Cont.

Int. J. Mol. Sci. 2020, 21, 142 7 of 22 Int. J. Mol. Sci. 2020, 21, 142 7 of 22

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Figure 3. FigureEffects 3. ofEffects nicotinic of nicotinic acid (NA) acid (NA) on the on lifespan the lifespan of C. of elegansC. elegansand and accessory accessoryaging aging markers,markers, including pharyngeal pumping, autofluorescence, and body bends. Nematodes were grown on plates including pharyngeal pumping, autofluorescence, and body bends. Nematodes were grown on plates containing various dosages of NA as described in Section 4. (a) 90 worms (n = 90) for each group were containing various dosages of NA as described in Section4.( a) 90 worms (n = 90) for each group were used, counting the number of surviving worms every two days to obtain the lifespan data. (b) On the used, counting the number of surviving worms every two days to obtain the lifespan data. (b) On the third, fifth, and seventh days of life, the pharyngeal pumping of nematodes (n = 10 worms) was third, fifth,determined. and seventh (c) On days the 10th of life, day theof life, pharyngeal the red autofluorescence pumping of of nematodes the nematodes (n = (n10 = 3 worms) worms) was determined.determined; (c) On the representative 10th day of images life, the and red the autofluorescence corresponding quantification of the nematodes results ( nare= 3shown. worms) (d) wasOn determined;the representative11th day of life, imagesbody bend and of thethe correspondingnematodes (n = 10 quantification worms) was determined. results are shown.Values (mean (d) On ± SD) the 11th day ofnot life, sharing body a bendcommon of the letter nematodes are significantly (n = 10 different worms) (p was < 0.05). determined. Scale bar is Values 100 μm. (mean SD) not ± sharing a common letter are significantly different (p < 0.05). Scale bar is 100 µm. 2.4. Effects of NA on the NAD+ Level, Lifespan, and Physiological Indexes of the sir-2.1 Mutant of C. 2.4. Effectselegans of NA on the NAD+ Level, Lifespan, and Physiological Indexes of the sir-2.1 Mutant of C. elegans The sir-2.1Themutants sir-2.1 mutants were grown were grown on the on plates the plates with with orwithout or without 600 600 nmol nmol NA NA/plate/plate on the surface. surface. After growthAfter forgrowth seven for days, seven the days, nematode the nematode homogenates homogenates were were collected collected and and used used toto determinedetermine the the + + NAD+ level.NAD The level. results The results showed showed that the that NAD the NAD+ level level was was significantly significantly increased increased inin thethe nematodesnematodes treated with NA (Figure 4a). However, NA did not significantly (p > 0.05) extend the lifespan of sir- treated with NA (Figure4a). However, NA did not significantly ( p > 0.05) extend the lifespan of 2.1 mutants (Figure 4b; Table S2). This result showed that the lifespan extension ability of NA sir-2.1 mutants (Figure4b; Table S2). This result showed that the lifespan extension ability of NA disappeared in sir-2.1 mutants, demonstrating that NA prolongs the lifespan of C. elegans via the Sir- disappeared2.1 signaling in sir-2.1 mutants,pathway. demonstratingMoreover, the increase that NA in prolongs pharyngeal the lifespanpumping of andC. elegansbody bendvia the and Sir-2.1 the signalingreduction pathway. in Moreover, autofluorescence the increase induced in pharyngealby NA also pumpingdisappeared and in bodysir-2.1 bend mutants and (Figure the reduction 4c–e), in autofluorescencesuggesting that induced the effects by NA of NA also on disappeared these aging accessory in sir-2.1 markersmutants are (Figure also mediated4c–e), suggesting by Sir-2.1. that the effects of NA on these aging accessory markers are also mediated by Sir-2.1.

Int. J. Mol. Sci. 2020, 21, 142 8 of 22 Int. J. Mol. Sci. 2020, 21, 142 8 of 22

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Figure 4. Cont. Int. J. Mol. Sci. 2020, 21, 142 9 of 22

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Figure 4. FigureEffects 4. ofEffects nicotinic of nicotinic acid (NA) acid on(NA) intracellular on intracellular NAD +NAD, lifespan,+, lifespan, pharyngeal pharyngeal pumping, pumping, autofluorescence,autofluorescence, and body and bends body ofbends the sir-2.1of the sir-2.1mutants. mutants. The The mutants mutants were were grown grown on onplates plates with or + or withoutwithout 600 nmol 600/ platenmol/plate NA. (NA.a) The (a) intracellularThe intracellular NAD NAD+ levels levels ( n(n= = 33 independentindependent experiments) experiments) and and (b) lifespan(b) lifespan (n = 90(n = worms). 90 worms). (c) On(c) On the the third, third, fifth, fifth, and and seventh seventh days days ofof life,life, pharyngeal pumping pumping (n = 10 worms) was performed. (d) On the 10th day of life, red autofluorescence (n = 3 worms) was (n = 10 worms) was performed. (d) On the 10th day of life, red autofluorescence (n = 3 worms) was measured. (e) On the 11th day of life, body bend (n = 10 worms) was determined. Values are expressed measured. (e) On the 11th day of life, body bend (n = 10 worms) was determined. Values are expressed as the mean ± SD; asterisks * indicate significant changes (p < 0.05) compared with the control mutants. as the mean SD; asterisks * indicate significant changes (p < 0.05) compared with the control mutants. Scale± bar is 100 μm. Scale bar is 100 µm.

Int. J. Mol. Sci. 2020, 21, 142 10 of 22 Int. J. Mol. Sci. 2020, 21, 142 10 of 22

+ 2.5.2.5. Saturating Saturating Concentration Concentration of of NAD NAD+ asas aa CosubstrateCosubstrate forfor SIRT1SIRT1 + WeWe found found that that NA NA could could signiﬁcantly significantly increase increase the the intracellular intracellular level level of of NAD NAD+in inC. C. elegans elegansand and Hs68Hs68 cells; cells; however,however, NANA couldcould only extend the the lifespan lifespan of of C.C. elegans elegans. As. As shown shown in inthe the Figure Figure 1a,b,1a,b, the + thesteady-state steady-state levels levels of ofintracellular intracellular NAD NAD+ in Hs68in Hs68 cells cells and and C. C.elegans elegans werewere approximately approximately 460 460 and and 55 µ + 55μM,M, respectively, respectively, which which are are quite quite different. diﬀerent. We We thus thus hypothesized hypothesized that that the theintracellular intracellular NAD NAD+ level + levelin Hs68 in Hs68 cells cellsis higher is higher than thanthe maximal the maximal concentration concentration of NAD of NAD+ neededneeded for SIRT1; for SIRT1; thus, NA thus, cannot NA + cannotincrease increase the SIRT1 the SIRT1 activity activity via viaan anincrease increase of of the the intracellular NADNAD+ level.level. TheThe saturatingsaturating + concentrationconcentration of of NAD NAD+for for SIRT1SIRT1 was was then then determined determined by by the the SIRT1 SIRT1 Fluorescence Fluorescence Resonance Resonance Energy Energy TransferTransfer (FRET)-based (FRET)-based screening screening assay assay kit kit (Cayman, (Cayman, Ann Ann Arbor, Arbor, MI, MI, USA). USA). The The results results showed showed that that + thethe enzyme enzyme activity activity reached reached the the maximum maximum level level when when the the NAD NAD+concentration concentrationwas was increased increased to to approximatelyapproximately 200 200µ μMM (Figure (Figure5). 5).

10000

1000

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10 Fluorescence

0.1 0 200 400 600 800 1000 3000 + μ NAD ( M)

+ FigureFigure 5. 5.SIRT1-saturating SIRT1-saturating NAD NAD+concentration concentrationin in vitro vitro.. SIRT1 SIRT1 activities activities were were determined determined in in reactions reactions + µ + n containingcontaining various various concentrations concentrations of NAD of NAD(1, 10,+ (1, 200, 10, 500, 200, 1000, 500, and 1000, 3000 andM NAD 3000 , μ=M3 NAD independent+, n = 3 experiments) using a commercialized SIRT1 Fluorescence Resonance Energy Transfer (FRET)-based independent experiments) using a commercialized SIRT1 Fluorescence Resonance Energy Transfer screening assay kit. (FRET)-based screening assay kit. 2.6. Effects of NA on the NAD+ Level, Lifespan, and Physiological Indexes of pme-1 Mutants of C. elegans 2.6. Effects of NA on the NAD+ Level, Lifespan, and Physiological Indexes of pme-1 Mutants of C. elegans The results showed that the NAD+ level was significantly increased in pme-1 mutants after + treatmentThe withresults NA showed (Figure 6thata). Thethe steadyNAD NADlevel +waslevel significantly in the pme-1 increasedmutants was in pme-1 86 1.3 mutantsµM, which after + ± wastreatment approximately with NA 156%(Figure higher 6a). The than steady the 55 NADµM levellevel ofin wild-typethe pme-1 mutantsC. elegans was, thus 86 ± confirming 1.3 μM, which an increasewas approximately in intracellular 156% NAD higher+ in thethanpme-1 the 55mutants. μM level However, of wild-type NA did C. notelegans significantly, thus confirming (p = 0.068) an + extendincrease the in lifespan intracellular of the NADpme-1 mutantsin the pme-1 (Figure mutants.6b; Table However, S3), suggesting NA did thatnot thesignificantly lifespan extension (p = 0.068) abilityextend of the NA lifespan was associated of the pme-1 with mutants the intracellular (Figure 6b; NAD Table+ level. S3), Moreover,suggesting the that increase the lifespan in pharyngeal extension + pumpingability of and NA body was associated bend and the with reduction the intracellular in autofluorescence NAD level. induced Moreover, by NA the essentially increase in disappeared pharyngeal inpumpingpme-1 mutants and body (Figure bend6c–e). and the reduction in autofluorescence induced by NA essentially disappeared in pme-1 mutants (Figure 6c–e).

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Figure 6. Cont.

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+ Figure 6.FigureEffects 6. ofEffects nicotinic of nicotinic acid (NA) acid on (NA) the levelon the of level intracellular of intracellular NAD+ NAD, lifespan,, lifespan, pharyngeal pharyngeal pumping,pumping, autofluorescence, autofluorescence, and body and bends body ofbendspme-1 ofmutants. pme-1 mutants. The mutants The mutants were were grown grown on plates on plates + with or withoutwith or 600 without nmol 600 NA nmol/plate. NA/plate. (a) The intracellular(a) The intracellular NAD+ NADlevels levels (n = 3 ( independentn = 3 independent experiments) experiments) and (b) lifespanand (b) (lifespann = 90 worms). (n = 90 worms). (c) Onthe (c) On third, the fifth, third, and fifth, seventh and seventh days ofdays life, of pharyngeal life, pharyngeal pumping pumping (n = 10 worms) was performed. (d) On the 10th day of life, red autofluorescence (n = 3 worms) was (n = 10 worms) was performed. (d) On the 10th day of life, red autofluorescence (n = 3 worms) was measured. (e) On the 11th day of life, body bend (n = 10 worms) was determined. Values are expressed measured. (e) On the 11th day of life, body bend (n = 10 worms) was determined. Values are expressed as the mean ± SD; asterisks * indicate significant changes (p < 0.05) compared with the control mutants. as the mean SD; asterisks * indicate significant changes (p < 0.05) compared with the control mutants. Scale± bar is 100 μm. Scale bar is 100 µm.

Int. J. Mol. Sci. 2020, 21, 142 13 of 22

2.7. Eﬀects of NA on the NAD+ Level, Lifespan, and Physiological Indexes in Normal C. elegans with an Increased IntracellularInt. J. Mol. Sci. NAD2020, 21+, 142Level 13 of 22

The eff2.7.ects Effects of NA of onNA wild-type on the NADC.+ Level, elegans Lifespan,with an and increased Physiological NAD Indexes+ level in Normal were tested C. elegans by pretreatment with an with NAD+Increasedin the NGM Intracellular plates. NAD Hashimoto+ Level et al. [17] reported that the addition of 10, 100, and 1000 µM + + NAD to NGMThe can effects extend of the NA nematodes’ on wild-type lifespan. C. elegans The with authors an increased found that NAD 100+ level and 1000were µtestedM NAD by in NGM hadpretreatment a similar lifespan with NAD extension+ in the NGM effect plates. in nematodes, Hashimoto suggesting et al. [17] reported that adding that the 100 additionµM NAD of +10,in NGM can achieve100, and 1000 the maximalμM NAD+ concentrationto NGM can extend of the the intracellularnematodes’ lifespan. NAD +Therequired authors forfound Sir-2.1. that 100 In and this study, we used1000 μ theM NAD same+ in method NGM had of a pretreatment similar lifespan with extension 100 µ efMfect NAD in nematodes,+ in NGM suggesting and found that thatadding the + + intracellular100 NAD μM +NADlevels in inNGMC. elegans can achievewere the increased maximal byconcentration approximately of the290% intracellular over those NAD in required the control for Sir-2.1. In this study, we used the same method of pretreatment with 100 μM NAD+ in NGM and to approximately 153 µM at a dosage of 600 nmol/plate of NA. Under these conditions, NA still found that the intracellular NAD+ levels in C. elegans were increased by approximately 290% over + significantlythose increased in the control NAD toin approximately nematodes (Figure 153 μM7a); at however,a dosage of the 600 lifespan nmol/plate extension of NA. ability Under ofthese NA in C. elegansconditions,disappeared NA still (Figure significantly7b; Table increased S4). Moreover, NAD+ in thenematodes increase (Figure in pharyngeal 7a); however, pumping the lifespan and body bendsextension and the ability reduction of NA in in autofluorescence C. elegans disappeared induced (Figure by NA7b; Table disappeared S4). Moreover, (Figure the7c–e). increase These in results demonstratepharyngeal thatpumping the intracellular and body bends NAD and+ levels the reduction of the nematodes in autofluorescence can influence induced the lifespanby NA extension abilitydisappeared of NA. (Figure 7c–e). These results demonstrate that the intracellular NAD+ levels of the nematodes can influence the lifespan extension ability of NA. (a) 200

c b 150 M) μ (

+ 100 NAD a 50

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Control 20 Pre-add NAD+ 100 μM Pre-add NAD+ 100 μM + NA 600 nmol/plate 0 0 2 4 6 8 10121416182022242628 Days

Figure 7. Cont. Int. J. Mol. Sci. 2020, 21, 142 14 of 22

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(e) 50 b b 40

a 20 Body bends / 30" 10

0 Pre-add NAD+ - + + NA - - +

Figure 7. EffFigureects of 7. nicotinic Effects of acid nicotinic (NA) onacid the (NA) level on of the intracellular level of intracellular NAD+, lifespan, NAD+, lifespan, pharyngeal pharyngeal pumping, autofluorescence,pumping, and autofluorescence, body bends in and wild-type body bendsC. elegans in wild-typegrown C. on elegans theNGM grown plates on the pretreated NGM plates with + + NAD+. C. eleganspretreatedwere with grown NAD . onC. elegans the plates were grown pretreated on the with plates 100 pretreatedµM NAD with+ 100in NGMμM NAD and in on NGM plates and on plates with or without 600 nmol NA/plate on NGM. (a) The intracellular NAD+ levels (n = 3 with or without 600 nmol NA/plate on NGM. (a) The intracellular NAD+ levels (n = 3 independent independent experiments) and (b) lifespan (n = 90 worms). (c) On the third, fifth, and seventh days of experiments)life, and pharyngeal (b) lifespan pumping (n = 90 ( worms).n = 10 worms) (c) On thewas third, performed. fifth, and(d) seventhOn the days10th day of life, of pharyngeallife, red pumping (nautofluorescence= 10 worms) ( wasn = 3 performed.worms) was measured. (d) On the(e) On 10th the day11th day of life, of life, red body autofluorescence bend (n = 10 worms) (n = 3 worms) waswas measured. determined. (e) Values On the (mean 11th ± daySD) not of life,sharing body a common bend (n letter= 10 are worms) significantly was determined.different (p < 0.05). Values (mean SD)Scale not bar sharing is 100 μ am. common letter are significantly different (p < 0.05). Scale bar is 100 µm. ± 3. Discussion3. Discussion To evaluateTo theevaluate CRM the potential CRM potential of nicotinic of nicotinic acid acid (NA), (NA), this study study aimed aimed to explore to explore the ability the and ability mechanism of NA to extend the lifespan of Hs68 cells and C. elegans. The results showed that NA and mechanism of NA to extend the lifespan of Hs68 cells and C. elegans. The results showed that cannot extend the lifespan of Hs68 cells; however, NA did significantly extend the lifespan of NA cannotnematodes. extend the NA lifespan ameliorated of Hs68 pharyngeal cells; however, pumpin NAg, body did significantlybend, and autofluorescence, extend the lifespan thus of nematodes.confirming NA ameliorated the health pharyngeal benefit and antiaging pumping, properties body bend, of NA and in C. autofluorescence, elegans. By contrast, thus the effects confirming of the health benefitNA on the and lifespan antiaging extension properties and physiological of NA in improvementsC. elegans. By disappeared contrast, the when eff theects intracellular of NA on the lifespan extensionNAD+ levels and were physiological increased by improvements two methods: disappeared(1) the use of the when pme-1 the mutants intracellular and (2) NADthe direct+ levels addition of NAD+ to NGM plates. These results demonstrated that the lifespan-extending ability of were increased by two methods: (1) the use of the pme-1 mutants and (2) the direct addition of NAD+ NA was tightly associated with the intracellular level of NAD+ in C. elegans. Importantly, we found to NGM plates.that the These saturating results concentration demonstrated of NAD+ thatfor SIRT1 the lifespan-extendingwas approximately 200 abilityμM; however, of NA the was steady- tightly + associated withstate concentration the intracellular of NAD level+ in ofHs68 NAD cells reachedin C. elegans up to .460 Importantly, μM, which was we substantially found that higher the saturating than concentrationthe ofmaximal NAD+ NADfor SIRT1+ concentration was approximately required for SIRT1. 200 µ TheseM; however, results demonstrated the steady-state that the concentration lifespan of NAD+ inextension Hs68 cells ability reached of NA updepends to 460 onµ whetherM, which the was intracellular substantially NAD+ level higher was than lower the than maximal the sirtuin- NAD+ concentrationsaturating required concentration. for SIRT1. These results demonstrated that the lifespan extension ability of NA A previous study indicated that the Michaelis constant (Km) of NAD+ for SIRT1 was depends on whether the intracellular NAD+ level was lower than the sirtuin-saturating concentration. approximately 390 μM [18] (note that the saturating concentration = 2 × Km); however, other studies + A previousreported study that indicated the Km or thatthe saturating the Michaelis concentration constant of (Km) NAD of+ for NAD SIRT1 forwere SIRT1 within was a similar approximately range 390 µM[18detected] (note thatin the the present saturating study. For concentration examples, Km= has2 beenKm); reported however, to be approximately other studies 95 reported [19,20] or that × the Km or the125 μ saturatingM [21]; the saturating concentration concentration of NAD has+ bforeen SIRT1reported were to be withinapproximately a similar 200–250 range μM detected [22–24]. in the presentOur study. results For examples,confirmed Kmthat hasthe beensaturating reported concentration to be approximately of NAD+ required 95 [19, 20for] orSIRT1 125 µwasM[ 21]; + the saturatingapproximately concentration 200–250 has μ beenM. On reported the other tohand, be approximatelywe found that the 200–250 steady-stateµM[ level22– of24 NAD]. Our in results C. confirmed that the saturating concentration of NAD+ required for SIRT1 was approximately 200–250 µM. On the other hand, we found that the steady-state level of NAD+ in C. elegans was approximately 55 µM. To the best of our knowledge, there is no data in the literature on the Km value or the saturating concentration of NAD+ for Sir-2.1 of C. elegans. There is no commercially available Sir-2.1 protein on the market; hence, the saturating concentration of NAD+ for Sir-2.1 was not determined in this Int. J. Mol. Sci. 2020, 21, 142 16 of 22 study. However, when the intracellular NAD+ level was increased to approximately 153 µM by adding 100 µM NAD+ to the NGM plates, the lifespan of C. elegans was not extended by NA, suggesting that the saturating concentration of NAD+ for Sir-2.1 may be approximately 150 µM. A previous study has shown that the steady-state concentration of NAD+ in the pme-1 mutant strains is approximately 1.5 times higher than that in wild-type C. elegans [25]; our results are consistent with these data. Moreover, the lifespan extension ability of NA had a marginal efficacy (i.e., p = 0.086) when the intracellular NAD+ level reached up to 86 µM in the pme-1 mutants, suggesting that the saturating concentration of NAD+ for Sir-2.1 may be higher than 86 µM. These results support our hypothesis that the intracellular NAD+ level is lower than the saturating concentration of NAD+ for Sir-2.1, and thus, NA can extend the lifespan of C. elegans. By contrast, the steady-state NAD+ level was substantially higher than the saturating concentration of NAD+ for SIRT1 in Hs68 cells, and thus, NA cannot extend the lifespan of Hs68 cells via the NAD+–SIRT1 signaling mechanism. Therefore, this study successfully answered the question of why NA can extend the lifespan of nematodes but cannot extend the lifespan of Hs68 cells. We have previously demonstrated that FK866 can induce a vitamin B3 deficiency, mimicking the situation in Hs68 cells [8]. We found that the intracellular NAD+ level can be significantly decreased in parallel to a significantly shortened lifespan in Hs68 cells treated with FK866. Interestingly, the FK866-induced lifespan shortening of Hs68 cells can be antagonized by cotreatment with NA. An FK866-induced decrease in SIRT1 activity in Hs68 cells was also antagonized by cotreatment with NA [8]. The results suggested that NA can extend the lifespan of Hs68 cells and activate SIRT1 when the intracellular NAD+ level is considerably reduced in Hs68 cells by treatment with FK866. These previous results support the mechanism proposed in the current study. Furthermore, numerous studies have demonstrated that human cells under conventional culture conditions have NAD+ levels generally higher than 200–250 µM[26–30]; for example, Hara et al. [26] reported that the steady-state levels of NAD+ are 503 104 µM in HEK293 cells, 546 46 µM in HeLa ± ± cells, and 597 90 µM in HL60 cells. However, regular cell culture medium contains sufficient vitamin ± + B3 to produce NAD . For example, the medium for Hs68 cells, i.e., Dulbecco’s modified Eagle medium (DMEM), contains 4 mg/L NAM. DMEM also contains 16 mg/L tryptophan, which may increase the amount of NAD+ via the de novo biosynthesis pathway [4]. Therefore, it is reasonable to assume that cultured cells may have higher levels of intracellular NAD+ than in vivo. This led to a question whether intracellular NAD+ in the human body is lower than the saturating concentration of NAD+ for SIRT1. It has been previously reported that under normal conditions, the concentration of NAD+ in human lymphocytes is 401 128 µM[31] and the concentration of NAD+ in the human brain tissue is ± 300 20 µM[32]. These results suggest that the intracellular NAD+ level is still substantially higher ± than the SIRT1-saturating concentrations. These results suggest that the potential of NA as a CRM in the human body may be realized only in individuals with lower intracellular NAD+, e.g., vitamin B3-deficient individuals or the elderly. However, it should be mentioned that the health benefits of vitamin B3 may be mediated by different the mechanisms rather than the NAD+–SIRT1 pathway. For example, it is known that there are seven different sirtuins (i.e., SIRT1-7) in mammals [4]. The Km values for NAD+ vary widely between sirtuins; for example, the Km for SIRT2 is 83 µM[33], for SIRT3 is 880 µM[34], for SIRT4 is 35 µM[35], for SIRT5 is 980 µM[36], and for SIRT6 is 26 µM[37]. Thus, there still are certain sirtuins with a saturating concentration obviously higher than 200–250 µM. Moreover, it is possible that an + increase in intracellular NAD by vitamin B3 may have certain health benefits via other mechanisms in addition to the effects on sirtuins. For example, a recent paper reported that NAM cannot prolong the lifespan of C57BL/6J mice; however, NAM still had certain health benefits via the hepatic glucose metabolism regulation and oxidative stress reduction [11]. The NAD+ signaling mechanisms relating to the therapeutic potential of NAD+-boosting molecules including NA, NAM, nicotinamide riboside (NR; + a new type of vitamin B3 molecule), and nicotinamide mononucleotide (NMN; an NAD precursor) have been reviewed elsewhere [38]. Int. J. Mol. Sci. 2020, 21, 142 17 of 22

4. Materials and Methods

4.1. Materials

All chemicals used were of analytical grade. NaCl, KCl, NaOH, MgCl2, Na2HPO4, KH2PO4, NaHCO3, Na2CO3, HCl, dimethylsulfoxide (DMSO), and formaldehyde were purchased from Merck (Darmstadt, Germany). NA, NAD+, potassium ferricyanide, potassium ferrocyanide, fluorescein, glutaraldehyde, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), alcohol dehydrogenase (ADH), N-ethyldibenzopyrazine ethyl sulfate (commonly referred to as phenazine ethosulfate), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal), ampicillin sodium salt, fluorodeoxyuridine (FUdR), and cholesterol were obtained from Sigma (St. Louis, MO, USA). ® Ethanol, glycerol, MgSO4, and CaCl2 were purchased from J.T. Baker (Phillipsburg, NJ, USA). Fluorescein di(β-D-galactopyranoside) (FDG) was purchased from Molecular Probes (Eugene, OR, USA). American bacteriological agar and yeast extract was obtained from Conda (Madrid, Spain). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), nonessential amino acids (NEAA), penicillin–streptomycin, and sodium pyruvate were obtained from HycloneTM (Boston, MA, USA). Vegetable peptone and tryptone were obtained from Fluka (Buchs, Switzerland). Human fibroblast Hs68 cells (ATCC® CRL-1635™) were purchased from the Cell Culture Center of the Food Industry Research and Development Institute (Hsinchu, Taiwan). The wild-type C. elegans strain N2 was a gift from the C. elegans core facility (National Tsing Hua University, Taiwan). The VC199 sir-2.1(ok343)VI and RB1042 pme-1(ok988) mutants were obtained from the Caenorhabditis Genetics Center (University of Minnesota, MN, USA).

4.2. Cell Culture Hs68 cells were routinely cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) in 75 cm2 flasks with 25 mM (4.5 mg/mL) glucose, 10% fetal bovine serum, and 1.0 mM pyruvate at 37 ◦C in a humidified incubator under 5% CO2. Hs68 cells is one of a series of the human foreskin fibroblast lines developed at the Naval Biosciences Laboratory (NBL) in Oakland, CA. Hs68 cells were obtained from an apparently normal Caucasian newborn male with a finite lifespan but may have a low level of aspartoacylase activity.

4.3. Determination of the Intracellular NAD+ Level in Hs68 Cells Intracellular NAD+ in Hs68 cells was determined by the acid extraction method with subsequent enzymatic cycling [8]. Brieﬂy, the cells were cultured in a 10 cm dish at 1.5 105 cells per dish. After × 24 h of cell attachment, the cells were incubated with NA for 24 h. Cells were trypsinized and collected at approximately 5 105/vial (pooled three dishes of cells). The cell pellet was extracted by adding × 500 µL ice-cold 7.0% perchloric acid (PCA). A portion of the extract (i.e., 100 µL) was neutralized with + an equal volume of a 1 M K2HPO4 solution. The level of NAD was determined using a 96-well microplate in 100 µL reaction mixture per well (n = 3). The reaction mixture contained 10 µmol Tris-HCl (pH 8.0), 0.4 µmol PES, 0.05 µmol MTT, 0.03 mg ADH, 60 µmol ethanol, and 20 µL cell extract. The rate of increase in absorbance at 570 nm within 8 min was detected by a microplate reader (Micro Qunat, BioTek, Winooski, VT, USA) and calibrated using commercial NAD+ dissolved in the neutralized acid solution in the micromolar range used as the standard. The intracellular NAD+ level (µM) = the measured NAD+ (µM) the dilution factor during neutralization (i.e., twofold) 500 µL/2.55 µL, × × where 500 µL is the volume of PCA solution and 2.55 µL is the cell volume of 5 105 cells (note: 1 106 × × of Hs68 cells is approximately 5.1 µL[5]).

4.4. Assay of Replicative Lifespan of Hs68 Cells The replicative lifespan of Hs68 cells was monitored by the cumulative-growth method [8]. Briefly, Hs68 cells were serially cultured in a 10 cm dish with 1.0 105 cells per dish. The cells were subcultured × exactly once per week, and the cell numbers were counted. The population doubling levels (PDLs) Int. J. Mol. Sci. 2020, 21, 142 18 of 22 were calculated as log2 (Nt/No), where Nt and No were defined as the total count of cells at the time of harvesting and seeding, respectively. Cumulative PDLs (CPDs) were obtained by summing the total PDLs before a given passage number. The replicative lifespan was determined when the cells were senescent, which was defined as an Nt/No ratio less than 1.5 for two consecutive passages. The precise level of CPDs of Hs68 cells was not specified by the supplier; hence, we defined the CPDs at the initial passage as zero and used the additional CPDs to represent the doubling levels after the initial passage.

4.5. Determination of the Senescence-Associated β-Galactosidase (SA-βG) Activity SA-βG activity was measured by the double-substrate assay [39]. Briefly, 1 104 cells were × cultured for 24 h in a 12-well culture plate. Then, the cells were fixed with PBS containing 2% formaldehyde and 0.2% glutaraldehyde (1 mL/well) for 5 min. After three washes, the fixed cells were incubated in a staining solution (1 mL/well) containing 2.45 mM X-Gal and 40 µM FDG in a humidified incubator at 37 ◦C for 24 h without CO2. The X-Gal-stained cells were imaged using a microscope. Alternatively, 100 µL of the supernatant was transferred to a 96-well plate for the determination of the fluorescein fluorescence by using a fluorimeter (Flx800, Bio-Tek, Winooski, VT, USA).

4.6. Handling Procedures for C. elegans Nematodes were maintained and propagated on NGM as described elsewhere [40] with some modifications. In brief, nematodes were regularly grown at 23 ◦C in 10 cm plates in 20 mL/plate NGM; 10 mg (wet weight) of Escherichia coli OP50 (used as nematode food) in LB medium were spread onto the surface of the NGM plate and dried under laminar flow. The synchronization, preparation of NGM, LB medium, and M9 buffer (mainly for worm washing), and other procedures were performed as described previously [40]. Amp/FUdR plates with or without NA were prepared and subsequently used for the lifespan assay. Amp/FUdR plates were prepared using 5 cm plates by adding 10 mL/plate NGM containing 100 µg/mL ampicillin and 50 µM FUdR (to avoid subsequent egg hatching) [40]. In addition, 10 mg wet weight of OP50 bacteria in 200 µL of LB medium with or with NA was spread onto the center of the top surface of an NGM plate. After the surface bacteria buffer was dried, the Amp/FUdR plates were then expose to UV at two doses of 9999 100 mJ/cm2 to kill the bacteria on the surface of the plates. × 4.7. Determination of the Intracellular NAD+ Level in C. elegans Intracellular NAD+ in C. elegans was determined by the acid extraction method with subsequent enzymatic cycling [8]. Briefly, nematodes were collected and washed two times with M9 buffer in 15 mL centrifuge tubes (centrifuged at 1500 rpm for 5 min). Nematodes were extracted with a 4 × volume of 7.0% ice-cold PCA with sonication. The extract (100 µL) was neutralized by an equal volume + of 1 M K2HPO4. The NAD concentration in the extract was determined by a procedure similar to that described for the cycling assay in Hs68 cells. The intracellular NAD+ level in nematodes was corrected for the dilution factor at neutralization (i.e., twofold) and nematode volume (4 volume of the PCA × solution). The intracellular NAD+ level in nematodes (µM) = the measured NAD+ (µM) the dilution × factor during neutralization (i.e., twofold) 5, where 5 is obtained from (the PCA solution volume × (µL) + the nematode volume (µL))/the nematode volume.

4.8. Assay of the Lifespan of C. elegans The lifespan of the nematodes was determined as described elsewhere [40] with slight modiﬁcations. Synchronized larvae at the L1 stage were gently picked onto a 5 cm Amp/FUdR plate (30 worms/plate) containing various dosages of NA on the center of the top surface of the plate and grown at 23 ◦C. The plates of all groups were replaced with new plates every four days, and the surviving nematodes were counted every two days. The worms were considered dead when they did not react to the touch of a platinum wire or lost their pharyngeal pumping. The recorded survival percentage was plotted by Sigma Plot 8.0 and the average, median, and maximum lifespan values were calculated in SPSS. Int. J. Mol. Sci. 2020, 21, 142 19 of 22

4.9. Assay of the Pharyngeal Pumping in C. elegans Pharyngeal pumping was determined as described previously [41,42]. On the third, ﬁfth, and seventh days of the nematode lifespan, each group included 10 randomly selected nematodes to record pharyngeal pumping with a charge coupled device (CCD) video camera under a microscope. The number of pharyngeal beats within 30 s was calculated based on the slow-motion playback.

4.10. Determination of the Autofluorescence in C. elegans Autofluorescence was determined as described previously [43,44]. On the 10th day of life, nematodes (n = 3) were randomly picked onto a slide coated with 2% agar pad (1 worm/slide). Nematodes were paralyzed with 20 mM sodium azide, and fluorescence of the nematodes was imaged by a ZEISS Axio Imager A2 fluorescence microscope (Zeiss, Göttingen, Germany) with a Rhod filter. Red autofluorescence was quantified by the ImageJ image analysis software (National Institutes of Health, Bethesda, MD, USA).

4.11. Assay of Body Bends of C. elegans Body bends were determined by the swimming test as described previously [41]. On the 11th day of life, each group included 10 randomly selected nematodes to determine body bends. After crawling on an NGM plate without OP50 for 30 s to remove excess E. coli from the worms, each nematode was placed in a well of a 24-well plate containing 1 mL of M9 buﬀer. The motility of nematodes was recorded with a CCD video camera under a microscope, and the number of body bends within 30 s was calculated based on the slow-motion playback. Body bends were deﬁned as the number of repeated twists at the center point of the nematode.

4.12. Determination of the SIRT1-Saturating Concentration of NAD+ In Vitro The saturating concentration of NAD+ as a cosubstrate for SIRT1 was determined by using a commercial SIRT1 FRET-based screening assay kit (Cayman, Ann Arbor, MI, USA). All of the determination procedures, including the amount of the SIRT1 protein, were performed according to the manufacturer instructions, except the NAD+ concentration per assay was varied from 10 to 3000 µM.

4.13. Statistical Analysis Data were analyzed using Student’s t-test or analysis of variance (ANOVA), followed by Duncan’s test for group mean comparisons using the SPSS v.14.0 software (SPSS, Inc., Chicago, IL, USA). The diﬀerences in the lifespan of the nematodes between the groups were analyzed using log-rank test. p-values less than 0.05 were considered statistically signiﬁcant.

5. Conclusions In summary, NA can extend the lifespan of C. elegans and improve aging markers, including pharyngeal pumping, autoﬂuorescence, and body bends, when the steady-state level of NAD+ is approximately 55 µM. The lifespan extension and aging improvement by NA of C. elegans disappear when the intracellular level of NAD+ is increased up to 153 µM. Moreover, the steady-state level of intracellular NAD+ is approximately 460 µM, which is substantially higher than the SIRT1-saturating concentration of 200–250 µM; thus, NA cannot extend the lifespan of Hs68 cells. These results demonstrate that the lifespan extension ability of NA depends on whether the intracellular level of NAD+ is lower than the sirtuin-saturating concentration in Hs68 cells and in C. elegans. These results also suggest that the CRM potential of NA should be limited to individuals with lower intracellular + NAD , e.g., in individuals with vitamin B3 deﬁciency or the elderly.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/1/142/s1. Int. J. Mol. Sci. 2020, 21, 142 20 of 22

Author Contributions: Conceptualization, N.-C.Y.; Formal analysis, Y.-H.C. and I.L.; Funding acquisition, N.-C.Y.; Investigation, N.-C.Y. and Y.-H.C.; Methodology, N.-C.Y., Y.-H.C. and I.L.; Supervision, N.-C.Y.; Writing—original draft, N.-C.Y.; Writing—review & editing, N.-C.Y. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by grants from the National Science Foundation (MOST 105-2320-B-040-017-MY3), Taiwan ROC. Acknowledgments: The sir-2.1 and pme-1 mutants were provided by the Caenorhabditis Genetics Center (CGC), which is funded by National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD010440). C. elegans strain N2 was a gift from the C. elegans core facility of Taiwan (National Tsing Hua University, Taiwan). Conflicts of Interest: The authors declare no conflict of interest.

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