Introduction

Dietary restriction (DR) or caloric restriction (CR) extends lifespan in a wide variety of animals [1]. This concept was first shown in rodents [2], and subsequent studies using monkeys showed that CR delays age-related mortality [3] and all-cause mortality [4]. A parallel study conducted at the National Institute of Aging showed that modest benefits in overall measures of health and function were observed in CR monkeys, although a significant difference in survival was not detected between control-fed and CR animals (p = 0.06) [5]. DR also exerts longevity effects in invertebrates such as fruit flies [6] and the nematode Caenorhabditis elegans [7, 8].

CR-mediated lifespan extension has been intensively studied in yeast. Lin et al. reported that CR (limiting glucose availability) extends the lifespan (the number of mother cell divisions) of this organism via a Sir2-dependent pathway [9]. This protein is a member of the sirtuins, a family of nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases [10]. However, it remains controversial whether Sir2 acts as a master regulator of CR-mediated lifespan extension [11]. In C. elegans, eat-2 weak mutant worms, which represent a genetic DR model due to insufficient food intake, exhibit Sir2 homolog (sir-2.1)-dependent lifespan extension [12], although several other studies have reported that DR extends lifespan through sir-2.1-independent mechanisms [13, 14]. Lifespan extension caused by DR in fruitflies also requires Sir2 [15]. In mammals, seven sirtuins (SIRT1-7) have been identified and multiple lines of evidence have indicated that sirtuins mediate the effects of CR in mammals [16]. For example, knockout mice studies have shown that increases in physiological activities during CR and the increased longevity induced by CR require SIRT1 [17–19]. CR induces the expression of a subset of sirtuins in mice [20–22] and also in human [23].

Although DR provides beneficial effects, the process could cause adverse side effects such as decreased fertility [24]. Candidate DR mimetics that provide the beneficial effects of DR through similar mechanisms without inducing undesirable consequences have been reported [24]. For instance, resveratrol extends the lifespan of nematodes and fruit flies in a sir-2.1/sir2-dependent manner without reducing fertility [25]. Resveratrol’s effects on lifespan are, however, less clear in mammals; for instance, although resveratrol prolongs the lifespan of obese mice [26], no evidence exists concerning longevity effects in non-obese mammals [27]. In mice, resveratrol delays age-related deterioration but does not extend lifespan [28].

Sesame seeds contain a class of propyl phenolic dimers known as lignans. Sesamin is one of the most abundant lignan in sesame seeds [29]. Sesamin has been reported to mediate biological effects such as the reduction of IgE production [30], inhibition of carcinogenesis [31], suppression of hypertension [32], and reduction of cholesterol levels [33] and fatty acid synthesis [34]. We have previously shown that sesamin supplementation extends lifespan in C. elegans [35]. Caenorhabditis elegans has been used extensively as an experimental animal model, especially for studies on senescence and the influence of food and nutrition. The appeal of this organism is due to its ease of cultivation, an abundance of genetic tools, and the short and reproducible lifespan [36].

In the present study, we examined the molecular mechanisms underlying the longevity effect of sesamin in C. elegans. Based on the results from microarray analyses that support the involvement of sir-2.1, we showed that several DR-related pathways mediate the effect of sesamin.

Materials and methods

Chemicals

Sesamin was purchased from Wako (Osaka, Japan). γ-Cyclodextrin (γCD) was obtained from Cyclochem (Kobe, Japan).

Bacterial strain and culture conditions

Escherichia coli OP50, which is used as the standard feed for C. elegans cultivation, was grown on tryptone soya agar (Nissui Pharmaceutical, Tokyo, Japan) at 37 °C. Cultured bacteria were scraped and weighed. Aliquots (100 mg wet weight) of bacteria were suspended in 0.5 ml M9 buffer (5 mM potassium phosphate, 1 mM CaCl2, 1 mM MgSO4), and used in the experimental assays.

Caenorhabditis elegans strains and culture conditions

The wild-type C. elegans strain Bristol N2 and its derivative mutant strains were obtained from the Caenorhabditis Genetics Center as follows: VC199 sir-2.1(ok434) IV, DR412 daf-15(m81)/unc-24(e138) IV, and RB754 aak-2(ok524) X. Caenorhabditis elegans strains were maintained using standard techniques [37]. For gene expression analyses and lifespan assays, animals were cultured as follows: eggs were prepared from adult C. elegans by exposure to a sodium hypochlorite/sodium hydroxide solution. The egg suspension was incubated in M9 buffer for one day at 25 °C to allow hatching and synchronization, and the resulting suspension of synchronized L1-stage worms was centrifuged at 156×g for 1 min. After removing the supernatant by aspiration, the remaining larvae were transferred onto mNGM plates (1.7% (w/v) agar, 50 mM NaCl, 1 mM CaCl2, 5 µg/mL cholesterol, 25 mM KH2PO4, 1 mM MgSO4) covered with 10 mg OP50. Transferred worms were cultured at 25 °C for two days until worms reached the young adult stage (referred to as 3-day-old animals).

Administration of sesamin

Sesamin was administered in the form of a γCD inclusion complex so that C. elegans could ingest the compounds; the complex was prepared and administered as previously described [35, 38] but with some modifications. Briefly, 500 µl sterile γCD water solution (230 mg/ml) was mixed with 50 µl sterile sesamin ethanol solution (2.5 mg/ml) and stirred with a rotary mixer for 12–24 h at ambient temperature. The resulting solid complex (inclusion complex) was collected by centrifugation, followed by suspension in 125 µl M9 buffer. An aliquot (12.5 µl) of this sesamin-γCD inclusion complex suspension was adjusted to 100 µl by the addition of M9 buffer, and the diluted complex was combined with 100 µl OP50 suspension. The resulting suspension was spread onto mNGM plates (100 µl/plate). The amount of sesamin in the sesamin-γCD inclusion complex was determined as follows: the complex was prepared from 300 µl sesamin solution and 3 ml γCD solution, freeze-dried, and weighed (4.0 mg). The freeze-dried complex was mixed with dimethyl sulfoxide (DMSO)-d 6 , and the nuclear magnetic resonance (NMR) spectra were recorded using a Bruker AVANCE III HD 600 spectrometer (600 MHz for 1H). The 1H-NMR spectra were obtained at 25 °C. To obtain quantitative information through integral-based calculations, the 1H experiments used a 90° pulse, non-spinning mode, and 60 s for the relaxation delay D1. NMR chemical shifts were referenced to the solvent peak δH 2.49 (residual DMSO-d 6 ). The NMR spectra for sesamin (Online Resource Fig. S1) and γCD (Online Resource Fig. S2) were used for the assignment of signals. From the integrated intensity of each signal in the spectrum of the sesamin-γCD inclusion complex (Online Resource Fig. S3), the molar ratio of sesamin to γCD in the inclusion complex was calculated to be approximately 1:1.36, namely, 4.0 mg sesamin-γCD inclusion complex contained approximately 669 µg sesamin, and one assay plate was considered to be supplemented with 5.75 µg sesamin.

Microarray analysis

Three-day-old worms were cultured for one day on mNGM plates covered with OP50 alone (control-fed group) or with OP50 supplemented with sesamin (sesamin-fed group). Microarray expression profiling was performed with control-fed and sesamin-fed worms. Approximately, 100 worms in each group were collected by a worm picker and soaked in RNAlater solution (Qiagen). Total RNA was isolated using the RNeasy Lipid Tissue kit (Qiagen).

DNA synthesis and microarray hybridization were performed by Kurabo Industries Ltd. RNA quality (RNA integrity number (RIN) >7) was confirmed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). A total of ~1 µg of RNA was used as the template for fluorescent labeling of cRNA. Labelled cRNAs were hybridized to the Affimetrix C. elegans Genome Array (containing 22,500 transcripts). Microarray data analyses were performed with the MAS5.0 (Microarray Suite statistical algorithm, Affymetrix). Differential expression was analyzed by the Comparison Analysis of MAS5.0 using the Wilcoxon’s Signed Rank test. Each probe set on the experiment array (sesamin-fed group) was compared to its counterpart on the baseline array (control-fed group), and a ‘Change p value’ was calculated. Probe sets that showed differential expression were assigned with ‘Change calls’ (Increase (p < 0.002), Marginal Increase (0.002 ≤ p < 0.002667), Marginal Decrease (0.997333 < p ≤ 0.998), or Decrease (p > 0.998)). The ‘Signal Log Ratio’ was computed using a one-step Tukey’s Biweight method by taking a mean of the log ratios of probe pair intensities across the two arrays (control-fed vs. sesamin-fed group). The final data extraction was performed using DNA Microarray Viewer ver. 2 (Kurabo Industries, Ltd., Osaka, Japan).

Reverse transcription and real-time PCR

Genomic DNA was removed and cDNA was synthesized using a QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR (quantitative PCR) was performed in a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA) using FastStart Universal SYBR Green Master (ROX) (Roche). All data were normalized to the cyc-1 gene. Samples from four independent replicates were analyzed. The primers used for real-time PCR were as follows: cyc-1, (F: Forward) 5′-CGTGGTTCAAGGATCTAAACG-3′ and (R: Reverse) 5′-ACCGAGTTCTCCAAAGCGTA-3′; abu-7/abu-8, (F) 5′-CGACAACTCCTGCACATCC-3′ and (R) 5′-GTAAGTTGGCTGGGCTTGTT-3′; abu-10, (F) 5′-CCAACAATCCCAACATTCGT-3′ and (R) 5′-TTGGCATACGCATTGGTTAG-3′; abu-13, (F) 5′-CTTGTTCAGCCAGTCATTCG-3′ and (R) 5′-CCATTAGCTTTGTTAAATTCTGTGG-3′; abu-14, (F) 5′-CGCTGACGAAGAGACTGTCA-3′ and (R) 5′-CGCAGCATGAGTTGGAGTT-3′; pqn-32, (F) 5′-CAGAGACCACAGGTTCAGCAC-3′ and (R) 5′-GCTGCAGTGGGATGTTGA-3′; pqn-73, (F) 5′-ATTTCCCGCATTGGATCAT-3′ and (R) 5′-CCTGATTAGGCCCACTTCC-3′.

Determination of C. elegans lifespan

Lifespan assays were performed as follows: synchronized 3-day-old animals (35 animals per plate) were placed on 5 cm mNGM plate covered with 10 mg OP50 alone or with sesamin supplementation and the plates were incubated at 25 °C. Animals were transferred daily to fresh plates for the first four days and thereafter transferred every second day. The numbers of live and dead animals were scored every day. An animal was considered dead when it failed to respond to a gentle touch with a worm picker. Animals that crawled off the plate or died from internal hatching were considered lost and not included in the analysis. Each assay was conducted in duplicate and repeated twice, except for the assays using sir-2.1 mutants (repeated three times). Worm survival was calculated by the Kaplan–Meier method, and survival differences were tested for significance using the log-rank test.

Mean lifespan (MLS) was estimated using the formula [39]

$$\text{MLS}=\frac{1}{N}\sum\limits_{j}{\frac{{{x}_{j}}+{{x}_{j+1}}}{2}}{{d}_{j}},$$

where d j is the number of worms that died in the age interval (x j to x j+1 ) and N is the total number of worms. The standard error (SE) of the estimated mean lifespan was calculated using the following equation:

$$\text{SE}=\sqrt{\frac{1}{N\left( N-1 \right)}\sum\limits_{{}}{{{\left( \frac{{{x}_{j}}+{{x}_{j+1}}}{2}-\text{MLS} \right)}^{2}}}{{d}_{j}}}.$$

Maximum lifespan was calculated as the mean lifespan of the longest living 15% of the worms in each group.

RNAi experiments

RNA interference (RNAi) was carried out by feeding animals dsRNA-producing bacteria as previously described [40] but with some modifications. Animals were treated by RNAi over two generations to ensure that knockdown effects were stable and efficient. Briefly, synchronized L1-stage worms were transferred to plates containing RNAi-bacteria grown on NGM containing 50 µg/mL ampicillin and 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG), and the plates were incubated for 2 days at 25 °C until the transferred animals developed into young adults (P0). Eggs were collected from P0 animals and synchronized L1-stage larvae (F1) were prepared. F1 animals were cultivated for 2 days at 25 °C under RNAi conditions until the worms developed into young adults (3-day-old animals), at which point the animals were used in the following assays.

Measurement of ATP

Three-day-old worms were cultured for six days on mNGM plates covered with OP50 alone or with OP50 supplemented with sesamin. Approximately, 400 worms in each group were harvested into 1 ml M9 buffer. Worm pellets were washed four times with M9 buffer, resuspended in lysis buffer and immediately frozen in liquid nitrogen. The frozen pellets were boiled for 15 min at 100 °C to release ATP and dilution buffer was added. Samples were centrifuged at 15,000×g for 5 min. The supernatant was diluted with dilution buffer and used for measurement of ATP with the ATP Bioluminescence Assay Kit HSII (Roche) according to the manufacturer’s instructions. For normalization of the luminescence signal, protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

Dietary restriction protocol

Synchronized 3-day-old animals were cultured at 25 °C on mNGM plates covered with 5 mg OP50 for the first two days (ad libitum conditions), 0.05 mg OP50 for the next three days (DR conditions), and 0.1 mg OP50 (DR conditions) thereafter. Animals were transferred daily to fresh plates for the first five days and subsequently transferred every second day.

Results

Microarray expression profiling for genes regulated by sesamin

To identify genetic pathways that may contribute to sesamin-mediated lifespan extension, we performed a microarray analysis. Comparing the transcriptional profiles of control-fed and sesamin-fed worms, 13 upregulated genes (log-fold change ≥1) and 217 downregulated genes (log-fold change ≤1) were identified (Online Resource Table S1, S2). Functional annotation using DAVID (https://david.ncifcrf.gov/summary.jsp) revealed that the enriched gene ontology (GO) terms for genes downregulated by sesamin supplementation frequently indicated ‘development’ or ‘morphogenesis’ (Table 1). ‘Determination of adult lifespan’ was also significantly enriched (p = 0.044, Table 1). Interestingly, one of the top GO terms was ‘endoplasmic reticulum-unfolded protein response’ (p = 0.038). Endoplasmic reticulum (ER)-unfolded protein response (UPR) genes have been shown to be negatively regulated by sir-2.1 and to mediate (at least partially) sir-2.1-dependent longevity [41]. Strikingly, the expression of a subset of abu (activated in blocked unfolded protein response) and pqn (prion-like Q/N proteins) genes, both of which encode protein members of a prion-like glutamine/asparagine-rich protein family that are thought to restore protein folding in ER [42], was decreased in sesamin-fed animals (Table 2, Online Resource Table S2). The real-time PCR results confirmed that expression of the ER UPR-related abu/pqn genes were reduced by sesamin supplementation (Table 2). Based on these observations, we hypothesized that the sir-2.1 pathway might be activated in worms that were fed sesamin.

Table 1 Gene ontology (GO) enrichment for genes downregulated by sesamin supplementation
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Table 2 Sesamin-fed animals exhibit decreased the ER UPR-related abu/pqn gene expression
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Caenorhabditis elegans lifespan extension caused by sesamin supplementation requires sir-2.1

Because the microarray analysis implied the involvement of sir-2.1, we examined whether the longevity effect of sesamin requires sir-2.1. First, the lifespan of RNAi-treated worms in the presence and absence of sesamin was measured. Control RNAi-treated animals exhibited a significant increase in lifespan in the presence of sesamin, whereas sir-2.1 RNAi-treated animals did not show a significantly different lifespan with sesamin supplementation (Fig. 1a). Second, we determined the lifespan of sir-2.1 deletion mutants. Again, sir-2.1 mutants did not show a significant change in lifespan with sesamin supplementation (Fig. 1b). These results strongly suggested that lifespan extension caused by sesamin requires sir-2.1 in C. elegans.

Fig. 1
figure 1

Sesamin extends C. elegans lifespan in a sir-2.1-dependent manner. a Survival curves of RNAi-treated animals in the presence and absence of sesamin. Sesamin significantly increased lifespan in control RNAi-treated animals but failed to extend lifespan in sir-2.1 RNAi-treated animals. Mean lifespan ± SE of control-fed (m cont) and sesamin-fed (m sesam) animals under the control RNAi condition was 19.9 ± 0.3 and 21.8 ± 0.3 days, respectively. m cont and m sesam under the sir-2.1 RNAi condition was 19.9 ± 0.4 and 20.3 ± 0.4 days, respectively. b Survival curves of control-fed and sesamin-fed sir-2.1 mutants. Loss of sir-2.1 abolished sesamin-induced lifespan extension. m cont and m sesam of the sir-2.1 mutants was 22.7 ± 0.4 and 23.2 ± 0.4 days, respectively. Survival differences were tested for significance using the log-rank test. ***P < 0.001. N.S. not significant. Detailed lifespan data and statistics are provided in Online Resource Table S3

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Longevity effect of sesamin requires the autophagic gene bec-1

Sirtuin-mediated effects in human cell lines have been reported to be mediated (at least in part) through autophagy [43]. Morselli et al. also showed that DR and resveratrol promote longevity through the sir-2.1-dependent induction of autophagy in C. elegans [43]. To determine whether the longevity effect of sesamin is mediated by the autophagy pathway, RNAi of the bec-1 gene, which encodes the C. elegans homolog of the key autophagic regulator ATG6, was tested for an effect on lifespan extension caused by sesamin supplementation. Notably, bec-1 RNAi-treated animals did not show a significant change in lifespan in the presence of sesamin compared with that in the absence of sesamin (Fig. 2a). These results suggested that the longevity effect of sesamin requires the autophagic gene bec-1.

Fig. 2
figure 2

Lifespan extension by sesamin supplementation requires DR-related genes. a Survival curves of bec-1/ATG6 RNAi-treated worms in the presence and absence of sesamin. m cont and m sesam was 16.7 ± 0.3 and 17.3 ± 0.4 days, respectively. b Survival curves of the daf-15/Raptor heterozygous mutants. m cont and m sesam was 21.3 ± 0.4 and 21.5 ± 0.3 days, respectively. c Survival curves of the aak-2/AMPK mutants. m cont and m sesam was 16.7 ± 0.3 and 17.6 ± 0.3 days, respectively. Statistical analyses were performed using the log-rank test. N.S. not significant. Detailed lifespan data and statistics are provided in Online Resource Table S3

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Lifespan extension with sesamin supplementation involves the TOR pathway

DR extends lifespan, at least in part, by downregulation of the target of rapamycin (TOR) pathway [44], and inhibition of TOR triggers autophagy in yeast and mammals [45]. In addition, we found that sinh-1, which encodes a component of the TOR complex, was downregulated by sesamin (online resource Table S2). This observation supports the idea that sesamin might extend lifespan by inhibiting TOR. To test this possibility, we assayed mutants heterozygous for the TOR-binding partner daf-15/Raptor. (This test employed daf-15 heterozygotes because daf-15 homozygotes do not reach maturity, and because the loss of one copy of daf-15 decreases pathway activity sufficiently to affect lifespan [46].) Lifespan extension with sesamin supplementation was suppressed in daf-15 heterozygotes (Fig. 2b), suggesting that the longevity effect of sesamin involves the TOR pathway.

Longevity caused by sesamin supplementation requires aak-2/AMPK

Given that several DR-related pathways (such as the SIR-2.1 and TOR pathways) were required for lifespan extension conferred by sesamin, we suspected that the fuel sensor AMP-activated protein kinase (AMPK) might be involved. To examine the involvement of AMPK, mutants of aak-2, which encodes a C. elegans AMPK α-subunit, were assayed for lifespan extension. We found that there was no significant difference in the lifespans of aak-2 mutants in the presence or absence of sesamin (Fig. 2c). AMPK is known to be activated upon an increase in the AMP-to-ATP ratio; we therefore compared ATP concentrations in worms grown with and without sesamin supplementation. We found no significant difference in ATP concentrations between sesamin-fed and control-fed worms (Online Resource Fig. S4). These results suggested that the longevity observed in sesamin-fed animals may be mediated by AMPK but does not involve a decrease in ATP levels.

Sesamin does not extend lifespan under DR conditions

Based on the above data, we hypothesized that sesamin might act as DR mimetic, exerting beneficial effects through a mechanism similar to that of DR. If so, sesamin supplementation should be ineffective under DR. Indeed, we found that sesamin did not prolong lifespan under DR. Specifically, under conditions of DR, sesamin-fed animals did not demonstrate a significantly longer lifespan than control animals (Fig. 3), an observation that contrasted with the increased longevity seen with sesamin supplementation when animals were given ad libitum access to food. These results supported the idea that sesamin mimics DR to extend lifespan in C. elegans. Notably, however, DR conditions significantly increased lifespan in both control-fed worms (p < 0.001, log-rank test) and sesamin-fed animals (p < 0.001, log-rank test). This observation suggested that sesamin supplementation is unlikely to prolong lifespan beyond that obtained with DR.

Fig. 3
figure 3

Sesamin does not extend lifespan under DR conditions. Survival curves of control-fed and sesamin-fed animals under ad libitum and DR conditions. Sesamin supplementation and DR conditions were initiated on Day 0 and Day 2, respectively. m cont and m sesam under ad libitum conditions was 18.9 ± 0.3 and 20.4 ± 0.3 days, respectively. m cont and m sesam under DR conditions was 22.0 ± 0.5 and 22.0 ± 0.5 days, respectively. Although sesamin-fed animals showed a significant increase in lifespan compared with control-fed animals under ad libitum conditions, the two groups did not exhibit significant differences in lifespan under DR conditions. Significant differences were assessed using the log-rank test. ***P < 0.001. N.S. not significant. Detailed lifespan data and statistics are provided in Online Resource Table S3.

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Discussion

The longevity response to DR is actively regulated by nutrient-sensing pathways involving sirtuins, TOR, AMPK, and insulin/IGF-1 (IIS) signaling and are conserved from yeast to mammals [14]. In this study, we showed that sesamin increases the lifespan of the nematode C. elegans through the sir-2.1/SIRT1, TOR, and aak-2/AMPK pathways. In addition, we have previously reported that sesamin-associated lifespan extension depended on the insulin/IGF-1 (IIS) pathway [35]. Therefore, lifespan extension by sesamin is likely to involve nearly every known DR-related nutrient-sensing pathways. Based on this evidence, we propose a working model, in which sesamin might extend lifespan by acting upstream of the aforementioned nutrient-sensing pathways (Fig. 4). DR-triggered SIR-2.1 activation induces autophagy and induction of autophagy promotes longevity in C. elegans [43]. DR also activates AAK-2/AMPK such that AMPK activates the DAF-16/FoxO transcription factor [13]. At the same time, DR suppresses the TOR pathway, resulting in the induction of autophagy [46] and the activation of DAF-16/FoxO [47]. SKN-1/Nrf also mediates the TOR-dependent longevity [47]. The IIS signaling pathway regulates DR-induced longevity through the suppression of DAF-16/FoxO and AAK-2/AMPK [48]. We have previously reported that the sesamin-associated extension of C. elegans lifespan depended on the DAF-16/FoxO and SKN-1/Nrf transcription factors [35]. The present study showed that lifespan extension by sesamin supplementation depends on SIR-2.1/SIRT1, a TOR component (DAF-15), AAK-2/AMPK, and a key regulator of autophagy (BEC-1). Considered together, sesamin might mimic a DR signal to promote longevity in C. elegans.

Fig. 4
figure 4

Working model for the longevity effect of sesamin via DR-related nutrient-sensing pathways. This study and our previous study demonstrated that sesamin-promoted longevity requires the IIR, SIRT1/SIR2-2.1, AMPK/AAK-2, TOR-binding partner DAF-15, SKN-1/Nrf, and an autophagic protein BEC-1. Because all of these proteins or pathways have been reported to mediate a DR signal, we propose a working model in which sesamin might extend lifespan by acting upstream of the DR-associated nutrient-sensing pathways. References are indicated in square brackets. Inductive pathways are shown as arrows, and suppressive pathways are shown as arrows with T-shaped heads

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It is unlikely that sesamin exerts its effects by causing DR-like decreases in body weight and fertility. Notably, we have previously demonstrated that nematode growth curves and the numbers of offspring were unaffected by sesamin supplementation [35]. Nevertheless, sesamin extends the lifespan of C. elegans through signaling pathways also employed by DR. These features are reminiscent of those of resveratrol, a candidate DR mimetic, which exerts beneficial aspects of DR without involving trade-off effects, such as decreased fertility, due to reduced calorie intake [25].

Well-known candidate DR mimetic compounds include resveratrol [49], 2-deoxyglucose (2DG) [50], and metformin [51]. Resveratrol has been reported to act through sir-2.1/SIRT [25, 41] and aak-2/AMPK [13]. Metformin has been proposed to extend lifespan in C. elegans and mice by activating AMPK [19, 52]. 2DG has been shown to act via aak-2/AMPK, but not via sir-2.1/SIRT1, in C. elegans [53]. The combination of our previous work and the current study shows that sesamin extends C. elegans lifespan through the sir-2.1/SIRT1, aak-2/AMPK, TOR, and IIS pathways. These results suggest that sesamin may act, at least in part, through mechanisms that are non-overlapping with known candidate DR mimetics.

It is important to mention that the role of Sir2 in CR-mediated lifespan extension has remained contentious, especially in yeast [11]. The initially proposed role of Sir2 was based on the finding that short-lived strains lacking Sir2 did not have extended lifespan under CR conditions [9]. However, the finding that CR fails to increase life span in a strain lacking Sir2 can be interpreted in two ways: (1) CR directly increases Sir2 activity or (2) yeast strains lacking Sir2, which have an approximately 50% reduction in mean replicative life-span potential, do not live long enough to respond to CR [11]. Several studies have shown that Sir2-independent CR-mediated lifespan extension in yeast [54, 55]. As is the case for C. elegans, lifespan extension by DR is either abrogated or not affected by sir-2.1 deletion, depending on the chosen method of DR [11]. Although it is undisputed that sir-2.1 is one of the mediators for DR-associated longevity [11, 14], the data should be interpreted with caution and follow-up studies will be needed to confirm the mechanism underlying sir-2.1-dependent lifespan extension by sesamin.

Sesamin’s direct target remains unclear, but several known pathways or proteins have been implicated in mediating the effects of sesamin. For instance, sesamin metabolites have been shown to activate the Nrf2/ARE (antioxidant response element) signaling pathway in rat pheochromocytoma PC12 cells [56]. Sesamin has also been shown to induce autophagy in colon cancer cells by reducing the tyrosine phosphorylation of EphA1 and EphB2 [57]. It is critical to identify the target(s) of sesamin and to examine potential longevity effects of sesamin in mammals, including primates and humans.