European Review for Medical and Pharmacological Sciences

2019; 23: 1826-1839

Sesamin suppresses aging phenotypes in adultmuscular and nervous systems and intestines ina Drosophila senescence-accelerated model
T.D. LE1, Y. NAKAHARA1, M. UEDA1, K. OKUMURA1, J. HIRAI1, Y. SATO2,
D. TAKEMOTO2, N. TOMIMORI2, Y. ONO2, M. NAKAI2, H. SHIBATA2, Y.H. INOUE1
Insect Biomedical Research Centre, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, Japan
Institute for Health Care Science, Suntory Wellness Ltd., Seika-cho, Soraku-gun, Kyoto, Japan

1
2

Tuan Dat Le and Yasuyuki Nakahara contributed equally to this work

Abstract. – OBJECTIVE: Sesamin is a ma-

jor lignan constituent of sesame and possesses
various health-promoting effects. Previous studies have demonstrated that sesamin extends thelifespan of Drosophila and Caenorhabditis elegans and corrects oxidative damage-related tissue dysfunction in mammals. To understandits anti-aging effects, we aimed to determine
whether sesamin restores tissue function hampered by oxidative damage and suppresses several aging-related phenotypes using Drosophilasenescence-accelerated models.
MATERIALS AND METHODS: We elucidated
the anti-aging effects of sesamin on several aging-related phenotypes in the muscle, brain andmidgut using the senescence-accelerated models (Sod1n1 mutant and Sod1-depleted flies) byimmunostaining experiments. We determinedthe expression levels of several anti-oxidativeand DNA repair genes using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR).
We also identified the metabolite of sesamin in
Drosophila by LC-MS/MS.
RESULTS: We confirmed that sesamin (0.35
and 2 mg/ml) extended the lifespan of the flymodels. As observed in mammals, it can be absorbed and metabolized by Drosophila adults.
The sesamin feeding suppressed the age-dependent impairment of locomotor activity andinhibited the accumulation of reactive oxygen
species (ROS) in their bodies. Sesamin delayed
the age-dependent accumulation of damaged
proteins in the muscle, partially suppressed theloss of dopaminergic neurons in adult brains displaying ROS accumulation, and suppressed theaccumulation of DNA damage and hyperproliferation of intestinal stem cells. Four antioxidative
genes and two DNA repair genes were simultaneously upregulated in sesamin-fed adults.
CONCLUSIONS: These observations represent the first direct evidence of the anti-aging ef-

1826fects of sesamin at the individual level. We propose that sesamin exerts anti-aging effects inthe muscles, brain and midgut by inducing antioxidative and DNA repair genes, resulting in extended lifespan in flies.
Key Words:
Sesamin, Anti-aging, Drosophila, Muscle, Intestine.

Introduction
Sesame seeds contain certain types of lignansthat play important roles in health promotion1. Sesamin is a major lignan in sesame seeds. Many invitro and in vivo studies have demonstrated its biological effects, including antioxidant2-6, anti-carcinogenic7,8 and anti-hypertensive effects9-11. Thus,
sesame seeds have been used as a traditional health food as well as a medicinal plant, consumed
as a supplement or remedy. After intake, sesaminis metabolized to SC1, (1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7dioxabicyclo-[3,3,0] octane, by cytochrome P450
in mammals3,12. Because SC1 displays potent antioxidant activity, it may be responsible, in part, forsome of the physiological activities of sesamin3,13,14.
A previous study10,15 using experimental animals
has demonstrated the anti-hypertensive effects ofsesamin and has suggested that sesamin feeding
prevents hypertension by inhibiting NADPH oxidase activity and the expression of its subunits.
Another study3 has reported that the catechol moiety of the sesamin molecule is responsible for theprotective effects of sesamin against oxidative damage in the rat liver. It has recently been reported

Corresponding Author: Yoshihiro H. Inoue, MD; e-mail: yhinoue@kit.ac.jp

Sesamin suppresses aging in Drosophila adults

that sesamin extends the mean lifespan and alleviates paraquat-induced decline in the survival rate of
Drosophila by interacting with antioxidant enzymes16. The compound also enhances host defense
and increases the average lifespan of Caenorhabditis elegans17. Although there is much biologicaland biochemical evidence demonstrating the anti-aging effects of sesamin in animals, a few studies have aimed to verify its role in hindering theaging process. Apart from simply measuring thelifespan of organisms, it is not easy to monitor the
progression of aging and evaluate the anti-aging
effects simultaneously. The fruit fly Drosophilamelanogaster serves as a useful genetic model due
to its high fecundity and short life cycle18,19. Furthermore, advances in genetic techniques have facilitated the experimental investigation. Drosophila has numerous advantages in drug discovery18,20,
particularly as an experimental model for studies
on aging. We previously established an experimental system that allowed the downregulation of
the Sod1 gene encoding the Cu/Zn superoxide dismutase that eliminates superoxide radicals among
reactive oxygen species (ROS)21,22. As normal
Drosophila adults age, they display impaired locomotor activity, accumulation of abnormal protein
aggregates containing polyubiquitinated proteinsin the muscle and loss of dopaminergic (DA) neurons in the brain22-25. Moreover, as the flies’ ages increase, intestinal stem cells increase in number inintestinal epithelial cells26-28. We showed that adults with depleted Sod1 and its hypomorphic mutant,
Sod1n1, exhibit not only a shortened lifespan but
also accelerated age-dependent phenotypes froman earlier stage. Therefore, compared with mouseand rat models, these flies represent a good senescence-accelerated model for the rapid evaluationof anti-aging effects22. In the present work, we aimed to elucidate the anti-aging effects of sesaminon several aging-related phenotypes in muscle,
brain and midgut using Drosophila senescence-accelerated models (Sod1n1 mutant and Sod1-depleted
flies). We also determined the expression levels of
several anti-oxidative genes and DNA repair genes
using quantitative Real Time-Polymerase Chain
Reaction (qRT-PCR). In addition, we analyzed themetabolism of sesamin in Drosophila.

Materials and Methods
Fly Stocks and Culture
Canton S was used as a wild-type stock. To examine the lifespan extension and anti-aging effects

of sesamin, a Drosophila senescence-accelerated
model, Sod1n1 mutant was used29. This mutant
shows a considerably declined SOD activity due
to an aminoacid substitution, without the reducedmRNA level29. Our previous work revealed that
this fly model displayed several aging phenotypes
faster than wild-type flies22. We used this homozygous mutant for a lifespan assay and climbing
assay as described in the following sections. For
examining aging phenotypes appeared in certain
adult tissues, we performed depletion of Sod1
gene at the adult stage. We used the Gal4/UAS
system, ectopic gene expression system in Drosophila for this purpose30,31. For inducing dsRNA
against the Sod1 mRNA, UAS-Sod1RNAiF103 stock
was used22. For depletion in muscle, Mef2-Gal4.
R3 (Mef-Gal4) was used as a Gal4 driver stock.
For depletion in adult brains, Actin5C-Gal4 wasused. We performed ubiquitous depletion of theSod1 gene at the adult stage and observed the aging-related phenotypes in adult brains. Gal4 driver stocks (Actin5C-Gal4 and Mef2-Gal4) wereobtained from the Bloomington Drosophila Stock
Center (Bloomington, IN, USA). We designatedMef2-Gal4>UAS-Sod1F103 flies (muscle-specific
Sod1 depletion) and Actin5C-Gal4>UAS-Sod1F103
flies (ubiquitous Sod1 depletion) thereafter. To
visualize intestine stem cells (ISCs) and theirdaughter cells, known as enteroblasts (EBs), in
adult midguts, a stock carrying esg-GAL4 and
UAS-GFP (esg>GFP), provided by the Drosophila Genetic Resource Center (Kyoto, Japan), wasused32. P{GstD1-GFP} (GstD-GFP) was used asa marker to visualize ROS accumulation22,33. All
fly stocks were maintained on a normal cornmeal
diet at 25°C. Every 1 L of normal cornmeal foodincluded 7.2 g agar, 100 g glucose, 40 g dried
yeast, and 40 g cornmeal. All ingredients weremixed well and boiled for 10 min. After coolingto 70°C, 5 mL of 10% parahydroxybenzonate dissolved in ethanol and 5 mL propionic acid wereadded.
Lifespan Assay
The lifespan extension effect of sesamin was
confirmed using our Drosophila senescence-accelerated model, Sod1n1 mutants. The lifespanassay was performed as previously described22,34.
To avoid differences in lifespan between matedand virgin females and to avoid the higher accidental mortality observed in females comparedwith males, male adults were selected for the lifespan assay. Young adults were collected within
24 h after eclosion. Twenty flies were reared in a1827

T.D. Le, Y. Nakahara, M. Ueda, K. Okumura, J. Hirai, Y. Sato, D. Takemoto, et al.

single plastic vial containing Drosophila instantmedium (Formula 4-24, Blue, Wako, Osaka, Japan). Sesamin (Nacalai Tesque, Kyoto, Japan) was
dissolved in dimethyl sulfoxide (DMSO), and thestock solution was diluted using distilled water toachieve final concentrations of 0.35 or 2 mg/mL
in the diet. As controls, DMSO (1% and 2%, respectively) alone was added to the instant food at
the corresponding concentrations. The assay was
repeated three to six times (using a total of 76-97
Sod1n1 mutant flies). Dead adults in each vial werescored every 12 h for Sod1n1 mutants. Food vials
were changed every 3 days. Survival curves wereanalyzed by the Kaplan-Meier test.
Climbing Assays
Climbing assays, that utilized a fly’s instinctof negative geotaxis, were performed according
to a previous protocol22 with slight modifications.
Briefly, newly eclosed Sod1n1 mutant adults werecollected and raised on a diet containing 2-mg/
mL sesamin or 2% DMSO alone. Twenty adultflies of the same age (0, 5, 8, or 10 days after eclosion) were gently tapped with a constant strength
to the bottom of a plastic culture tube (100 mm inheight and 15 mm in diameter), and the numberof flies that climbed up to a marked point (50 mm
in height, half the height from the bottom of thetube) after 6 s were counted and scored as follows:
10 points for climbing over the point, 5 points for
climbing up between the line and the bottom of
the tube, and 0 points for staying at the bottom of
the tube throughout. The average scores for each
fly were calculated, and the mean scores of fiverepeated trials (a total of 100-200 flies) were presented22,35.
Absorption and Metabolism
Five to seven-day-old wild-type flies were collected and fed a diet supplemented with 2 mg/mL
sesamin dissolved in 2% DMSO or 2% DMSO
alone for 7 days. The flies were then maintainedon a diet without sesamin for 30 min to drain the
undigested diet from the gut. The wings and legs
were removed from the flies before homogenizing them to prevent incorporating any sesaminthat may have adhered to the body surface. After
briefly rinsing with Phosphate-Buffered Saline
supplemented with 0.1% Triton X-100 (PBST), theflies were flash-frozen in liquid nitrogen. Frozen
Drosophila adults were suspended in 200 μL of
0.1 M KCl and homogenized using a TissueLyser (QIAGEN Sciences Inc., Germantown, MD,USA) for 60 s. Sesamin and its metabolites were1828

extracted from Drosophila homogenates using
the method described by Bligh-Dyer36. Extracts
were analyzed according to the method reported
by Tomimori et al37 using an ACQUITY UPLC
system (Waters Corp., Milford, MA, USA) coupled to a Quattro micro MS (Mass Spectrometry)system (IET; Mundelein, IL, USA). UV detection
was performed at 280 nm. The limit of detection
was 0.05 μg/mL in both sesamin and SC1. The
MS spectrometer was operated in full scan andmultiple reaction monitoring modes.
Immunostaining Procedures
For muscle immunohistochemistry, we collected newly eclosed flies with a muscle-specific Sod1
depletion and raised them for 5, 20 or 30 days on
diets supplemented with 2 mg/mL sesamin or 2%
DMSO. For dissection of the indirect flight muscles, the thoraces of the adult flies were cut along
a longitudinal midline in relaxing buffer (0.1 M
KCl, 20 mM Tris-HCl, pH 7.2, 1 mM MgCl2, and
1 mM EDTA), as described22. After removal of
cuticles, a thin block of the thorax, containing thedorsal longitudinal muscles in indirect flight muscle, was dissected from the abdomen. The muscle samples were fixed in 4% paraformaldehyde
for 30 min washed with PBST, and blocked with
10% normal goat serum. To identify abnormal
protein aggregates, a mouse antibody reactive toboth mono- and polyubiquitinated proteins (EnzoLife Sciences, Farmingdale, NY, USA; 1:300)
was used. The samples were incubated with primary antibody overnight at 4°C. After washingwith PBST, samples were incubated with Alexa
Fluor 488-conjugated secondary antibodies. For
simultaneous detection of myofibrils, we usedrhodamine-phalloidin (Molecular Probes, Eugene, OR, USA; 1:80) at room temperature andwashed the samples with PBST. One image per
fly was observed, and the pixels in one optic field(4.0 × 10-2 mm2) were measured using a confocal
microscope.
To visualize the DA neurons by immunohistochemistry in the brains of adult flies, whole brains
were dissected from the flies with a ubiquitousdepletion of Sod1 on day 5, as described previously22. To collect an intact whole adult brain, thehead capsule was carefully removed using one
pair of fine forceps to avoid damage to the brain
and optic lobes. The isolated brains were thenfixed in 3.7% formaldehyde for 15 min and incubated with anti-tyrosine hydroxylase primary antibody (Millipore, Billerica, MA, USA) overnightat 4°C. After washing, samples were incubatedSesamin suppresses aging in Drosophila adults

with Alexa Fluor 594-conjugated secondary antibodies. The number of visualized DA neurons in
the PLL1, PPM1/2, PPM3, PPL2 and VUM clusters in the adult brains were counted.
To evaluate the aging phenotypes in the adultmidgut, the accumulation of DNA damage andhyperproliferation of ISCs and EBs, which occur
to regenerate the damaged epithelial cells, wasassessed28,38. Anti-γH2AvD immunostaining was
performed to detect DNA damage foci in ISCsand EBs that were recognized by the esg>GFP
reporter. To simplify the experiments, flies carrying the wild-type allele of Sod1 were subjected to
extrinsic oxidative stress by feeding them paraquat, an agent known to induce free radical production, instead of utilizing the Sod1n1 mutants.
In previous studies32, metformin was used as ananti-aging drug that suppressed DNA damage
accumulation and, consequently, stem cell hyperproliferation in Drosophila adult midguts. Therefore, metformin was compared with sesamin. For
midgut immunohistochemistry, newly eclosed
esg>GFP flies were collected, aged on a standard
fly food for 33 days, divided into three groups andfed instant medium supplemented with 1.5 mg/
mL metformin, 2 mg/mL sesamin, or 2% DMSO
alone for 7 days at 28°C. Flies were transferred to
new food every 2 days. For the paraquat-feeding
assay, 40-day-old flies were placed in empty vials
in 10 mM paraquat (methyl viologen; Sigma-Aldrich, St. Louis, MO, USA) with 5% sucrose. For
immunohistochemical analysis of the digestive
tract, we prepared the midguts from adult flies as
described previously34. After incubating for 1820 h, the abdominal ventral epidermis of the flies
was cut along the midline using a micro scissor.
The epidermis was consequently opened in PBS
by a pair of fine forceps to collect the internal digestive organs. The midgut was carefully isolated
from the organs by removing the foregut, hindgut
and malpighian tubes to avoid damage to a tubular structure of the gut. The isolated midguts were
fixed at room temperature for 30 min in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO,
USA). After washing with PBST, samples were
blocked with 10% normal goat serum and incubated overnight with the primary antibody rabbit
anti-γH2AvD (1:300; Rockland Immunochemical
Inc., Gilbertsville, PA, USA) at 4°C. After extensive washing with PBST, samples were incubatedwith Alexa Fluor 594-conjugated secondary antibodies, washed with PBST and mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA). For a quantitative analysis

of cell numbers in the adult midgut, the number
of DAPI-positive (total cells), esg-positive cells(corresponding to ISCs and EBs), and γH2AvDpositive cells in an area of the posterior midgut
were counted using Image J software (NIH,
Bethesda, MD, USA). The results were presented as a percentage of the number of γH2AvDpositive cells among esg-positive cells or esg-positive cells among total cells.
All immunostained samples were mounted in
Vectashield (Vector Laboratories, Burlingame,
CA, USA) and observed using an Olympus laser
scanning confocal microscope (Fv10i, Olympus,
Tokyo, Japan). Brightness and contrast of entire
images were adjusted using Fv10i software. Image analysis was performed using Photoshop andImage J (NIH, Bethesda, MD, USA).
Quantitative Reverse Transcription
Polymerase Chain Reaction Analysis(qRT-PCR)
Quantitative Reverse Transcription Polymerase
Chain Reaction Analysis (qRT-PCR) was performed
to quantitate mRNA levels of four genes encoding
antioxidative enzymes (catalase, Sod1, Sod2, andJafrac1/Thioredoxin peroxidase 1), two genes encoding DNA repair enzymes (Gadd45 and CG9272),
and the GstD1 gene. Total RNA was extracted using
TRIzol reagent (Invitrogen, Carlsbad, CA, USA)
from newly eclosed (within 24 h) Sod1n1 mutant
adults fed with instant medium with or without
2-mg/mL sesamin for 7 days. cDNA was synthesized from total RNA using a PrimeScript II Fidelity
RT-PCR Kit (TaKaRa, Otsu, Shiga, Japan) and an
oligo dT primer. Real Time-PCR was performed
using the FastStart Essential DNA Green Master(Roche, Basel, Switzerland) and a Light Cycler Nano instrument (Roche, Basel, Switzerland). qPCR
primers were as follows: RP49-FW, 5’-TTCCTGGTGCACAACGTG-3’, RP49-RV, 5’-TCTCCTTGCGCTTCTTGG3’, α-tubulin84B-FW, 5’-GCTTCCTCATCTTCCACTCG-3’, α -tubulin84B-RV,
5’-GCTTGGACTTCTTGCCGTAG-3’,
Catalase-FW, 5’-TTTCTCCTGGTGCCATTACC-3’, Catalase-RV, 5’-CTGCAGCAGGATAGGTCCTC-3’,
SOD1-FW, 5’-AGGTCAACATCACCGACTCC-3’,
SOD1-RV, 5’-GTTGACTTGCTCAGCTCGTG-3’,
SOD2-FW,
5’-TGGCCACATCAACCACAC-3’,
SOD2-RV,
5’-TTCCACTGCGACTCGATG-3’,
Gadd45-FW,
5’-GATCCCTCTTCTGCCTGATG-3’, Gadd45-RV, 5’-TCGTCCACCTTGATCACGTA-3’, Jafrac1-FW, 5’-ACCGAGATCATTGCGTTCTC-3’, Jafrac1-RV, 5’-AAGTGGGTGAACTGGCTGTC-3’, GstD1-FW, 5’-AAGATCAATCCC1829

T.D. Le, Y. Nakahara, M. Ueda, K. Okumura, J. Hirai, Y. Sato, D. Takemoto, et al.

CAGCACAC-3’, GstD1-RV, 5’-GGTCTTGCCGTACTTCTCCA-3’, CG9272-FW, 5’-GGTTCGGCTACTGTGGTCAT-3’, CG9272-RV, 5’-TATTTGGGGTTCCACCTCCT-3’. All qRT-PCR experiments were performed in triplicate, and the results
were taken as the average of three replicates in eachgroup. The ∆∆Ct method was used to determine thedifferences in target gene expression relative to thereference Rp49 gene expression.
Evaluation of Oxidative Stress
The newly eclosed adults ubiquitously depleted
for Sod1 (Actin5C>Sod1RNAi) were respectively
raised on a diet supplemented with or without 2
mg/mL sesamin for 5 and 10 days. The GstD1
gene is used as a marker for estimating the extent
of ROS accumulation because its expression is
induced in response to the accumulation of oxidative stress.
Statistical Analysis
A survival curve was calculated following
Kaplan-Meier survival estimation and analyzed bylog-rank test between the sesamin-treated groupand the appropriate control group. For the comparisons of the two groups we used the Student’s t-test.
One-way ANOVA with post-hoc Tukey’s test wasapplied to assess the differences in more than two
groups. Two-way ANOVA with post-hoc Student’s
t-test was performed to compare the mean diffe-

rences between groups that were split into two
independent variables. Statistical analyses were
performed using IBM SPSS statistics 25 software(IBM, Armonk, NY, USA). Data were consideredsignificant at p-values < 0.05.

Results
Sesamin Feeding Extended the Lifespanof Drosophila Adults
As continuous administration of sesamin towild-type Drosophila adults extended their life
span in a previous study (see introduction), we
first confirmed the lifespan extension effect ofsesamin using our Drosophila senescence-accelerated model, Sod1n1 mutants, in which ROS
accumulation was amplified. Sod1n1 mutant
adults displayed a shortened adult lifespan,
and the mean time required for 50% lethality
in the presence of 1% and 2% DMSO was 7.1
and 6.6 days, respectively, and the longest adult
lifespans were 12.5 and 12.5 days on average,
respectively (Figure 1). The lifespan of Drosophila adults fed with 2 mg/mL sesamin was
significantly extended compared with that offlies fed with 2% DMSO ( p < 0.001), and themean time required for 50% lethality was 11.6
days in the former. Feeding of 0.35 mg/mL sesamin also extended the lifespan compared to

Figure 1. Sesamin extends lifespan in the Drosophila senescence-accelerated model, Sod1n1 mutant adult flies. Lifespancurves in adult flies fed with or without 0.35 mg/mL sesamin in 1% DMSO (n = 80 and 97, respectively), and with and without
2 mg/mL sesamin in 2% DMSO (n = 80 and 76, respectively). The results represent the average of four repeated experiments.
Curves were plotted using Kaplan-Meier survival analysis. A log-rank test was performed for each pair of adults fed with thecontrol diet and adults fed with the sesamin diet. Note that a significant lifespan extension was observed in both cases, whenflies were fed with 0.35 mg/mL (p = 0.004, log-rank test) and 2 mg/mL sesamin (p < 0.001, log-rank test).

1830

Sesamin suppresses aging in Drosophila adults

Figure 2. Drosophila adults fed with sesamin retain a higher climbing activity than control diet-fed adults. The locomotor activity of the Drosophila senescence-accelerated
model, Sod1n1 mutant flies, fed with a diet supplemented with
2% DMSO (white bars) or 2 mg/mL sesamin dissolved in 2%
DMSO (black bars) was examined at 0, 5, 8, and 10 days after
eclosion using a climbing assay. The y-axis shows the scoresfor flies that climbed above a line drawn at a certain heightfrom the bottom, within a predetermined period (see Materials and Methods), (n = 100-200 flies from five repeated assays). Data are shown as mean ± SE. There was no differencein the group-time interaction (p = 0.551).

feeding with 1% DMSO ( p = 0.004). Feedingof 2 mg/mL sesamin was more effective in extending adult lifespan than feeding with othersesamin concentrations; therefore, we used thisfinal concentration for further examination of
the anti-aging effects of sesamin.
Age-Dependent Loss of Locomotor
Activity was Suppressed in Drosophila
Adults Fed with Sesamin
We examined whether the lifespan extensionin adults fed with sesamin was a consequence of
aging suppression. We performed a climbing assay to quantitate adult locomotor activity (Figure2). The score derived from the climbing assay forcontrol Sod1n1 mutant flies decreased with aging(Figure 2). However, feeding with 2 mg/mL sesamin significantly improved this decline of locomotor activities with aging (p = 0.039). These
results indicate that age-dependent impairment oflocomotor activity was suppressed in Drosophila
adults fed with sesamin.
Sesamin Suppressed the Age-Dependent
Accumulation of Abnormal Protein
Aggregates in Indirect Flight Muscles
As we showed that sesamin can suppress theaging-related decrease in locomotor activities,
we examined whether sesamin suppressed theaccumulation of abnormal protein aggregates inadult indirect flight muscles. As flies age, protein aggregates containing damaged proteins
accumulate in their muscles. We performed immunostaining in the indirect flight muscles of amuscle-specific Sod1 depletion (Mef2>Sod1RNAi). Representative confocal microscopy images showing polyubiquitinated protein (Figure3a-f) and merged images with myofibrils (Figure
3a′-f′), and presented the average data for polyubiquitinated protein (Figure 3g). We showed anincrease in protein aggregates with aging in theindirect flight muscles of 5-, 20-, and 30-day-old
flies fed with the control diet (62, 333, and 459
pixels, respectively). In contrast, we found a significant decrease in the number and size of aggregates in muscles from 5-, 20-, and 30-day-old
flies fed with the sesamin diet (p < 0.001 for 47
pixels; p < 0.001 for 218 pixels, and p < 0.001 for
276 pixels, respectively) (Figure 3g). These observations indicated that the sesamin diet markedly suppressed the accumulation of protein
aggregates that increased with aging.
Expression of GstD1 (a Marker Genefor Oxidative Stress) was Partially
Suppressed in Drosophila Adults
Fed with Sesamin
As ROS accumulation is associated with theprogression of aging, we examined whether thesesamin diet influenced ROS accumulation. As
ubiquitous Sod1-depleted flies fed with the DMSO
diet aged, we observed more intense GFP fluorescence along their whole body, especially in theabdomens and thoraxes (Supplementary Figure1a-c). In contrast, ubiquitous Sod1-depleted flies
fed with the sesamin diet displayed less intense
GFP signals (Supplementary Figure 1d, e) in 5and 10-day-old flies. Thus, induction of the oxidative stress marker gene, GstD1, was partially suppressed in Drosophila adults fed with sesamin.
The Sesamin Diet Partially Suppressed
the Age/Oxidative Stress-Dependent Lossof DA Neurons in Drosophila Adults
To further understand the mechanisms underlying the role of sesamin in alleviating theage-dependent loss of locomotor activity, we examined whether it could also suppress the loss of
DA neurons in Drosophila adult brains harboring
ROS accumulation. We confirmed that the loss of
DA neurons occurred in each of the five typicalclusters in adult brains prepared from 5-day-old
control ubiquitous Sod1-depleted adults (Figure
1831

T.D. Le, Y. Nakahara, M. Ueda, K. Okumura, J. Hirai, Y. Sato, D. Takemoto, et al.

Figure 3. Sesamin suppresses the accumulation of polyubiquitinated protein aggregates in the myofibrils of the indirectflight muscles. Immunostaining of the indirect flight muscles dissected from flies with a muscle-specific depletion of Sod1
(mef2<Sod1RNAiF103) using an anti-ubiquitin-conjugated antibody (red) a-f, and phalloidin staining for F-actin (green) a′-f′.
Adult flies were collected within 24 h after eclosion, aged for 5 days a, d, 20 days b, e, and 30 days c, f, on a diet supplemented
with 2% DMSO alone a-c, or 2 mg/mL sesamin dissolved in 2% DMSO d-f. Scale bar represents 10 µm g, The average pixel
numbers of the sum of protein aggregates containing polyubiquitinated proteins per single confocal optic fields (4.0 × 10-2mm2) in control (white bars) and sesamin (black bars) fed flies are shown on the y-axis. Data are shown as mean ± SE. At least
10 images of muscle samples prepared from at least 10 flies were observed. Two-way ANOVA with post-hoc Student’s t-test
were applied to compare the mean differences. ***p < 0.001 (Student’s t-test).

Figure 4. Suppression of the oxidative stress-dependent loss of DA neurons in the brains of Sod1n1 mutant adult flies fed withsesamin. Adult whole brains were dissected from 0- and 5-day-old Sod1n1 mutant flies that were collected within 24 h after
eclosion and raised on a diet supplemented with either 2% DMSO (control) or 2 mg/mL sesamin dissolved in 2% DMSO (sesamin). Immunostaining of the brains using an anti-tyrosine hydroxylase antibody to visualize DA neurons was performed andobserved using a confocal microscope. The number of DA neurons in each of the five main neuronal clusters (PPL1, PPM1/2,
PPM3, PPL2 and VUM) was counted and is summarized as a bar graph. Data are shown as mean ± SE. Statistical analysis ineach cluster was performed by one-way ANOVA with post-hoc Tukey’s test. Different letters indicate significant differences
between groups (p < 0.05). There were no differences in PPM1/2, PPM3, and PPL2 clusters by one-way ANOVA.

4). Sesamin feeding suppressed the decline of DA
neurons to the same levels as the 0-day-old flies.
In particular, PPL1, the largest DA neurons clu1832

ster in the brain, was significantly retained in flies
fed with sesamin compared with that in flies fed
with the control diet (p < 0.05; Figure 4).

Sesamin suppresses aging in Drosophila adults

Figure 5. Sesamin inhibits DNA damage accumulation and hyperproliferation of ISCs and EBs in intestinal epithelia in aged
flies fed with or without paraquat. Representative confocal microscopy images show DNA damage accumulation in ISCs and
EBs without a′-c′, and with 10 mM paraquat 5e′-g′, and hyperproliferation of ISCs and EB cells without 5a′′-c′′, and with 10
mM paraquat 5e′′-g′′, in the midgut of aged adults (40 days old). ISCs and their daughter EBs were visualized using esg>GFP
reporter (green) and DAPI (blue). Scale bar represents 10 µm d. The average percentages of cells showing γH2AvD immunostaining signals in their nuclei among the ISCs and EBs, labelled as esg+ cells, observed per single optic field using confocal
microscopy are shown on the y-axis. At least 35 confocal images of midgut samples prepared from at least seven flies wereobserved for each condition. H, The average percentages of ISCs and EBs among the total number of intestinal cells observed
per single optic field in confocal microscope are shown on the y-axis. 25 images of the midgut samples prepared from at least
five flies were observed in each condition. Data are shown as mean ± SE. Statistical analyses were performed using a one-way
ANOVA with post-hoc Tukey’s test. Different letters indicate significant differences between groups (p < 0.05).

Sesamin Suppressed DNA Damage
Accumulation and Hyperproliferation ofIntestine Stem Cells in the Adult Midgut
As flies age, intestinal stem cells (ISC) proliferate more frequently to compensate for damaged
epithelial cells in the adult midgut27-29,32, we examined whether sesamin and metformin showedanti-aging effects in the midguts from adults
carrying Sod1+ allele. Representative confocal
microscopy images indicating DNA damage accumulation (Figure 5a′-c′, 6e′-g′), IPC hyperproliferation (Figure 5a′′-c ′′, e′′ -g′′), and the averagedata of each (Figure 6d, h) are shown. Metformin
was used as a drug having the anti-aging effectthat suppresses aging phenotype in the adultmidgut, although its effects on other aging phenotypes have not been examined. Metformin fe-eding decreased the frequency of cells exhibiting
nuclear DNA damage foci from 41% to 31% (p <0.05; Figure 5aʹ, bʹ, d) and from 58% to 49% under
extrinsic oxidative stress conditions (p < 0.05; Figure 5eʹ, fʹ, d). Metformin feeding also decreased
the frequency of hyperproliferation of the total
intestinal epithelial cells from 30% to 24% (p <0.05; Figure 5a′′, b′′, h) and from 37% to 35% under extrinsic oxidative stress conditions (p < 0.05;
Figure 6e′′, f′′, h). The sesamin diet also decreased
the frequency of nuclei exhibiting DNA damage
foci to 31% (p < 0.05; Figure 5a′, c′, d) and 48%
under extrinsic oxidative stress conditions (p <0.05; Figure 5e′, g′, d). Moreover, sesamin feeding
significantly decreased the hyperproliferation to26% (p < 0.05; Figure 5a′′, c′′, h) and 31% underoxidative stress conditions (p < 0.05; Figure 5e′′,1833

T.D. Le, Y. Nakahara, M. Ueda, K. Okumura, J. Hirai, Y. Sato, D. Takemoto, et al.

Figure 6. Quantitation of mRNA of seven anti-oxidative response-related genes in the Drosophila senescence-accelerated
model, Sod1n1 mutant adults fed with or without sesamin. Relative mRNA levels of each gene in adults fed with a diet containing sesamin (light gray bars) compared with those in adults fed with a diet without sesamin (control, dark gray bars) were
calculated using Real Time-qRT-PCR analysis. Total RNA was isolated from adults fed with a diet containing 2 mg/mL sesamin and from those fed with a diet containing 2% DMSO as a control for 7 days. A, α-tubulin was used as a housekeeping
gene, b-d, f, genes for antioxidative enzymes: catalase b, Sod1 c, Sod2 d, and Jafrac1 e. DNA repair genes, Gadd45 f, andCG9272 g. H, GstD1, expression of which is induced in response to oxidative stress. The qRT-PCR analysis to quantitate theamount of mRNA of each target gene was repeated three times. Statistical analysis was performed using Student’s t-test. ns,
not significant; *p < 0.05; **p < 0.01.

g′′, h). These results indicate that sesamin can remarkably suppress DNA damage accumulationand the IPC hyperproliferation with aging, andthe effects were comparable to those of metformin.

result is consistent with the observation that theinduction of GstD1-GFP was suppressed by thesesamin diet (Supplementary Figure 1). No significant differences were observed in levels of thehousekeeping gene α-tubulin.

Six Genes Encoding Enzymes that
Remove Oxidative Stress wereUpregulated in Drosophila Adults
Fed with Sesamin
The mRNA levels of four antioxidative enzymes (catalase, SOD1, SOD2, and Jafrac1) increased by 20%-30% in Sod1n1 mutant flies fed withsesamin compared with those in adults fed withthe control diet (p < 0.01; Figure 6). The mRNAlevels of the genes encoding two DNA repair
factors (Gadd45 and CG9272) also increased by
20%-30% in adult flies fed with sesamin. In contrast, GstD1 expression decreased by 30%. This

Sesamin was Absorbed and Metabolized
in Drosophila Adults
We confirmed whether sesamin was absorbed
and metabolized in the wild-type Drosophila
adult body. HPLC chromatograms of the Drosophila extracts are shown in Figure 7. Sesamin(black arrow) and its metabolite peak (indicatedby Peak 1 arrow) were detected in extracts fromadults fed with sesamin. MS in the negative ion
mode for peak 1 showed a deprotonated molecularion of m/z 341 [M-H]-. Furthermore, the retention
time of peak 1 was identical to that of the authentic SC1 standard with multiple reaction monito-

1834

Sesamin suppresses aging in Drosophila adults

Figure 7. HPLC chromatogram of Drosophila extracts. The HPLC chromatogram of extract of wild-type adults fed with a,control diet supplemented with 2% DMSO and b, diet supplemented with 2 mg/mL sesamin dissolved in 2% DMSO for 7 days.

ring at m/z 341.2 > 176.2. These data suggest thatsesamin was absorbed and metabolized to SC1 in
Drosophila adults, as observed in mammals.

Discussion
Sesamin not only promotes lifespan extension
in Drosophila adults16 but also suppresses several

types of aging phenotypes, such as age-dependentaccumulation of aggregates containing damaged
proteins in the muscle and loss of DA neurons inadult brains. We also showed that a sesamin dietsuppressed stem cell hyperproliferation, a consequence of DNA damage accumulation in intestinal epithelial cells. These results in Drosophila
are consistent with previous findings showing thatsesamin has suppressive effects that restore tissue

Supplementary Figure 1. Suppression of GstD1 expression correlates with ROS accu-mulation in sesamin-fed Drosophila
adult flies. Stereofluorescence microscopy images of Drosophila adult (lateral view of flies). Induction of GstD1 expression
was monitored using a GFP reporter gene under the control of the GstD1 enhancer, a marker for ROS accumulation. To avoid
collecting autofluorescence from ovaries, we selected male flies carrying a GstD-GFP reporter. Flies with a ubiquitous Sod1
depletion (Actin5C>Sod1RNAi, GstD-GFP) (adults with a Sod1 depletion) were reared at 28°C for 0 days a, 5 days b, d, and 10
days c, e, after eclosion with 2% DMSO b, c, or on 2-mg/mL sesamin dissolved in 2% DMSO d, e. A distinctive reduction in
the intensity of GFP fluorescence was observed in adults fed with sesamin, compared with that in the control adults. Scale bars
represent 250 µm.

1835

T.D. Le, Y. Nakahara, M. Ueda, K. Okumura, J. Hirai, Y. Sato, D. Takemoto, et al.

dysfunction or damage in several animal diseasemodels. For example, sesamin inhibited the decline in exercise capacity and mitochondrial function impairment in the skeletal muscle of micewith high-fat diet-induced diabetes14. Furthermore, it displayed a hepatoprotective effect against
oxidative stress and apoptosis in mice exposed to
CCl439. In addition, it also suppressed behavioral
dysfunctions and exhibited neuroprotective effects in chemically induced Parkinson’s disease
rat and mouse models via inhibition of oxidative
stress and apoptosis in neuronal cells40,41. However, these effects of sesamin in animal models do
not necessarily indicate its potential anti-aging effects in mammalian models and humans. Current
studies on aging use a variety of model organisms
from yeast and worms to mice. However, it is noteasy to extrapolate the results derived from yeast and worms to higher organisms. Nonetheless,aging studies using mice are time-consuming andcost-intensive. On the other hand, Drosophila
provides a useful tool to advance studies on aging
and evaluate chemicals and natural products with
anti-aging activities quickly and efficiently18,19.
The Drosophila senescence-accelerated models
used in this study enable a detailed observationof several aging phenotypes in only 2 weeks22. As
our previous study indicated that Sod1-depleted
adults showed extra ROS accumulation and thatthe flies displayed several aging phenotypes at an
earlier adult stage, we believed that the aging-accelerated phenotypes, which appeared in Sod1n1
mutant having a lower Sod1 activity are associated with ROS accumulation. The Drosophila
results represent the first evidence regarding theanti-aging effects of sesamin in animal muscle,
brain and intestine.
We showed that two chemical compounds,
metformin and sesamin, have anti-aging effects
that delayed the progression of aging phenotypes
in Drosophila adult guts. A previous study32 hasreported that metformin suppresses the characteristic features of aged cells in the Drosophila
midgut, such as DNA damage accumulation andstem cell hyperproliferation. After we establisheda reliable experimental system for monitoring theanti-aging effects, we investigated whether sesamin delayed the progression of aging phenotypesof Drosophila midgut. Sesamin also inhibitedage- and oxidative stress-inducible DNA damage
and the hyperproliferation of intestine stem cells. The present work demonstrated that sesaminis a potential anti-aging drug as well as metformin32. To further confirm the finding, it is also fa1836

scinating to examine whether sesamin can show
a suppression effect on tissue damage by bacterial liposaccharide-induced ROS42. In contrast
to published data demonstrating that metformin
activates AMP-activated kinase (AMPK)43, nostudy has shown the biochemical data suggestingthat sesamin can activate AMPK. Therefore, it
is reasonable to speculate that an entirely different mechanism is responsible for the anti-aging
effects of sesamin. A previous DNA microarraystudy demonstrated that sesamin affected thetranscription of various genes, including those
encoding antioxidative enzymes in a rat model44.
In addition, sesamin feeding reportedly induced
the antioxidative enzyme activities such as superoxide dismutase, glutathione S-transferase, andglutathione peroxidase in the liver of mice models2,45. A recent Drosophila study16 reported thatsesamin influenced the expression of antioxidant
genes, such as Sod1, Sod2, catalase, and Rpn11.
Our qRT-PCR analysis also confirmed that thesesamin diet resulted in the simultaneous upregulation of antioxidative genes, including Sod1,
Sod2, and catalase, as well as of DNA repair
genes. Consistent with our observations, dietary
sesamin suppressed ROS accumulation in the fly
body. A possible transcription factor involved in
the oxidative stress response is Nrf2/Cnc-Keap133.
Hamada et al13 showed that an SC1, anti-oxidative
catechol metabolite, enhanced Nrf2 activation in
cultured PC12 cells. In this work, sesamin metabolite, SC1, was detected in the adult bodies fed
with sesamin. Therefore, sesamin feeding could
induce the antioxidative enzyme activities via induction of Nrf2/keap1 system in Drosophila adults. Another possible transcription factor involvedin the oxidative stress response is Foxo transcription factor46-49. To further understand the molecular mechanisms underlying the anti-aging effects
of sesamin, it is necessary to investigate whether
sesamin feeding can activate Nrf2 or Foxo in the
Drosophila model.
Sesamin was reportedly metabolized into SC1
in mammalian bodies by a cytochrome P450 enzyme12. Similarly, 90 P450-related genes including
pseudogenes have been identified in Drosophila
genome50,51. Although a Drosophila counterpartof the sesamin-metabolizing enzyme has notbeen characterized yet, a presence of SC1 in sesamin-feeding adults strongly suggests that thecounterpart enzyme(s) function in Drosophila asshown in mammals. In this experiment, extracts
from the whole body were used as HPLC samples
for sesamin and its metabolite analysis, because

Sesamin suppresses aging in Drosophila adults

Drosophila is too small for analyzing sesaminand its metabolites from each organ. Therefore,
sesamin which remained without being absorbed
in the gastrointestinal tract and/or sesamin adhering to the body surface may have been detected,
and it is difficult to compare sesamin with SC1
peak. Further studies are needed as to how efficiently the sesamin was metabolized in the bodyof Drosophila.
Our data on Drosophila indicate that sesaminpossesses anti-aging effects that suppress age-dependent muscle impairment. A previous study22
showed that the protein aggregates that accumulate in the muscle with aging are associated withmalformed mitochondria, damaged by oxidative
stress. Maharjan et al52 demonstrated that sesamin can rejuvenate mitochondria damaged byoxidative stress in cultured cells. Takada et al14
also reported that sesamin prevented the decline
in exercise capacity via maintenance of mitochondrial function and attenuation of oxidative stressin skeletal muscle. Studies10,14,15 using experimental animals have also demonstrated that sesaminfeeding inhibited NADPH oxidase activity andthe expression of its subunits, resulting in suppression of superoxide generation. Therefore, theanti-aging effects of sesamin in the muscle may
involve inhibition of oxidative stress in the mitochondria by the suppression of superoxide generation. Another possibility is that this compound
may stimulate the induction of autophagy to remove damaged mitochondria. A previous study53
has reported that sesamin can induce autophagy
in cancer cell lines. Although there is no direct
evidence of a relationship between this compoundand oxidative stress-induced autophagy, sesamin
extends the lifespan of C. elegans by activating
autophagy54. Furthermore, other studies55,56 haveshown that oxidative stress could decrease proteasomal activity in vitro and in vivo; therefore, it ispossible that sesamin regulates protein homeostasis by inhibiting oxidative stress-mediated proteasomal impairment during aging.
It was previously reported22 that apoptosis is
involved in the age-dependent loss of DA neurons
in Drosophila adult brains. Using the same experimental system, we showed that a sesamin dietpartially suppressed the loss of DA neurons. A
previous study13,57 has reported that a metaboliteof sesamin inhibits oxidative stress-induced cell
death in PC12 cells. Our findings regarding theneurons in Drosophila adults fed with sesamin
are consistent with this result suggesting that sesamin has an antioxidative effect on the neuronalprecursor cell line. Therefore, it is possible thatthe antioxidative activity of sesamin underlies its
suppressive effects regarding the age-dependent
loss of neurons.

Conclusions
We evaluated the anti-aging effects of sesaminusing the Drosophila senescence-accelerated models. Sesamin suppressed several aging-related
phenotypes that appeared in muscle, brains andintestines of Drosophila adults. These observations are the first direct evidence of the anti-aging
effects of sesamin at an individual level. Sesaminalso upregulated the expression of several anti-oxidative and DNA repair genes. We surmisethat sesamin exerts anti-aging effects in several
tissues by inducing anti-oxidative and DNA repair
genes, thus extending the lifespan of Drosophila.
Conflict of Interest
The Authors declare that they have no conflict of interest.

Acknowledgments

We acknowledge the Bloomington Stock Center and Drosophila Genetic Resource Center for providing the fly
stocks.

Statement of Interests

LTD, NY, UM, OK, HJ, and IYH received research grants
from Suntory Wellness Ltd and Joint Research Program ofAdvanced Insect Research Promotion Centre in Kyoto Institute of Technology (2017008). SY, TD, TN, OY, NM and SH
are employees of Suntory Wellness Ltd., which is a manufacturer of foods that contain sesamin.

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