The phytoestrogen prunetin affects body composition and improves fitness and lifespan in male Drosophila melanogaster Stefanie Piegholdta, Gerald Rimbacha, Anika E. Wagnera* a
Institute of Human Nutrition and Food Science, Christian-Albrechts-University Kiel, HermannRodewald-Strasse 6-8, D-24118 Kiel, Germany
* Corresponding author. Tel +49 431 880 5313. Fax +49 431 880 2628. E-mail address: [email protected]
Running title: Prunetin improves fitness & lifespan in Drosophila
Abbreviations: 20-OH-E, 20-hydroxyecdysone; 17bE, 17β-Estradiol; E(2)17G, β-Estradiol 17-(β-Dglucuronide); EcR, ecdysone receptor; EER, estrogen-related receptor; ER, estrogen receptor; Fulv, fulvestrant; IMD, immune deficiency; LXR, liver X receptor; NF-κB, nuclear factor κB; p-AMPK: phosphorylated AMP (adenosine monophosphate)-activated protein kinase; prun, prunetin; RXR, retinoid X receptor; usp, ultraspiracle
Dietary isoflavones, a group of secondary plant compounds exhibiting phytoestrogenic properties, are
primarily found in soy. Prunetin, a representative isoflavone, was recently identified to affect cell signaling
in cultured cells, however, in vivo effects remain elusive. In this study, the model organism Drosophila
melanogaster was used to investigate the effects of prunetin in vivo with respect to lifespan, locomotion,
body composition, metabolism and gut health. Adult flies were chronically administered a prunetin-
supplemented diet. Prunetin improved median survival by +3.0 days and climbing activity by +54% in
males. Notably, in comparison with females, male flies exhibited lower climbing activity, which could be
reversed by prunetin intake. Furthermore, prunetin-fed males exhibited increased expression of the
longevity gene Sir2 (+22%), as well as elevated AMPK activation (+51%) and triglyceride levels (+29%)
while glucose levels were decreased (-36%). As females are long-lived compared with their male
counterparts and exhibit higher triglyceride levels, prunetin apparently “feminizes” male flies via its
estrogenicity. We conclude that the lifespan-prolonging effects of prunetin in the male fruit fly depend on
changes in AMPK-regulated energy homeostasis via male “feminization”. Collectively, we identified
prunetin as a plant bioactive compound capable of improving health status and survival in male Drosophila
Keywords: isoflavone, climbing activity, survival, metabolism
Diet plays an important role in health and in the prevention of chronic diseases (1). The traditional Asian
diet is rich in fruits, vegetables and legumes, including soy. Soy is the most important dietary source of
isoflavones, and prunetin is one representative of the isoflavone group that exhibits potent bioactivity (2).
Although it has been demonstrated in cultured cells in vitro that prunetin may affect cell signaling, little is
known about its bioactivity in vivo. We investigated whether prunetin affects health and lifespan in the
model organism Drosophila melanogaster since inflammation, stress response, barrier function and
metabolism have been described as crucial determinants of longevity. In this context, Drosophila
melanogaster is an appropriate model for investigating the effects of plant bioactives on metabolism,
inflammation and aging because genes affecting common biological processes and molecular functions are
evolutionary conserved. Thereby Drosophila exhibits orthologs of a majority of mammalian genes.
Furthermore, numerous Drosophila protein sequences are similar to those of mammals (3). Additionally,
the fruit fly possesses a complex and dynamic gut that is similar in structure and organization to the
mammalian gut (4). Similarly, insect immune function has much in common with the innate immune
response of mammals, and the fruit fly is a distinguished model for investigating innate immunity
(reviewed in (5, 6)).
Prunetin is a phytoestrogen and therefore may exert estrogenic effects in the fruit fly. Drosophila growth,
metamorphosis, reproduction and aging are controlled by fly steroid hormones known as ecdysteroids (7);
innate immunity also depends on ecdysteroid expression (8). Lifespan and metabolic homeostasis are
directly linked to the presence of defined levels of fly steroid hormones (9, 10). Furthermore, physical
activity is a marker of health in Drosophila melanogaster and is also positively associated with longevity
(11, 12). As active 20-hydroxyecdysone (20-OH-E; (13)) shares structural similarities with mammalian
estrogens and with the plant-derived phytoestrogen prunetin (Fig. 1), comparative examinations of lifespan
and body composition were performed to assess the existence of a putative feminization effect. Further,
premature mortality has been associated with increased AMP expression (14), which is related to changes
in intestinal immune response, possibly via alterations in the expression of Relish (Rel), a NF-κB family
ortholog in the fruit fly (15). As prunetin significantly improves intestinal epithelial barrier function and
reduces NF-κB transactivation in CaCo-2 cells in vitro (16), we aimed to investigate whether and how
prunetin affects gut health and longevity in Drosophila melanogaster in vivo.
Therefore, the flies were fed a standard diet supplemented with prunetin either alone or in combination
with fulvestrant, a pure antiestrogen that selectively down-regulates estrogen receptor (ER) α expression in
vitro (17) and in vivo in humans (18) and mice (19) and also inhibits ERα and ERβ transcription and
(trans-)activation in vitro (20, 21) and in vivo (19). Furthermore, 17β-estradiol (17bE), the most effective
estradiol with anabolic ability in humans (22), was included in the fly food as 17β-estradiol-glucuronide
(E(2)17G), which has been shown to be transported by the Drosophila multidrug resistance-associated
protein, a membrane-bound ABC transporter (23). Besides survivorship, climbing activity (which is a
marker for fitness and health) and body composition, underlying mechanisms referring to prunetin-
mediated effects on survival and fitness of the male fruit fly were investigated. Therefore, qRT-PCR (stress
response and longevity associated genes e.g. Rel, Sirtuin2) and Western Blotting analyses (AMPK
activation, which is of major regulatory importance for energy homeostasis) were performed. Prunetin was
identified as a novel, food-derived, potent plant bioactive compound capable of improving the health and
survival of male Drosophila melanogaster w1118 and combatting aging.
Materials and methods
Fly strains and husbandry
The wild type strain w1118 (Bloomington Drosophila Stock Center #5905) was used for lifespan
experiments, as well as for RT-qPCR analysis and immunofluorescence measurements. The Rel-deficient
strain w1118; RelE38 es, which lacks all four Rel transcription start sites (15), was used for lifespan
experiments. Drosophila stocks were maintained at 25°C and 60% humidity under a 12/12 h light/dark
cycle in an incubator (Memmert, Germany) on standard medium consisting of 6.0% cornmeal, 2.5%
inactive dry yeast (Dutscher Scientific, Grays, UK), 1.0% Agar Type II (100 mesh; Apex via Genesee
Scientific, San Diego, CA/USA), 5.5% dextrose and 3.0% sucrose (Carl Roth, Germany). Experimental
food was prepared according to (24) with modifications. Tegosept (0.3% [w/v]) (Apex via Genesee
Scientific) and propionic acid (0.3% [v/v]) (Carl Roth) were added as preservatives.
Test compounds and inhibitor
Prunetin (≥98%) was purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Prunetin
was dissolved in dimethyl sulfoxide (DMSO; Carl Roth) and stored as a 50 mM stock solution at -80°C.
For experimental treatments, fly food was supplemented with 25 µM prunetin or 0.05% DMSO ([v/v];
vehicle control), unless otherwise indicated. β-estradiol 17-(β-D-glucuronide) sodium salt (E(2)17G cpd)
and the ER antagonist fulvestrant were obtained from Sigma. Stock solutions were prepared in DMSO at
concentrations of 25 mg/ml (E(2)17G) and 5 mg/ml (fulvestrant) and stored at -80°C. Fly food was
supplemented with E(2)17G or fulvestrant at final concentrations of 25 nM or 250 nM. Due to solubility
limitations of the above-mentioned substances, the DMSO concentration of the control food was adjusted
to 0.15% [v/v] in following experiments.
To determine how prunetin affected lifespan, synchronized flies were allowed to mate for two days past
eclosure (according to (25)). Two-day-old w1118 imagoes were separated according to sex, transferred to
experimental vials containing standard medium (consisting of cornmeal, agarose, yeast and sucrose) and
supplemented either with prunetin (25 µM) or DMSO (control). The dietary prunetin concentration of 25
µM, as used in this study, represents an appropriate dose resulting in isoflavone concentrations of up to 50
µM in (intestinal) chyme in mammals receiving a standard serving of respective phytoestrogen-rich dietary
foods (e.g. soy, lima and butter beans) (26). Furthermore, higher dietary (phyto-)estrogen concentrations
(≥100 µM) may be toxic (9). The flies were transferred to fresh medium three times a week. The number
of dead flies was recorded on each day of transfer until all flies were dead. Each group was made up of
four biological replicates containing 20 flies each (n=80 per group). Three independent experiments were
performed. To assess the impact of prunetin on lifespan in relation to Rel expression, age-matched, male
w1118; RelE38 es flies were similarly treated with either prunetin or DMSO. Studies on the importance of
prunetin’s estrogenic properties on lifespan were performed using the w1118 strain and included the
administration of either E(2)17G (25 nM) or a combination of prunetin (25 µM) and fulvestrant (250 nM).
25 flies per vial in 9-12 biological replicates were investigated per group.
Gustatory assay with sulforhodamine B (food intake)
Flies were separated according to sex two days past eclosure. They were reared on food containing either
prunetin (25 µM) or DMSO for five days. Their medium was changed twice. Subsequently, the flies were
transferred onto the appropriate medium containing 0.2% [w/v] sulforhodamine B (Acid Red 52; Sigma)
for 500 min. As a negative control, the medium was not supplied with Acid Red. The flies were not starved
prior to their exposure to colored food to avoid falsification of quantification due to restriction-induced
food intake. Abdomen redness was documented via stereoscopic white light pictures (Leica, Wetzlar,
Germany; (27)). The flies were frozen at -80°C until quantitatively analyzed. For quantification of food
intake, 20 flies per group were subjected to fluorescent measurement according to (28) with modifications.
In brief, frozen flies were homogenized with an Ultra-Turrax (IKA, Staufen, Germany) in phosphate-
buffered saline (PBS; GIBCO via Thermo Fisher Scientific) + 1% Triton-X100 ([v/v]; Sigma). The
resultant homogenates were centrifuged, and fluorescence of the supernatant was measured at an extinction
wavelength of 535/25 nm and an emission wavelength of 590/20 nm in a Tecan Infinite200 microplate
reader (Tecan, Crailsheim, Germany). A standard curve was prepared via serial dilution of a 40-µg aliquot
of the initial food preparation homogenized in PBS/Triton-X100. Food consumption was calculated based
on the dilutions and on fly numbers. Intake of prunetin-supplemented food was normalized to ingestion of
control food. The gustatory assay was repeated three times.
Negative geotaxis assay (climbing activity)
Evaluating the climbing activity of Drosophila melanogaster is a common method of assessing locomotor
activity in the flies. Climbing speed was determined by performing a RING assay (Rapid Iterative
Negative Geotaxis) according to (29) and (30) with modifications. In brief, flies were fed either a control
or a prunetin-supplemented diet for 30 days. Ten flies per group were transferred to empty vials. Following
this, the vials were quickly tapped three times to knock the flies to the bottom, thereby inducing climbing.
A snapshot was taken after two seconds. The flies were subjected to the climbing assay for ten successive
rounds. The height of the vial (and thus the maximum climbing distance) was divided into four equal
segments, and the flies in each segment were allocated a defined climbing score ranging from 1 to 4. Flies
that did not climb were assigned a score of 0. The distance that was overcome by the flies of each
experimental group was calculated by averaging the climbing scores of all flies in a given vial. The
experiment was repeated three times at the same time point (7 h light) to mitigate possible effects caused
by circadian rhythm (29). The climbing scores of all measurements per group were averaged and
normalized to the average climbing score of the control group.
Dissection of midguts
Flies were dissected one after another. Therefore, each fly was successively anesthetized with CO2,
surface-disinfected with 70% clean ethanol ([v/v]) and fixed in PBS on a SYLGARD 184 (Sigma)-covered
petri dish. Following removal of the head, the abdomen was opened, and the whole gut, comprising the
hindgut, Malpighian tubules, crop and cardia, was isolated (31). The midgut was then separated from the
aforementioned organs and was preserved in TriFast reagent (peqlab, Erlangen, Germany) and kept on ice
for subsequent RNA isolation.
Weighing of male Drosophila melanogaster
Flies were fed either prunetin-containing (25 µM) or control food for 10 days and 30 days, as described
above. At least 15 anaesthetized flies were transferred to a pre-weighed empty vial. The vials were re-
weighed with the flies, and the mean weight of a single fly was calculated. The flies were subsequently
frozen at -80°C for further analyses. The experiment was repeated three times with three biological
replicates per group and time point.
Total RNA was extracted with TriFast reagent (peqlab, Erlangen, Germany) from whole flies (10 per
sample) and from dissected midguts (without Malpighian tubules, cardia and crop, according to (31); 20-25
per sample) according to the manufacturer’s protocol. Whole flies were homogenized in a TissueLyser II
prior to RNA isolation. RNA concentration and purity were determined via NanoDrop measurements
(NanoDrop2000c; ThermoScientific, Waltham, MA/USA). RT-qPCR was performed using a SensiFast
SYBR No-ROX One-Step Kit (whole fly homogenates; Bioline, London, UK) and SensiMix SYBR No-
ROX Kit (midgut homogenates; Bioline) on a Rotor-Gene 6000 real-time PCR cycler (Corbett/Qiagen).
cDNA was synthesized with a Tetro cDNA Synthesis Kit (Bioline) according to the manufacturer’s
instructions on a TPersonal 48 thermocycler (Biometra GmbH, Goettingen, Germany). Relative mRNA
quantification was calculated using a standard curve. Target gene expression (see Tab. 1) was normalized
to the expression of the housekeeping gene alpha-Tubulin at 84B. At least three independent experiments
were performed for each application. Estrogen effects on mRNA expression levels were investigated in
whole flies (ten per sample) reared on control or E(2)17G- or prunetin+fulvestrant-supplemented food
(five biological replicates per group) as described above.
Determination of protein, triglyceride and glucose levels (referred to as body composition) in whole fly
Flies were fed either a control diet or diets supplemented with prunetin, E(2)17G or prunetin+fulvestrant as
described above. Five flies per sample (three samples per replicate) were homogenized in PBS/Triton-
X100 (1%) in a TissueLyser II and subsequently centrifuged. The resultant supernatants were diluted in
homogenization buffer and subjected to either Pierce Bicinchoninic acid (BCA, protein; Thermo Fisher
Scientific), Fluitest TG (triglycerides) or Fluitest GLU (glucose) assay kits (Analyticon Biotechnologies
AG, Lichtenfels, Germany). All assays were performed according to the manufacturer’s instructions.
Protein, triglyceride and glucose levels were normalized to fly weight.
Flies were fed either a control diet or diets supplemented with prunetin, E(2)17G or prunetin+fulvestrant as
described above. Five flies per sample were homogenized in RIPA buffer (50 mmol/l Tris, 150 mmol/l
NaCl, 0.5% sodium deoxycholate [v/v], 0.1% SDS [w/v], 1% NP40 [v/v], pH=7.4) containing proteinase
(Sigma) and phosphatase inhibitors (Roche Applied Sciences, Mannheim, Germany) in a TissueLyser II.
The resultant lysates were centrifuged, and protein concentrations were determined by a BCA Protein
Assay. A total of 40 µg of each sample was heated with loading buffer and separated on a 12% Mini-
PROTEAN TGX Stain-Free gel (Bio-Rad, Munich, Germany). Then, the samples were transferred onto a
PVDF membrane (Bio-Rad) and blocked with 5% [w/v] skim milk dissolved in Tris-buffered saline +
0.05% [v/v] Tween-20 at room temperature for 1 h. The membranes were probed overnight with antibodies
against phosphorylated AMPK (p-AMPK #2535; Cell Signaling, Germany), AMPK (#80039; Abcam,
Cambridge, UK) and α-Tubulin (#2125; Cell Signaling), followed by incubation with the corresponding
secondary antibodies (anti-rabbit; anti-mouse (Bio-Rad)) at room temperature for 1 h. The membranes
were stripped (Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer’s
instructions. Bands were visualized with ECL substrate (Thermo Fisher Scientific) in a ChemiDoc XRS
system using Quantity One Software (version 4.6.3; Bio-Rad). Density analyses were performed with
Image Lab software (version 4.1; Bio-Rad).
To calculate survival rates, DLife software (Winchecker version 3.0; (25)) was used. Values are given as
the mean and were statistically evaluated via a Log-Rank Test based on R (i386 version 3.1.0). For RT-
qPCR, gustatory assay, body composition and negative geotaxis assay, values are given as the mean +
SEM, except when otherwise indicated. The data were analyzed for normality of distribution
(Kolmogorov-Smirnov and Shapiro-Wilk). Mean comparisons were carried out using a 2-sided Student’s t-
test in cases of normally distributed data. Otherwise, a non-parametric Mann-Whitney-U test was used.
Statistical analysis was performed with SPSS (version 19; SPSS Inc., Munich, Germany). Significance was
assumed at p-values