The phytoestrogen prunetin affects body composition ...

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with fulvestrant, a pure antiestrogen that selectively down-regulates estrogen receptor (ER) α expression in. 49 vitro (17) and in vivo in humans (18) and mice ...

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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

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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

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Abstract

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Dietary isoflavones, a group of secondary plant compounds exhibiting phytoestrogenic properties, are

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primarily found in soy. Prunetin, a representative isoflavone, was recently identified to affect cell signaling

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in cultured cells, however, in vivo effects remain elusive. In this study, the model organism Drosophila

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melanogaster was used to investigate the effects of prunetin in vivo with respect to lifespan, locomotion,

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body composition, metabolism and gut health. Adult flies were chronically administered a prunetin-

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supplemented diet. Prunetin improved median survival by +3.0 days and climbing activity by +54% in

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males. Notably, in comparison with females, male flies exhibited lower climbing activity, which could be

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reversed by prunetin intake. Furthermore, prunetin-fed males exhibited increased expression of the

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longevity gene Sir2 (+22%), as well as elevated AMPK activation (+51%) and triglyceride levels (+29%)

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while glucose levels were decreased (-36%). As females are long-lived compared with their male

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counterparts and exhibit higher triglyceride levels, prunetin apparently “feminizes” male flies via its

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estrogenicity. We conclude that the lifespan-prolonging effects of prunetin in the male fruit fly depend on

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changes in AMPK-regulated energy homeostasis via male “feminization”. Collectively, we identified

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prunetin as a plant bioactive compound capable of improving health status and survival in male Drosophila

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melanogaster.

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Keywords: isoflavone, climbing activity, survival, metabolism

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Introduction

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Diet plays an important role in health and in the prevention of chronic diseases (1). The traditional Asian

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diet is rich in fruits, vegetables and legumes, including soy. Soy is the most important dietary source of

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isoflavones, and prunetin is one representative of the isoflavone group that exhibits potent bioactivity (2).

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Although it has been demonstrated in cultured cells in vitro that prunetin may affect cell signaling, little is

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known about its bioactivity in vivo. We investigated whether prunetin affects health and lifespan in the

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model organism Drosophila melanogaster since inflammation, stress response, barrier function and

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metabolism have been described as crucial determinants of longevity. In this context, Drosophila

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melanogaster is an appropriate model for investigating the effects of plant bioactives on metabolism,

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inflammation and aging because genes affecting common biological processes and molecular functions are

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evolutionary conserved. Thereby Drosophila exhibits orthologs of a majority of mammalian genes.

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Furthermore, numerous Drosophila protein sequences are similar to those of mammals (3). Additionally,

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the fruit fly possesses a complex and dynamic gut that is similar in structure and organization to the

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mammalian gut (4). Similarly, insect immune function has much in common with the innate immune

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response of mammals, and the fruit fly is a distinguished model for investigating innate immunity

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(reviewed in (5, 6)).

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Prunetin is a phytoestrogen and therefore may exert estrogenic effects in the fruit fly. Drosophila growth,

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metamorphosis, reproduction and aging are controlled by fly steroid hormones known as ecdysteroids (7);

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innate immunity also depends on ecdysteroid expression (8). Lifespan and metabolic homeostasis are

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directly linked to the presence of defined levels of fly steroid hormones (9, 10). Furthermore, physical

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activity is a marker of health in Drosophila melanogaster and is also positively associated with longevity

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(11, 12). As active 20-hydroxyecdysone (20-OH-E; (13)) shares structural similarities with mammalian

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estrogens and with the plant-derived phytoestrogen prunetin (Fig. 1), comparative examinations of lifespan

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and body composition were performed to assess the existence of a putative feminization effect. Further,

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premature mortality has been associated with increased AMP expression (14), which is related to changes

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in intestinal immune response, possibly via alterations in the expression of Relish (Rel), a NF-κB family

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ortholog in the fruit fly (15). As prunetin significantly improves intestinal epithelial barrier function and

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reduces NF-κB transactivation in CaCo-2 cells in vitro (16), we aimed to investigate whether and how

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prunetin affects gut health and longevity in Drosophila melanogaster in vivo.

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Therefore, the flies were fed a standard diet supplemented with prunetin either alone or in combination

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with fulvestrant, a pure antiestrogen that selectively down-regulates estrogen receptor (ER) α expression in

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vitro (17) and in vivo in humans (18) and mice (19) and also inhibits ERα and ERβ transcription and

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(trans-)activation in vitro (20, 21) and in vivo (19). Furthermore, 17β-estradiol (17bE), the most effective

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estradiol with anabolic ability in humans (22), was included in the fly food as 17β-estradiol-glucuronide

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(E(2)17G), which has been shown to be transported by the Drosophila multidrug resistance-associated

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protein, a membrane-bound ABC transporter (23). Besides survivorship, climbing activity (which is a

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marker for fitness and health) and body composition, underlying mechanisms referring to prunetin-

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mediated effects on survival and fitness of the male fruit fly were investigated. Therefore, qRT-PCR (stress

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response and longevity associated genes e.g. Rel, Sirtuin2) and Western Blotting analyses (AMPK

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activation, which is of major regulatory importance for energy homeostasis) were performed. Prunetin was

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identified as a novel, food-derived, potent plant bioactive compound capable of improving the health and

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survival of male Drosophila melanogaster w1118 and combatting aging.

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Materials and methods

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Fly strains and husbandry

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The wild type strain w1118 (Bloomington Drosophila Stock Center #5905) was used for lifespan

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experiments, as well as for RT-qPCR analysis and immunofluorescence measurements. The Rel-deficient

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strain w1118; RelE38 es, which lacks all four Rel transcription start sites (15), was used for lifespan

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experiments. Drosophila stocks were maintained at 25°C and 60% humidity under a 12/12 h light/dark

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cycle in an incubator (Memmert, Germany) on standard medium consisting of 6.0% cornmeal, 2.5%

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inactive dry yeast (Dutscher Scientific, Grays, UK), 1.0% Agar Type II (100 mesh; Apex via Genesee

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Scientific, San Diego, CA/USA), 5.5% dextrose and 3.0% sucrose (Carl Roth, Germany). Experimental

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food was prepared according to (24) with modifications. Tegosept (0.3% [w/v]) (Apex via Genesee

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Scientific) and propionic acid (0.3% [v/v]) (Carl Roth) were added as preservatives.

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Test compounds and inhibitor

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Prunetin (≥98%) was purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Prunetin

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was dissolved in dimethyl sulfoxide (DMSO; Carl Roth) and stored as a 50 mM stock solution at -80°C.

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For experimental treatments, fly food was supplemented with 25 µM prunetin or 0.05% DMSO ([v/v];

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vehicle control), unless otherwise indicated. β-estradiol 17-(β-D-glucuronide) sodium salt (E(2)17G cpd)

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and the ER antagonist fulvestrant were obtained from Sigma. Stock solutions were prepared in DMSO at

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concentrations of 25 mg/ml (E(2)17G) and 5 mg/ml (fulvestrant) and stored at -80°C. Fly food was

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supplemented with E(2)17G or fulvestrant at final concentrations of 25 nM or 250 nM. Due to solubility

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limitations of the above-mentioned substances, the DMSO concentration of the control food was adjusted

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to 0.15% [v/v] in following experiments.

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Lifespan experiments

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To determine how prunetin affected lifespan, synchronized flies were allowed to mate for two days past

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eclosure (according to (25)). Two-day-old w1118 imagoes were separated according to sex, transferred to

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experimental vials containing standard medium (consisting of cornmeal, agarose, yeast and sucrose) and

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supplemented either with prunetin (25 µM) or DMSO (control). The dietary prunetin concentration of 25

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µM, as used in this study, represents an appropriate dose resulting in isoflavone concentrations of up to 50

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µM in (intestinal) chyme in mammals receiving a standard serving of respective phytoestrogen-rich dietary

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foods (e.g. soy, lima and butter beans) (26). Furthermore, higher dietary (phyto-)estrogen concentrations

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(≥100 µM) may be toxic (9). The flies were transferred to fresh medium three times a week. The number

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of dead flies was recorded on each day of transfer until all flies were dead. Each group was made up of

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four biological replicates containing 20 flies each (n=80 per group). Three independent experiments were

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performed. To assess the impact of prunetin on lifespan in relation to Rel expression, age-matched, male

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w1118; RelE38 es flies were similarly treated with either prunetin or DMSO. Studies on the importance of

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prunetin’s estrogenic properties on lifespan were performed using the w1118 strain and included the

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administration of either E(2)17G (25 nM) or a combination of prunetin (25 µM) and fulvestrant (250 nM).

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25 flies per vial in 9-12 biological replicates were investigated per group.

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Gustatory assay with sulforhodamine B (food intake)

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Flies were separated according to sex two days past eclosure. They were reared on food containing either

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prunetin (25 µM) or DMSO for five days. Their medium was changed twice. Subsequently, the flies were

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transferred onto the appropriate medium containing 0.2% [w/v] sulforhodamine B (Acid Red 52; Sigma)

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for 500 min. As a negative control, the medium was not supplied with Acid Red. The flies were not starved

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prior to their exposure to colored food to avoid falsification of quantification due to restriction-induced

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food intake. Abdomen redness was documented via stereoscopic white light pictures (Leica, Wetzlar,

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Germany; (27)). The flies were frozen at -80°C until quantitatively analyzed. For quantification of food

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intake, 20 flies per group were subjected to fluorescent measurement according to (28) with modifications.

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In brief, frozen flies were homogenized with an Ultra-Turrax (IKA, Staufen, Germany) in phosphate-

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buffered saline (PBS; GIBCO via Thermo Fisher Scientific) + 1% Triton-X100 ([v/v]; Sigma). The

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resultant homogenates were centrifuged, and fluorescence of the supernatant was measured at an extinction

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wavelength of 535/25 nm and an emission wavelength of 590/20 nm in a Tecan Infinite200 microplate

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reader (Tecan, Crailsheim, Germany). A standard curve was prepared via serial dilution of a 40-µg aliquot

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of the initial food preparation homogenized in PBS/Triton-X100. Food consumption was calculated based

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on the dilutions and on fly numbers. Intake of prunetin-supplemented food was normalized to ingestion of

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control food. The gustatory assay was repeated three times.

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Negative geotaxis assay (climbing activity)

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Evaluating the climbing activity of Drosophila melanogaster is a common method of assessing locomotor

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activity in the flies. Climbing speed was determined by performing a RING assay (Rapid Iterative

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Negative Geotaxis) according to (29) and (30) with modifications. In brief, flies were fed either a control

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or a prunetin-supplemented diet for 30 days. Ten flies per group were transferred to empty vials. Following

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this, the vials were quickly tapped three times to knock the flies to the bottom, thereby inducing climbing.

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A snapshot was taken after two seconds. The flies were subjected to the climbing assay for ten successive

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rounds. The height of the vial (and thus the maximum climbing distance) was divided into four equal

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segments, and the flies in each segment were allocated a defined climbing score ranging from 1 to 4. Flies

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that did not climb were assigned a score of 0. The distance that was overcome by the flies of each

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experimental group was calculated by averaging the climbing scores of all flies in a given vial. The

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experiment was repeated three times at the same time point (7 h light) to mitigate possible effects caused

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by circadian rhythm (29). The climbing scores of all measurements per group were averaged and

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normalized to the average climbing score of the control group.

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Dissection of midguts

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Flies were dissected one after another. Therefore, each fly was successively anesthetized with CO2,

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surface-disinfected with 70% clean ethanol ([v/v]) and fixed in PBS on a SYLGARD 184 (Sigma)-covered

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petri dish. Following removal of the head, the abdomen was opened, and the whole gut, comprising the

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hindgut, Malpighian tubules, crop and cardia, was isolated (31). The midgut was then separated from the

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aforementioned organs and was preserved in TriFast reagent (peqlab, Erlangen, Germany) and kept on ice

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for subsequent RNA isolation.

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Weighing of male Drosophila melanogaster

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Flies were fed either prunetin-containing (25 µM) or control food for 10 days and 30 days, as described

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above. At least 15 anaesthetized flies were transferred to a pre-weighed empty vial. The vials were re-

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weighed with the flies, and the mean weight of a single fly was calculated. The flies were subsequently

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frozen at -80°C for further analyses. The experiment was repeated three times with three biological

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replicates per group and time point.

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RT-qPCR analysis

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Total RNA was extracted with TriFast reagent (peqlab, Erlangen, Germany) from whole flies (10 per

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sample) and from dissected midguts (without Malpighian tubules, cardia and crop, according to (31); 20-25

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per sample) according to the manufacturer’s protocol. Whole flies were homogenized in a TissueLyser II

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prior to RNA isolation. RNA concentration and purity were determined via NanoDrop measurements

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(NanoDrop2000c; ThermoScientific, Waltham, MA/USA). RT-qPCR was performed using a SensiFast

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SYBR No-ROX One-Step Kit (whole fly homogenates; Bioline, London, UK) and SensiMix SYBR No-

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ROX Kit (midgut homogenates; Bioline) on a Rotor-Gene 6000 real-time PCR cycler (Corbett/Qiagen).

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cDNA was synthesized with a Tetro cDNA Synthesis Kit (Bioline) according to the manufacturer’s

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instructions on a TPersonal 48 thermocycler (Biometra GmbH, Goettingen, Germany). Relative mRNA

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quantification was calculated using a standard curve. Target gene expression (see Tab. 1) was normalized

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to the expression of the housekeeping gene alpha-Tubulin at 84B. At least three independent experiments

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were performed for each application. Estrogen effects on mRNA expression levels were investigated in

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whole flies (ten per sample) reared on control or E(2)17G- or prunetin+fulvestrant-supplemented food

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(five biological replicates per group) as described above.

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Determination of protein, triglyceride and glucose levels (referred to as body composition) in whole fly

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lysates

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Flies were fed either a control diet or diets supplemented with prunetin, E(2)17G or prunetin+fulvestrant as

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described above. Five flies per sample (three samples per replicate) were homogenized in PBS/Triton-

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X100 (1%) in a TissueLyser II and subsequently centrifuged. The resultant supernatants were diluted in

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homogenization buffer and subjected to either Pierce Bicinchoninic acid (BCA, protein; Thermo Fisher

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Scientific), Fluitest TG (triglycerides) or Fluitest GLU (glucose) assay kits (Analyticon Biotechnologies

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AG, Lichtenfels, Germany). All assays were performed according to the manufacturer’s instructions.

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Protein, triglyceride and glucose levels were normalized to fly weight.

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Western blotting

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Flies were fed either a control diet or diets supplemented with prunetin, E(2)17G or prunetin+fulvestrant as

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described above. Five flies per sample were homogenized in RIPA buffer (50 mmol/l Tris, 150 mmol/l

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NaCl, 0.5% sodium deoxycholate [v/v], 0.1% SDS [w/v], 1% NP40 [v/v], pH=7.4) containing proteinase

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(Sigma) and phosphatase inhibitors (Roche Applied Sciences, Mannheim, Germany) in a TissueLyser II.

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The resultant lysates were centrifuged, and protein concentrations were determined by a BCA Protein

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Assay. A total of 40 µg of each sample was heated with loading buffer and separated on a 12% Mini-

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PROTEAN TGX Stain-Free gel (Bio-Rad, Munich, Germany). Then, the samples were transferred onto a

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PVDF membrane (Bio-Rad) and blocked with 5% [w/v] skim milk dissolved in Tris-buffered saline +

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0.05% [v/v] Tween-20 at room temperature for 1 h. The membranes were probed overnight with antibodies

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against phosphorylated AMPK (p-AMPK #2535; Cell Signaling, Germany), AMPK (#80039; Abcam,

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Cambridge, UK) and α-Tubulin (#2125; Cell Signaling), followed by incubation with the corresponding

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secondary antibodies (anti-rabbit; anti-mouse (Bio-Rad)) at room temperature for 1 h. The membranes

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were stripped (Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer’s

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instructions. Bands were visualized with ECL substrate (Thermo Fisher Scientific) in a ChemiDoc XRS

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system using Quantity One Software (version 4.6.3; Bio-Rad). Density analyses were performed with

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Image Lab software (version 4.1; Bio-Rad).

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Statistical analyses

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To calculate survival rates, DLife software (Winchecker version 3.0; (25)) was used. Values are given as

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the mean and were statistically evaluated via a Log-Rank Test based on R (i386 version 3.1.0). For RT-

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qPCR, gustatory assay, body composition and negative geotaxis assay, values are given as the mean +

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SEM, except when otherwise indicated. The data were analyzed for normality of distribution

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(Kolmogorov-Smirnov and Shapiro-Wilk). Mean comparisons were carried out using a 2-sided Student’s t-

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test in cases of normally distributed data. Otherwise, a non-parametric Mann-Whitney-U test was used.

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Statistical analysis was performed with SPSS (version 19; SPSS Inc., Munich, Germany). Significance was

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assumed at p-values

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