1
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
2
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
3
1
Abstract
2
Dietary isoflavones, a group of secondary plant compounds exhibiting phytoestrogenic properties, are
3
primarily found in soy. Prunetin, a representative isoflavone, was recently identified to affect cell signaling
4
in cultured cells, however, in vivo effects remain elusive. In this study, the model organism Drosophila
5
melanogaster was used to investigate the effects of prunetin in vivo with respect to lifespan, locomotion,
6
body composition, metabolism and gut health. Adult flies were chronically administered a prunetin-
7
supplemented diet. Prunetin improved median survival by +3.0 days and climbing activity by +54% in
8
males. Notably, in comparison with females, male flies exhibited lower climbing activity, which could be
9
reversed by prunetin intake. Furthermore, prunetin-fed males exhibited increased expression of the
10
longevity gene Sir2 (+22%), as well as elevated AMPK activation (+51%) and triglyceride levels (+29%)
11
while glucose levels were decreased (-36%). As females are long-lived compared with their male
12
counterparts and exhibit higher triglyceride levels, prunetin apparently “feminizes” male flies via its
13
estrogenicity. We conclude that the lifespan-prolonging effects of prunetin in the male fruit fly depend on
14
changes in AMPK-regulated energy homeostasis via male “feminization”. Collectively, we identified
15
prunetin as a plant bioactive compound capable of improving health status and survival in male Drosophila
16
melanogaster.
17
Keywords: isoflavone, climbing activity, survival, metabolism
18
4
19
Introduction
20
Diet plays an important role in health and in the prevention of chronic diseases (1). The traditional Asian
21
diet is rich in fruits, vegetables and legumes, including soy. Soy is the most important dietary source of
22
isoflavones, and prunetin is one representative of the isoflavone group that exhibits potent bioactivity (2).
23
Although it has been demonstrated in cultured cells in vitro that prunetin may affect cell signaling, little is
24
known about its bioactivity in vivo. We investigated whether prunetin affects health and lifespan in the
25
model organism Drosophila melanogaster since inflammation, stress response, barrier function and
26
metabolism have been described as crucial determinants of longevity. In this context, Drosophila
27
melanogaster is an appropriate model for investigating the effects of plant bioactives on metabolism,
28
inflammation and aging because genes affecting common biological processes and molecular functions are
29
evolutionary conserved. Thereby Drosophila exhibits orthologs of a majority of mammalian genes.
30
Furthermore, numerous Drosophila protein sequences are similar to those of mammals (3). Additionally,
31
the fruit fly possesses a complex and dynamic gut that is similar in structure and organization to the
32
mammalian gut (4). Similarly, insect immune function has much in common with the innate immune
33
response of mammals, and the fruit fly is a distinguished model for investigating innate immunity
34
(reviewed in (5, 6)).
35
Prunetin is a phytoestrogen and therefore may exert estrogenic effects in the fruit fly. Drosophila growth,
36
metamorphosis, reproduction and aging are controlled by fly steroid hormones known as ecdysteroids (7);
37
innate immunity also depends on ecdysteroid expression (8). Lifespan and metabolic homeostasis are
38
directly linked to the presence of defined levels of fly steroid hormones (9, 10). Furthermore, physical
39
activity is a marker of health in Drosophila melanogaster and is also positively associated with longevity
40
(11, 12). As active 20-hydroxyecdysone (20-OH-E; (13)) shares structural similarities with mammalian
41
estrogens and with the plant-derived phytoestrogen prunetin (Fig. 1), comparative examinations of lifespan
42
and body composition were performed to assess the existence of a putative feminization effect. Further,
43
premature mortality has been associated with increased AMP expression (14), which is related to changes
44
in intestinal immune response, possibly via alterations in the expression of Relish (Rel), a NF-κB family
45
ortholog in the fruit fly (15). As prunetin significantly improves intestinal epithelial barrier function and
46
reduces NF-κB transactivation in CaCo-2 cells in vitro (16), we aimed to investigate whether and how
47
prunetin affects gut health and longevity in Drosophila melanogaster in vivo.
48
Therefore, the flies were fed a standard diet supplemented with prunetin either alone or in combination
49
with fulvestrant, a pure antiestrogen that selectively down-regulates estrogen receptor (ER) α expression in
50
vitro (17) and in vivo in humans (18) and mice (19) and also inhibits ERα and ERβ transcription and
51
(trans-)activation in vitro (20, 21) and in vivo (19). Furthermore, 17β-estradiol (17bE), the most effective
52
estradiol with anabolic ability in humans (22), was included in the fly food as 17β-estradiol-glucuronide
53
(E(2)17G), which has been shown to be transported by the Drosophila multidrug resistance-associated
54
protein, a membrane-bound ABC transporter (23). Besides survivorship, climbing activity (which is a
55
marker for fitness and health) and body composition, underlying mechanisms referring to prunetin-
5
56
mediated effects on survival and fitness of the male fruit fly were investigated. Therefore, qRT-PCR (stress
57
response and longevity associated genes e.g. Rel, Sirtuin2) and Western Blotting analyses (AMPK
58
activation, which is of major regulatory importance for energy homeostasis) were performed. Prunetin was
59
identified as a novel, food-derived, potent plant bioactive compound capable of improving the health and
60
survival of male Drosophila melanogaster w1118 and combatting aging.
61
Materials and methods
62
Fly strains and husbandry
63
The wild type strain w1118 (Bloomington Drosophila Stock Center #5905) was used for lifespan
64
experiments, as well as for RT-qPCR analysis and immunofluorescence measurements. The Rel-deficient
65
strain w1118; RelE38 es, which lacks all four Rel transcription start sites (15), was used for lifespan
66
experiments. Drosophila stocks were maintained at 25°C and 60% humidity under a 12/12 h light/dark
67
cycle in an incubator (Memmert, Germany) on standard medium consisting of 6.0% cornmeal, 2.5%
68
inactive dry yeast (Dutscher Scientific, Grays, UK), 1.0% Agar Type II (100 mesh; Apex via Genesee
69
Scientific, San Diego, CA/USA), 5.5% dextrose and 3.0% sucrose (Carl Roth, Germany). Experimental
70
food was prepared according to (24) with modifications. Tegosept (0.3% [w/v]) (Apex via Genesee
71
Scientific) and propionic acid (0.3% [v/v]) (Carl Roth) were added as preservatives.
72
Test compounds and inhibitor
73
Prunetin (≥98%) was purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Prunetin
74
was dissolved in dimethyl sulfoxide (DMSO; Carl Roth) and stored as a 50 mM stock solution at -80°C.
75
For experimental treatments, fly food was supplemented with 25 µM prunetin or 0.05% DMSO ([v/v];
76
vehicle control), unless otherwise indicated. β-estradiol 17-(β-D-glucuronide) sodium salt (E(2)17G cpd)
77
and the ER antagonist fulvestrant were obtained from Sigma. Stock solutions were prepared in DMSO at
78
concentrations of 25 mg/ml (E(2)17G) and 5 mg/ml (fulvestrant) and stored at -80°C. Fly food was
79
supplemented with E(2)17G or fulvestrant at final concentrations of 25 nM or 250 nM. Due to solubility
80
limitations of the above-mentioned substances, the DMSO concentration of the control food was adjusted
81
to 0.15% [v/v] in following experiments.
82
Lifespan experiments
83
To determine how prunetin affected lifespan, synchronized flies were allowed to mate for two days past
84
eclosure (according to (25)). Two-day-old w1118 imagoes were separated according to sex, transferred to
85
experimental vials containing standard medium (consisting of cornmeal, agarose, yeast and sucrose) and
86
supplemented either with prunetin (25 µM) or DMSO (control). The dietary prunetin concentration of 25
87
µM, as used in this study, represents an appropriate dose resulting in isoflavone concentrations of up to 50
88
µM in (intestinal) chyme in mammals receiving a standard serving of respective phytoestrogen-rich dietary
89
foods (e.g. soy, lima and butter beans) (26). Furthermore, higher dietary (phyto-)estrogen concentrations
6
90
(≥100 µM) may be toxic (9). The flies were transferred to fresh medium three times a week. The number
91
of dead flies was recorded on each day of transfer until all flies were dead. Each group was made up of
92
four biological replicates containing 20 flies each (n=80 per group). Three independent experiments were
93
performed. To assess the impact of prunetin on lifespan in relation to Rel expression, age-matched, male
94
w1118; RelE38 es flies were similarly treated with either prunetin or DMSO. Studies on the importance of
95
prunetin’s estrogenic properties on lifespan were performed using the w1118 strain and included the
96
administration of either E(2)17G (25 nM) or a combination of prunetin (25 µM) and fulvestrant (250 nM).
97
25 flies per vial in 9-12 biological replicates were investigated per group.
98
Gustatory assay with sulforhodamine B (food intake)
99
Flies were separated according to sex two days past eclosure. They were reared on food containing either
100
prunetin (25 µM) or DMSO for five days. Their medium was changed twice. Subsequently, the flies were
101
transferred onto the appropriate medium containing 0.2% [w/v] sulforhodamine B (Acid Red 52; Sigma)
102
for 500 min. As a negative control, the medium was not supplied with Acid Red. The flies were not starved
103
prior to their exposure to colored food to avoid falsification of quantification due to restriction-induced
104
food intake. Abdomen redness was documented via stereoscopic white light pictures (Leica, Wetzlar,
105
Germany; (27)). The flies were frozen at -80°C until quantitatively analyzed. For quantification of food
106
intake, 20 flies per group were subjected to fluorescent measurement according to (28) with modifications.
107
In brief, frozen flies were homogenized with an Ultra-Turrax (IKA, Staufen, Germany) in phosphate-
108
buffered saline (PBS; GIBCO via Thermo Fisher Scientific) + 1% Triton-X100 ([v/v]; Sigma). The
109
resultant homogenates were centrifuged, and fluorescence of the supernatant was measured at an extinction
110
wavelength of 535/25 nm and an emission wavelength of 590/20 nm in a Tecan Infinite200 microplate
111
reader (Tecan, Crailsheim, Germany). A standard curve was prepared via serial dilution of a 40-µg aliquot
112
of the initial food preparation homogenized in PBS/Triton-X100. Food consumption was calculated based
113
on the dilutions and on fly numbers. Intake of prunetin-supplemented food was normalized to ingestion of
114
control food. The gustatory assay was repeated three times.
115
Negative geotaxis assay (climbing activity)
116
Evaluating the climbing activity of Drosophila melanogaster is a common method of assessing locomotor
117
activity in the flies. Climbing speed was determined by performing a RING assay (Rapid Iterative
118
Negative Geotaxis) according to (29) and (30) with modifications. In brief, flies were fed either a control
119
or a prunetin-supplemented diet for 30 days. Ten flies per group were transferred to empty vials. Following
120
this, the vials were quickly tapped three times to knock the flies to the bottom, thereby inducing climbing.
121
A snapshot was taken after two seconds. The flies were subjected to the climbing assay for ten successive
122
rounds. The height of the vial (and thus the maximum climbing distance) was divided into four equal
123
segments, and the flies in each segment were allocated a defined climbing score ranging from 1 to 4. Flies
124
that did not climb were assigned a score of 0. The distance that was overcome by the flies of each
7
125
experimental group was calculated by averaging the climbing scores of all flies in a given vial. The
126
experiment was repeated three times at the same time point (7 h light) to mitigate possible effects caused
127
by circadian rhythm (29). The climbing scores of all measurements per group were averaged and
128
normalized to the average climbing score of the control group.
129
Dissection of midguts
130
Flies were dissected one after another. Therefore, each fly was successively anesthetized with CO2,
131
surface-disinfected with 70% clean ethanol ([v/v]) and fixed in PBS on a SYLGARD 184 (Sigma)-covered
132
petri dish. Following removal of the head, the abdomen was opened, and the whole gut, comprising the
133
hindgut, Malpighian tubules, crop and cardia, was isolated (31). The midgut was then separated from the
134
aforementioned organs and was preserved in TriFast reagent (peqlab, Erlangen, Germany) and kept on ice
135
for subsequent RNA isolation.
136
Weighing of male Drosophila melanogaster
137
Flies were fed either prunetin-containing (25 µM) or control food for 10 days and 30 days, as described
138
above. At least 15 anaesthetized flies were transferred to a pre-weighed empty vial. The vials were re-
139
weighed with the flies, and the mean weight of a single fly was calculated. The flies were subsequently
140
frozen at -80°C for further analyses. The experiment was repeated three times with three biological
141
replicates per group and time point.
142
RT-qPCR analysis
143
Total RNA was extracted with TriFast reagent (peqlab, Erlangen, Germany) from whole flies (10 per
144
sample) and from dissected midguts (without Malpighian tubules, cardia and crop, according to (31); 20-25
145
per sample) according to the manufacturer’s protocol. Whole flies were homogenized in a TissueLyser II
146
prior to RNA isolation. RNA concentration and purity were determined via NanoDrop measurements
147
(NanoDrop2000c; ThermoScientific, Waltham, MA/USA). RT-qPCR was performed using a SensiFast
148
SYBR No-ROX One-Step Kit (whole fly homogenates; Bioline, London, UK) and SensiMix SYBR No-
149
ROX Kit (midgut homogenates; Bioline) on a Rotor-Gene 6000 real-time PCR cycler (Corbett/Qiagen).
150
cDNA was synthesized with a Tetro cDNA Synthesis Kit (Bioline) according to the manufacturer’s
151
instructions on a TPersonal 48 thermocycler (Biometra GmbH, Goettingen, Germany). Relative mRNA
152
quantification was calculated using a standard curve. Target gene expression (see Tab. 1) was normalized
153
to the expression of the housekeeping gene alpha-Tubulin at 84B. At least three independent experiments
154
were performed for each application. Estrogen effects on mRNA expression levels were investigated in
155
whole flies (ten per sample) reared on control or E(2)17G- or prunetin+fulvestrant-supplemented food
156
(five biological replicates per group) as described above.
157
Determination of protein, triglyceride and glucose levels (referred to as body composition) in whole fly
158
lysates
8
159
Flies were fed either a control diet or diets supplemented with prunetin, E(2)17G or prunetin+fulvestrant as
160
described above. Five flies per sample (three samples per replicate) were homogenized in PBS/Triton-
161
X100 (1%) in a TissueLyser II and subsequently centrifuged. The resultant supernatants were diluted in
162
homogenization buffer and subjected to either Pierce Bicinchoninic acid (BCA, protein; Thermo Fisher
163
Scientific), Fluitest TG (triglycerides) or Fluitest GLU (glucose) assay kits (Analyticon Biotechnologies
164
AG, Lichtenfels, Germany). All assays were performed according to the manufacturer’s instructions.
165
Protein, triglyceride and glucose levels were normalized to fly weight.
166
Western blotting
167
Flies were fed either a control diet or diets supplemented with prunetin, E(2)17G or prunetin+fulvestrant as
168
described above. Five flies per sample were homogenized in RIPA buffer (50 mmol/l Tris, 150 mmol/l
169
NaCl, 0.5% sodium deoxycholate [v/v], 0.1% SDS [w/v], 1% NP40 [v/v], pH=7.4) containing proteinase
170
(Sigma) and phosphatase inhibitors (Roche Applied Sciences, Mannheim, Germany) in a TissueLyser II.
171
The resultant lysates were centrifuged, and protein concentrations were determined by a BCA Protein
172
Assay. A total of 40 µg of each sample was heated with loading buffer and separated on a 12% Mini-
173
PROTEAN TGX Stain-Free gel (Bio-Rad, Munich, Germany). Then, the samples were transferred onto a
174
PVDF membrane (Bio-Rad) and blocked with 5% [w/v] skim milk dissolved in Tris-buffered saline +
175
0.05% [v/v] Tween-20 at room temperature for 1 h. The membranes were probed overnight with antibodies
176
against phosphorylated AMPK (p-AMPK #2535; Cell Signaling, Germany), AMPK (#80039; Abcam,
177
Cambridge, UK) and α-Tubulin (#2125; Cell Signaling), followed by incubation with the corresponding
178
secondary antibodies (anti-rabbit; anti-mouse (Bio-Rad)) at room temperature for 1 h. The membranes
179
were stripped (Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer’s
180
instructions. Bands were visualized with ECL substrate (Thermo Fisher Scientific) in a ChemiDoc XRS
181
system using Quantity One Software (version 4.6.3; Bio-Rad). Density analyses were performed with
182
Image Lab software (version 4.1; Bio-Rad).
183
Statistical analyses
184
To calculate survival rates, DLife software (Winchecker version 3.0; (25)) was used. Values are given as
185
the mean and were statistically evaluated via a Log-Rank Test based on R (i386 version 3.1.0). For RT-
186
qPCR, gustatory assay, body composition and negative geotaxis assay, values are given as the mean +
187
SEM, except when otherwise indicated. The data were analyzed for normality of distribution
188
(Kolmogorov-Smirnov and Shapiro-Wilk). Mean comparisons were carried out using a 2-sided Student’s t-
189
test in cases of normally distributed data. Otherwise, a non-parametric Mann-Whitney-U test was used.
190
Statistical analysis was performed with SPSS (version 19; SPSS Inc., Munich, Germany). Significance was
191
assumed at p-values
