To construct GMR-GAL4/Y; UAS-hDuox2/?, virgin female GMR-GAL4 flies containing two copies of the GMR-GAL4 gene on the X chromosome were mated with male hDuox2 flies containing two copies
Trang 1A Drosophila model for the screening of bioavailable NADPH
oxidase inhibitors and antioxidants
Nguyen Thi Tu Anh• Maiko Nishitani•
Shigeharu Harada• Masamitsu Yamaguchi•
Kaeko Kamei
Received: 16 October 2010 / Accepted: 28 January 2011 / Published online: 11 February 2011
Ó Springer Science+Business Media, LLC 2011
Abstract NADPH oxidase is the major source of
non-mitochondrial cellular reactive oxygen species (ROS), and
also is reported to be a major cause of various diseases
including atherosclerosis and hypertension In order to
screen a new curative reagent that can suppress NADPH
oxidase activity, we developed a Drosophila melanogaster
fly that would overexpress human Dual oxidase 2
(hDuox2), a member of the NADPH oxidase family, as a
screening model These flies (GMR-GAL4/UAS-hDuox2)
had a high generation of ROS in the posterior region of the
eye discs along with an easily recognizable rough-eye
phenotype, which is an ideal and convenient marker for
further screening steps Moreover, the hDuox2-induced
rough-eye phenotype can be rescued by feeding with a
culture medium containing mulberry leaves (MLs), which
reportedly have an antimetabolic effect Some
commer-cially available antioxidants such as quercetin-3-O-D
-glu-coside or quercetin-3-O-glucose-600-acetate, or the naringin
contained in MLs and other herbs, also have shown a
similar suppressing effect on the rough-eye phenotype Our
results suggest that flavonoid glycoside is absorbed from
the intestine and functions in the body of D melanogaster
as it does in mammalian models such as rats Thus, the
GMR-GAL4/UAS-hDuox2 fly line is a promising model
for the screening of novel drugs such as NADPH oxidase
inhibitors and/or antioxidants
Keywords NADPH oxidase Drosophila melanogaster Metabolic syndrome model Screening Antioxidant NADPH oxidase inhibitor
Introduction Reactive oxygen species (ROS) play a critical role in the oxidative stress that causes various diseases, including atherosclerosis and hypertension [1 3] In recent years, it has become clear that ROS are produced by specific enzymes, including NADPH oxidase, by transferring one electron from NADPH to molecular oxygen [4] The NADPH oxidase family exists, with conserved functions,
in a wide variety of cell types from many organisms, including mammals, nematodes, fruit flies, green plants, fungi and slime molds [5] Inappropriate activation of NADPH enzymes may contribute to tissue dysfunction such as damage to the liver and pancreatic b-cells that leads to progression from the metabolic syndrome to Type
2 diabetes [6] Recently, Sugimoto et al discovered that inhibition of ROS generation via the suppression of NADPH oxidase expression in white adipose tissues and liver by the administration of mulberry leaves (MLs) ameliorated metabolic disorders in a mouse model [7] As well, several studies have revealed that MLs contain many antioxidants, particularly quercetin-3-(6-malonyglucoside), which is the most abundant flavonoid glycoside that has attenuated atherosclerotic lesion development in LDL receptor-deficient mice [8] These studies indicated that NADPH oxidase is an important target of antimetabolic drugs that may be present in MLs In order to screen new antimetabolic substances via the suppression of NADPH activity using MLs and other substances, the development
of a metabolic syndrome model is needed Drosophila
N T T Anh M Nishitani S Harada M Yamaguchi
K Kamei (&)
Department of Applied Biology, Kyoto Institute of Technology,
Matsugasaki Sakyo-ku, Kyoto 606-8585, Japan
e-mail: kame@kit.ac.jp
DOI 10.1007/s11010-011-0743-3
Trang 2melanogaster might be a viable candidate for studies of
metabolic syndrome because it shares most of the same
basic metabolic functions with vertebrates Many
analo-gous organ systems that control nutrient uptake, storage
and metabolism in humans are present in fruit flies [9] In
addition, their rapid growth, inexpensive costs of breeding,
and small genome size facilitates screening steps
Human dual oxidases (hDuox1 and hDuox2), members of
the NADPH oxidase family, are expressed in the thyroid, the
airway epithelial cells and in a wide variety of tissues such as
brain, cerebellum, mammary gland, muscle, nervous,
pla-centa, and testis The genes encoding for these two enzymes
are located on chromosome 15, and share 83% sequence
similarity [10] In the thyroid gland, Duox enzymes produce
hydrogen peroxide, which is essential for the oxidation step
of iodide in thyroid hormone production The important role
of Duox2 in this process was proven by the identification of
patients who suffer from hypothyroidism due to mutations in
the Duox2 gene [11] Besides, both in human and other
organisms, dual oxidases are novel H2O2sources that are
indispensable in host defense against infection and
extra-cellular matrix modification [12–14]
Recently, the gut antimicrobial activities of flies, in
which Drosophila NADPH oxidase (dDuox) had been
silenced, could be markedly ameliorated by the
introduc-tion of the hDuox2 gene, indicating that hDuox2 is
func-tional in Drosophila [13] Therefore, in order to construct
an in vivo screening system for new antimetabolic
sub-stances via the suppression of NADPH activity, we utilized
the GAL4-UAS expression system to provide ectopic
expression of hDuox2 in D melanogaster [15] GAL4 is a
yeast transcriptional activator that can activate the
tran-scription of any gene after its introduction into
Drosoph-ila—if the gene is preceded by a GAL4 upstream activating
sequence (UAS) that consists of five optimized GAL4
binding sites [16] In this study, a transgenic fly with a
GMR (glass multiple reporter)-GAL4 gene on chromosome
X was crossed with a fly with a UAS-hDuox2 gene on
chromosome 2 Since the GMR element causes high-level
expression of GAL4 protein in cells from the posterior to
the morphogenetic furrow, we were able to obtain an
expression of hDuox2 in the posterior area of the eye discs
This system has the advantage of allowing evaluation of
hDuox2 activity by simple inspection of the eye phenotype
Furthermore, we evaluated the use of flies that
overex-press hDuox2 as models for the screening of antimetabolic
substances The evaluation was carried out by feeding MLs
and their flavonoids to transgenic Drosophila Mulberry
leaves are reported to have an antimetabolic effect due to
their content of flavonoids with antioxidant activities The
effectiveness of MLs and these substances was evaluated
based on their ability to suppress the hDuox2-induced
rough-eye phenotype
Materials and methods Materials
Quercetin was purchased from Nacalai Tesque (Kyoto, Japan) The rutin, epigallocatechin gallate, and naringin were acquired from Wako Pure Chemical (Osaka, Japan) Quercetin-3-O-D-glucoside, quercetin-3-O-glucose-600 -ace-tate, and kaempherol all were acquired from Extrasynthese (France)
Mulberry leaves The mulberry trees were cultured in a mulberry plantation
at the Center for Bioresource Field Science, Kyoto Institute
of Technology, using a standard Japanese method The
‘‘Shin-Ichinose’’ mulberry (Morus alba L.) race was used Mulberry leaves were harvested and immediately dried by air flush at 180°C for 7 s The average diameter of the dried powder was 20 lm
Fly stocks Fly stocks were maintained at 25°C The UAS-hDuox2 fly line that carries the human Duox2 gene on second chromo-some was kindly provided by Dr Won-Jae Lee, of Ewha Woman’s University, South Korea [13] The GMR-GAL4/ UAS-DREF line was used as a control strain [17] The transgenic fly line carrying GMR-GAL4 on the X chromo-some (strain number 16) has been described previously [17] Establishment of transgenic flies
Virgin females were used for every crossing step to insure purity
To construct GMR-GAL4/Y; UAS-hDuox2/?, virgin female GMR-GAL4 flies containing two copies of the GMR-GAL4 gene on the X chromosome were mated with male hDuox2 flies containing two copies of the UAS-hDuox2 gene on the second chromosome In the next generation, all male flies carry a single copy of both GMR-GAL4 and hDuox2 (GMR-GMR-GAL4/Y; UAS-hDuox2/?)
To construct the GMR-GAL4; UAS-hDuox2 line, virgin female GMR-GAL4 flies containing two copies of the GMR-GAL4 gene on the X chromosome and a second-balancer chromosome CyO (GMR-GAL4; Sp/CyO) were crossed with male UAS-hDuox2 flies containing two cop-ies of the UAS-hDuox2 gene on the second chromosome The second chromosome balancer CyO suppresses recombination with their homologs, carries dominant markers (curly-wing phenotype), and causes a lethal effect when it is homozygous In the next generation, all male
Trang 3flies surely contain one copy of GMR-GAL4 on the X
chromosome These male flies were further selected by
curly-wing phenotype to get one copy of UAS-hDuox2 and
one second chromosome balancer, CyO (GMR-GAL4/Y;
UAS-hDuox2/CyO) These flies crossed again with female
GMR-GAL4; Sp/CyO Subsequently, we could select
female GAL4; UAS-hDuox2/CyO and male
GMR-GAL4/Y; UAS-hDuox2/CyO These two fly lines were
mated together to finally establish fly lines containing two
copies of GMR-GAL4 on the X chromosome and two
copies of UAS-hDuox2 on the second chromosome
(GMR-GAL4; UAS-hDuox2)
Culture medium
The medium buffer contained 1% yeast extract and 0.5%
agar in phosphate-buffered saline (PBS)
ML-supple-mented culture medium for feeding was prepared by
mix-ing 750 ll medium buffer with 0.15 g instant medium
Formula 4–24Ò (Carolina Biological Supply Co., USA)
and 10, 20, or 30% (w/w) of MLs Antioxidant stocks were
prepared by being dissolved in 99% ethanol
Antioxidant-supplemented mediums were prepared by dissolving
anti-oxidant stocks into medium buffer before mixing with
instant medium Formula 4–24Òto obtain a final 3% (w/w)
concentration A blue coloring agent contained in the
instant medium facilitated recognition of whether the
lar-vae consumed the tested medium
Feeding method
Three males and three females of the control (GMR-GAL4;
UAS-DREF) or the hDuox2-overexpressing flies
(GMR-GAL4; UAS-hDuox2) were, respectively, mated to lay
eggs on culture mediums containing various concentrations
of mulberry powder or antioxidants for 1 day Newborn
larvae were continuously fed on the same mediums for
10 days at 25°C to reach the adult phase
Scanning electron microscopy
Adult flies were anesthetized, mounted on stages, and
observed using a VE-7800 (Keyence Inc., Osaka, Japan)
scanning electron microscope in high vacuum mode In
every experiment, the eye phenotype of at least five adult
flies of each line was simultaneously examined by scanning
electron microscopy, and these experiments were repeated
three times In the experiments, no significant variation in
eye phenotype among the five individuals was observed
The rough area of the eyes was circled as an index of the
effect on the rough-eye phenotype
In vivo ROS detection Eye discs from third instar larvae were dissected in PBS, and then incubated with 10 lM CM-H2DCFDA (5-(and-6)-carboxy-20, 70-dichlorodihydrofluorescein diacetate, acetyl ester) (Molecular Probes, Invitrogen) for 5 min After washing with PBS, samples were fixed in 1% para-formaldehyde for 5 min, washed three times with PBS, and then mounted in Vectashield Mounting Medium (Vector laboratories) Preparations were examined under a fluo-rescence BX-50 microscope (Olympus, Tokyo, Japan) equipped with a cooled CCD camera (ORCA-ER; Ham-amatsu Photonics K.K., Shizuoka, Japan)
Immunohistochemistry Third instar larvae were dissected in PBS and eye discs were fixed in 4% paraformaldehyde–PBS for 30 min at 25°C After washing with 0.3% Triton X-100 in PBS (PBST), samples were blocked for 30 min at 25°C with 0.15% PBST containing 1% bovine serum albumin Sam-ples were then incubated with rabbit anti-hDuox2 antibody (Abcam) at a 1:100 dilution and 4°C for 16 h After extensive washing with PBST, the samples were incubated with goat anti-rabbit IgG Alexa FluorTM 488 (Molecular Probes, Invitrogen) at a 1:400 dilution for 2 h at 25°C, further washed with PBST and PBS, then mounted in Vectashield Mounting Medium (Vector laboratories) Preparations were examined under a fluorescence BX-50 microscope (Olympus, Tokyo, Japan) equipped with a cooled CCD camera (ORCA-ER; Hamamatsu Photonics K.K., Shizuoka, Japan)
Results Overexpression of human Duox2 disrupted normal eye development
The hDuox2 transgenic flies were successfully used to rescue the defect in antimicrobial infection activity in the Drosophila NADPH oxidase (dDuox) RNAi strain, indi-cating that hDuox2 is functional in Drosophila [13] In this study, we utilized the GMR-GAL4 driver strain to provide ectopic expression of hDuox2 from the posterior to the morphogenetic furrow of a Drosophila eye [17] Under scanning electron microscope, the eyes of flies carrying a single copy of GMR-GAL4 (GMR-GAL4/Y; ?) appeared
as a normal phenotype (Fig 1a), while those of flies car-rying a single copy of both GMR-GAL4 and hDuox2 (GMR-GAL4/Y; UAS-hDuox2/?) had a mild rough-eye phenotype (Fig.1b) When the copy number of both GMR-GAL4 and hDuox2 were increased to two (GMR-GMR-GAL4;
Trang 4UAS-hDuox2, Fig.1d), the compound eyes of these flies
had many fused ommatidia and became more severely
rough than those of flies containing two copies of
GMR-GAL4 only (GMR-GMR-GAL4; ?, Fig.1c)
To further confirm the overexpression of hDuox2 in the
compound eyes, we performed immunostaining of eye
imaginal discs from third instar larvae using anti-hDuox2
polyclonal antibody Only in GMR-GAL4; UAS-hDuox2,
an ectopic hDuox2 signal from the posterior region to the
morphogenetic furrow in eye discs was detected (Fig.2b)
The GMR-GAL4 line that was used as the negative control
showed no signal (Fig.2a) Immunostaining of eye
imag-inal discs of both GMR-GAL4; ? and GMR-GAL4;
UAS-hDuox2 using only goat anti-rabbit IgG Alexa FluorTM488
were also carried out to further exclude background effects
(Fig.2c, d, respectively)
Overexpression of hDuox2 increased ROS generation
Overexpression of hDuox2 can lead to an increased
gen-eration of ROS To detect intracellular ROS in
hDuox2-overexpressing flies, we used the non-fluorescent substrate
CM-H2DCFDA, which can be oxidized by ROS to become
an intracellular green fluorescent product With no
treat-ment with substrate CM-H2DCFDA, no detectable signal
was observed in eye imaginal discs for either control flies
GMR-GAL4 or hDuox2-overexpressing flies, as shown in Fig.3a and b, respectively In contrast, upon treatment with substrate CM-H2DCFDA, hDuox2-overexpressing flies showed stronger fluorescent signals from the posterior region to the morphogenetic furrow in eye discs (Fig.3d)
as compared with that of the control GMR-GAL4 fly line (Fig.3c) This indicates that ROS generation was induced
by the overexpression of hDuox2 in eye discs
Mulberry leaves could suppress the hDuox2-induced rough-eye phenotype
In previous research, metabolic disorder was ameliorated in obese db/db mice fed MLs, at least partially by the sup-pression of oxidative stress by both antioxidative flavonoid scavenging of ROS and by the inhibition of NADPH oxi-dase expression [7,18] Therefore, in this study, to evaluate the use of GMR-GAL4/UAS-hDuox2 as a metabolic syn-drome model, we fed these flies 10, 20, and 30% (w/w) MLs To exclude effects of MLs other than on hDuox2 activity, flies overexpressing DREF, a transcription factor,
by GMR-GAL4 with approximate rough-eye phenotype were used as a control [17] As shown in Fig.4a–d, MLs had no effect on the eye phenotype of DREF-over-expressing flies, indicating that MLs exerted no effect on GAL4 expression However, the rough-eye phenotype of
Fig 1 Adult compound eyes observed by scanning electron
micros-copy a The eyes of flies carrying a single copy of GMR-GAL4
(GMR-GAL4/Y; ?) appeared as a normal phenotype; b flies carrying
a single copy of both GMR-GAL4 and hDuox2 (GMR-GAL4/Y;
UAS-hDuox2/?) showed a mild rough-eye phenotype; c flies
carrying two copies of GMR-GAL4 (GMR-GAL4; ?) had many fused ommatidia; d flies carrying two copies of both GMR-GAL4 and hDuox2 (GMR-GAL4; UAS-hDuox2) showed a more severe rough-eyes phenotype Magnification is 2009 for left panels and 7009 for right panels Flies were reared at 25°C
Trang 5GMR-GAL4/UAS-hDuox2 was suppressed effectively
when feeding with a culture medium containing 20% MLs
(Fig.4g) The extent of suppression was more increased as
the concentration of MLs was increased up to 30% in the
medium (Fig.4h) The suppressive effects can be realized
by comparing the rough areas of eyes marked by dot circles
in Fig.4 These results suggest that the
GMR-GAL4/UAS-hDuox2 fly is a potential model for the screening of new
antimetabolic substances
Antioxidants could suppress the hDuox2-induced
rough-eye phenotype
Some phenolic compounds that have antioxidative
activi-ties, including quercetin, rutin, kaempherol,
epigallocate-chin gallate, quercetin-3-O-D-glucoside,
quercetin-3-O-glucose-600-acetate, and naringin, were tested for the ability
to suppress the hDuox2-induced rough-eye phenotype The oral administration of quercetin, rutin, kaempherol, and epigallocatechin gallate did not affect the rough-eye phenotype of hDuox2-overexpressing flies (Fig.5b–e) However, flavonoid glycosides such as quercetin-3-O-D -glucoside, quercetin-3-O-glucose-600-acetate, and naringin could significantly suppress the hDuox2-induced rough-eye phenotype (Fig.5f–h)
Discussion
In this study, we crossed UAS-hDuox2 with GMR-GAL4 driver to establish a fly line overexpressing hDuox2 The eyes of these flies appeared severely rough compared with
Fig 2 Immunostaining of eye imaginal discs with anti-hDuox2
antibody Eye imaginal discs were reacted with rabbit anti-hDuox2
antibody followed by anti-rabbit IgG Alexa FlourTM 488 antibody
(a, b) a Immunostaining of eye discs of GMR-GAL4; ? showed no
detectable signal; b notably, in GMR-GAL4; UAS-hDuox2, an
ectopic hDuox2 signal from the posterior region to the morphogenetic
furrow in eye discs was detected Immunostaining of eye imaginal discs of both GMR-GAL4; ? (c) and GMR-GAL4; UAS-hDuox2 (d) with only the anti-rabbit IgG Alexa FlourTM488 antibody showed
no detectable signal Flies were reared at 28°C The arrowheads indicate morphogenetic furrows in the eye discs
Trang 6Fig 3 Detection of ROS in eye
imaginal discs of third instar
larvae Without substrate
CM-H2DCFDA, there were no
detectable signals in either
GAL4; ? (a) or
GMR-GAL4; UAS-hDuox2 flies (b).
However, by incubation with
substrate CM-H2DCFDA, a
weak fluorescent signal in
control flies (GMR-GAL4; ?)
(c) was detected Remarkably, a
strong fluorescent signal was
detected in
hDuox2-overexpressing flies
(GMR-GAL4; UAS-hDuox2) (d) Flies
were reared at 25°C The
posterior of the discs are on the
right The arrowheads indicate
morphogenetic furrows in the
eye discs
Fig 4 Effect of MLs on the hDuox2-induced rough-eye phenotype.
Scanning electron micrographs of adult compound eyes at 2009
magnification of GMR-GAL4; UAS-DREF (a–d) and GMR-GAL4;
UAS-hDuox2 (e–h) GMR-GAL4; UAS-DREF flies fed culture
mediums containing 10% (b), 20% (c), or 30% (d) MLs showed no
effect on the rough-eye phenotype as compared to flies not fed MLs
(a) However, compared to flies not fed MLs (e), the rough-eye phenotype of hDuox2-overexpressing flies was suppressed when fed culture mediums containing 10% (f), 20% (g), or 30% (h) MLs The suppressive effects can be realized by comparing rough areas of the eyes marked by dot circles Flies were reared at 25°C
Trang 7GMR-GAL4 flies By immunohistochemical analysis with
anti-hDuox2 antibody, we proved that these flies expressed
an abundant amount of hDuox2 protein in the posterior
region to the morphogenetic furrow in eye discs
Subse-quently, the production of ROS in this region also
increased indicating that the hDuox2 enzyme functioned in
these flies These results showed that
GMR-GAL4/UAS-hDuox2 flies induced high oxidative stress in the posterior
region of the eye discs that led to an easily recognizable
rough-eye phenotype in adults
We continued to feed this fly line with MLs and several
antioxidative flavonoids to evaluate the ability of the line to
be used as a model for the screening of antimetabolic
substances The results show that, with increasing
con-centration of MLs in the culture medium, the suppressive
effect of MLs became more prominent Similar results
were obtained when culture medium containing 3% (w/w)
flavonoid glycoside, quercetin-3-O-D-glucoside,
quercetin-3-O-glucose-600-acetate, or naringin was fed to the fly
However, it is noteworthy that flavonoids without a
glycosidic ring, quercetin, rutin, kaempherol, and epigal-locatechin gallate exerted no effect on the hDuox2-induced rough-eye phenotype This differential effect could be explained by the differences between flavonoids and their glycosides in intestinal absorption and bioavailability due
to chemical structures and water solubility raised from attached glycoside rings that had been described in previ-ous human and rat studies [19–21] Our results might indicate that the absorption of antioxidants in the intestine
of D melanogaster is similar to mammalian models such
as rats
In this study, we used flavonoids as ROS scavengers to evaluate the fly as a screening tool, and, as well, the transgenic fly can be used to screen for NADPH oxidase inhibitors However, our screening system cannot distin-guish between ROS scavengers and NADPH oxidase inhibitors Therefore, further ROS scavenging assay using chemicals and/or kinetic assay of ROS generation by NADPH oxidase should be carried out Despite this limi-tation, Drosophila has many advantages as a screening
Fig 5 Effect of flavonoids on the hDuox2-induced rough-eye
phenotype Scanning electron micrographs of adult compound eyes
at 2009 magnification of GMR-GAL4; UAS-hDuox2 Untreated
GMR-GAL4; UAS-hDuox2 flies showed a severe rough-eye
pheno-type (a) Treatment with 3% (w/w) quercetin (b), rutin (c),
kaempherol (d), and epigallocatechin gallate (e) showed no detectable changes in compound eyes However, treatment with 3% (w/w) quercetin-3-O- D -glucoside (f), quercetin-3-O-glucose-6 00 -acetate (g), and naringin (h) strongly suppressed the hDuox2-induced rough-eye phenotype Flies were reared at 25°C
Trang 8system for new drugs Drosophila is easier to
inexpen-sively rear in the laboratory, has a shorter generation time,
and produces much more progenies when compared with
mice or rats Therefore, the use of our model is
recom-mended for the first simultaneous screenings of a great
number of samples In addition, because of the similar
basic metabolic functions and analogous organs between
Drosophila and vertebrates [9], the
GMR-GAL4/UAS-hDuox2 fly line constructed in our study is a promising
model for the screening of bioavailable novel drugs
Acknowledgments We are grateful to Dr Won-Jae Lee, Ewha
Woman’s University, South Korea for the UAS-hDuox2 stock.
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