Minibrain/Dyrk1a Regulates Food Intake through the
Sir2-FOXO-sNPF/NPY Pathway in Drosophila and
Seung-Hyun Hong1., Kyu-Sun Lee1,2., Su-Jin Kwak1, Ae-Kyeong Kim1, Hua Bai3, Min-Su Jung4,
O-Yu Kwon5, Woo-Joo Song4, Marc Tatar3, Kweon Yu1,2*
1Aging Research Centre, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea, 2Functional Genomics Program, University of Science and
Technology (UST), Daejeon, Korea, 3Department of Ecology and Evolutionary Biology, Brown University, Providence, Rhode Island, United States of America, 4Institute for
Brain Science and Technology, FIRST Research Group, Inje University, Busan, Korea, 5Department of Anatomy, School of Medicine, Chungnam National University,
Feeding behavior is one of the most essential activities in animals, which is tightly regulated by neuroendocrine factors.
Drosophila melanogaster short neuropeptide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY)
regulate food intake. Understanding the molecular mechanism of sNPF and NPY signaling is critical to elucidate feeding
regulation. Here, we found that minibrain (mnb) and the mammalian ortholog Dyrk1a target genes of sNPF and NPY
signaling and regulate food intake in Drosophila melanogaster and mice. In Drosophila melanogaster neuronal cells and
mouse hypothalamic cells, sNPF and NPY modulated the mnb and Dyrk1a expression through the PKA-CREB pathway.
Increased Dyrk1a activated Sirt1 to regulate the deacetylation of FOXO, which potentiated FOXO-induced sNPF/NPY
expression and in turn promoted food intake. Conversely, AKT-mediated insulin signaling suppressed FOXO-mediated sNPF/
NPY expression, which resulted in decreasing food intake. Furthermore, human Dyrk1a transgenic mice exhibited decreased
FOXO acetylation and increased NPY expression in the hypothalamus, as well as increased food intake. Our findings
demonstrate that Mnb/Dyrk1a regulates food intake through the evolutionary conserved Sir2-FOXO-sNPF/NPY pathway in
Drosophila melanogaster and mammals.
Citation: Hong S-H, Lee K-S, Kwak S-J, Kim A-K, Bai H, et al. (2012) Minibrain/Dyrk1a Regulates Food Intake through the Sir2-FOXO-sNPF/NPY Pathway in
Drosophila and Mammals. PLoS Genet 8(8): e1002857. doi:10.1371/journal.pgen.1002857
Editor: Pankaj Kapahi, Buck Institute, United States of America
Received January 18, 2012; Accepted June 7, 2012; Published August 2, 2012
Copyright: ? 2012 Hong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the grants from the Research Foundation of Korea (2009-0080870 and 2009-0073680), Korea Healthcare Technology R&D
Project (A092004), and KRIBB Research Initiative Program. Work from the laboratory of MT was supported by funds from the National Institutes of Health (USA)
(R01 AG024360). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Neuropeptides regulate a wide range of physiological processes
in animals. In mammals, NPY is widely distributed in the brain
and involved in various physiological functions including food
intake. In the mammalian brain, the hypothalamus is the center
for controlling food intake. The hypothalamic injection of NPY in
the rat brain induces hyperphagia and obesity. In the hypothal-
amus, the arcuate nucleus (ARC) that contains orexigenic NPY
and AgRP expressing neurons and anorexigenic POMC neurons
senses hormonal levels of insulin and leptin and regulates food
intake . In Drosophila, sNPF, a functional homolog of NPY
produced in sNPFnergic neurons of the fly brain, regulates food
intake and growth . Recently, we reported that sNPF and sNPF
receptor (sNPFR1) regulate body growth through evolutionary
conserved ERK-mediated insulin signaling in Drosophila and rat
insulinoma cells .
Drosophila Minibrain (Mnb) and its mammalian ortholog Dual
specificity tyrosine-phosphorylation-regulated kinase 1a (Dyrk1a)
are highly expressed in the neural tissues [4,5,6]. The Dyrk1a gene
has been implicated in Down Syndrome (DS) [5,7] and the
expression level of Dyrk1a is increased in DS patients and Ts65Dn
mice, a mouse model of Down syndrome [4,8]. Mutations of
mnb and Dyrk1a in Drosophila and mammals show neural phe-
notypes like defects in neuroblasts proliferation and brain
development [6,9]. Human patients with truncated mutations in
the Dyrk1a gene also show microcephaly [10,11]. To date,
however, the effects of mnb and Dyrk1a upon food intake have
not been described.
FoxO1 modulates food intake by regulation of orexigenic Argp
and anorexigenic Pomc genes in the hypothalamus of mice. In the
ARC of hypothalamic neurons, FoxO1 is localized in the nuclei
during fasting and in the cytoplasm by feeding . Sirtuin1
(Sirt1), the mammalian ortholog of Drosophila Silent information
regulator 2 (Sir2), in the ARC also regulates food intake . The
Sirt1 protein level increases during fasting. Sirt1 inhibition by the
hypothalamic knock-out in the AgRP neurons decreases food
intake . In N43 hypothalamic cells, pharmacological inhibition
of Sirt1 increases anorexigenic POMC expression but co-treatment
with Sirt1 inhibitor and FoxO1 siRNA does not , suggesting
PLoS Genetics | www.plosgenetics.org1August 2012 | Volume 8 | Issue 8 | e1002857
that Sirt1-mediated FoxO1 deactylation is involved in the
regulation of POMC mRNA and food intake.
In this study, we identified mnb and Dyrk1a as target genes of
sNPF and NPY signaling, respectively, and describe a molecular
mechanism of how Mnb and Dyrk1a regulate food intake in
Drosophila and mice.
sNPF Targets mnb to Regulate Food Intake in Drosophila
To find genes affected by sNPF signaling, we performed a DNA
microarray analysis using the Affymetrix Drosophila Genome 2.0
Array GeneChip with mRNA extracted from Drosophila neuronal
BG2-c6 cells treated with sNPF peptide. Among the 159 genes
with at least a two-fold change, mRNA of mnb increased 34-fold
compared to the control (Table S1). To test whether the
expression of mnb is dependent on sNPF signaling in vivo, we
examined the expression levels of mnb in sNPF and sNPFR1
mutants. When sNPF was overexpressed in sNPFnergic neurons
with the sNPF-Gal4 driver  (sNPF.sNPF, sNPF.2XsNPF), mnb
mRNA increased 4 to 5-fold compared with the sNPF-Gal4.
mRNA of mnb decreased by less than half when sNPF was inhibited
(sNPF.sNPF-Ri) or by an sNPF mutant (sNPFc00448) (Figure 1A and
Figure S1A). When sNPFR1 was overexpressed via a sNPFR1-
Gal4 driver (Figure S2) (sNPFR1.sNPFR1), mnb mRNA was
increased 3-fold compared with the sNPFR1-Gal4 control.
When sNPFR1 was inhibited (sNPFR1.sNPFR1-Ri) or suppressed
(sNPFR1.sNPFR1-DN), mnb mRNA was decreased by more than
50% (Figure 1A and Figure S1A). Like mnb mRNA, Mnb pro-
teins were also increased in sNPF or sNPFR1 overexpression
with the sNPF-Gal4 or sNPFR1-Gal4 driver, (sNPF.2XsNPF,
sNPFR1.sNPFR1) while reduced in an sNPF mutant (sNPFc00448)
or sNPFR1 inhibition (sNPFR1.sNPFR1-Ri) compared with the
sNPF-Gal4 or sNPFR1-Gal4 control (Figure S3A). However,
the numbers of Mnb expression neurons (asterisks) are consis-
tent in the
(sNPFR1.sNPFR1), sNPFR1 inhibition (sNPFR1.sNPFR1-Ri),
and an sNPFc00448mutant (Figure S3B–S3F). These results indicate
that sNPF-sNPFR1 signaling regulates mnb mRNA and protein
expression in Drosophila.
To understand how Mnb protein may interact with the sNPFR1
receptor, we immunostained fly adult brains with Mnb and
sNPFR1 antibodies. The Mnb antibody produced strong and
weak staining in neuronal cells (Figure 1H, 1K, red) while the
sNPFR1 receptor antibody stained many neurons (Figure 1I, 1L,
green). Among the strongly stained Mnb neurons, cell bodies of
symmetrically localized median neurons behind the antennal lobe
show overlap with the antibody against sNPFR1 (Figure 1J, 1M,
arrows). At least ten neuronal cell bodies in median neurons were
stained with the both antibodies. This coincidence suggests that at
least part of Mnb function may be regulated by sNPF-sNPFR1
Since sNPF signaling regulates food intake and growth, and
growth is regulated by ERK-mediated insulin signaling , we
hypothesized that sNPF may regulate food intake through the mnb
gene. To assess this hypothesis, we used the CAFE´assay  to
measure feeding in mnb mutant adults. Because homozygous mnb
deletion mutants (mnbd305and mnbd419) generated by the imprecise
excisions of the P-element (Figure S4A) are lethal (as are
homozygous Dyrk1a mutant mice) we analyzed mnb overexpression
and hypomorphs generated by RNAi. mnb overexpression in
sNPFR1 neurons (sNPFR1.mnb) increased cumulative food
consumption compared to the sNPFR1-Gal4 control whereas
inhibiting mnb (sNPFR1.mnb-Ri) decreased cumulative food
consumption (Figure 1C), indicating that mnb expression in
sNPFR1 neurons can regulate food intake. Likewise, we measured
the amount of food intake by the amount of digested dye from
colored food. Overexpression of mnb in sNPFR1 neurons
(sNPFR1.mnb and sNPFR1.2Xmnb) increased consumed dye up
to 57% compared with that of the sNPFR1-Gal4 control whereas
mnb inhibition (sNPFR1.mnb-Ri) or the mnb mutant (mnbG1767)
decreased this intake by 30% (Figure 1B and Figure S1B). As
expected, levels of mnb mRNA and protein were markedly reduced
by mnb inhibition and by the mnbG1767mutant relative to the
sNPFR1-Gal4 and w- controls (Figure S4B, S4C). Since sNPFR1
signaling in the insulin producing cells (IPCs) regulates body
growth through insulin signaling , we examined the effect of
mnb in IPCs upon food intake. However, food intake was not
affected by mnb overexpression in IPCs driven via Dilp2-Gal4
(Dilp2.mnb and Dilp2.2Xmnb) or by mnb inhibition in IPCs
(Dilp2.mnb-Ri) (Figure 1B). Expression of mnb in sNPFR1 neurons
but not in IPCs (Figure 1D–1G) is sufficient to regulate food
To determine the consequences of mnb control upon food intake
we measured the body weight of young adults from mutant and
control. Overexpression of mnb in sNPFR1 neurons (sNPFR1.mnb)
increased body weight relative to that of sNPFR1-Gal4 controls,
similar to the effect seen when sNPFR1 is overexpressed
(sNPFR1.sNPFR1). On the contrary, body weight is decreased
when mnb is repressed in sNPFR1 neurons (sNPFR1.mnb-Ri) and
mnbG1767mutant (Figure S4D). The amounts of food intake in the
mutants were similar when they were normalized to body mass or
to the number of flies (Figure S4E).
Since mnb is involved in neural development [6,9], we restricted
mnb expression in the adult stage using the tub-GAL80ts inducible
system  and tested food intake. mnb overexpression (sNPFR1-
Gal4+tubGal80ts.mnb, sNPFR1-Gal4+tubGal80ts.2Xmnb) and mnb
inhibition (sNPFR1-Gal4+tubGal80ts.mnb-Ri) flies were cultured in
the 22uC permissive temperature until adulthood to suppress
sNPFR1-Gal4 expression by the tubGal80ts. Then, these adult flies
were shifted to the 30uC restrictive temperature in which the
tubGal80ts cannot suppress sNPFR1-Gal4. In the permissive
Feeding behavior is one of the most essential activities in
animals. Abnormal feeding behaviors cause metabolic
syndromes including obesity and diabetes. Neuropeptides
regulate feeding behavior in animals from nematode to
human. Here, we presented molecular genetic evidences
of how neuropeptides regulate food intake using fruit fly
and mouse model systems. Drosophila short neuropetide F
(sNPF) and the mammalian functional homolog neuro-
peptide Y (NPY) are produced from neurons in the brain of
fruit fly and mouse, respectively. These neuropeptides
turned on the minibrain, in mammals also called Dyrk1a, a
target gene through the PKA-CREB pathway. Then, this
Mnb/Dyrk1a enzyme activated Sir2/Sirt1 enzyme, which
activated FOXO transcriptional factor, turning on the
expression of a sNPF/NPY target gene. The increased
sNPF/NPY increased food intake in fruit flies and mice. On
the contrary, increased food intake induced insulin and
activated insulin signaling. When insulin signaling is
activated, FOXO transcriptional factor inhibited expression
of a sNPF/NPY target gene. The inhibited sNPF/NPY
reduced food intake. These findings indicate that FOXO
transcription factor acts as a gatekeeper for fasting–
feeding transition by regulating sNPF/NPY expression in
Drosophila and mammals.
Mnb/Dyrk1a Regulates Food Intake
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Figure 1. Expression and distribution of Drosophila mnb in adults in relation to sNPF, sNPFR1, and feeding. (A) mnb mRNA prepared from
fly heads was measured by RT-qPCR. mnb mRNA was increased relative to sNPF-Gal4 and sNPFR1-Gal4 controls when sNPF and sNPFR1 was
overexpressed in sNPFnergic neurons (sNPF.sNPF, sNPF.2XsNPF) and in sNPFR1 neurons (sNPFR1.sNPFR1). mnb mRNA was decreased when sNPF
and sNPFR1 were inhibited (sNPF.sNPF-Ri, sNPFc00448, sNPFR1.sNPFR1-Ri, sNPFR1.sNPFR1-DN). (B) Food consumption measured by the colormetric
assay. Relative to sNPFR1-Gal4 control, mnb overexpression in sNPFR1 neurons (sNPFR1.mnb, sNPFR1.2Xmnb) increased feeding whereas mnb
suppression (sNPFR1.mnb-Ri, mnbG1767) decreased feeding. Overexpression or inhibition of mnb in the insulin producing cells with the Dilp2-Gal4
driver (Dilp2.mnb, Dilp2.2Xmnb, Dilp2.mnb-Ri) did not change the feeding. (C) Food consumption measured by CAFE´assay. Relative to the
sNPFR1-Gal4 (open triangle) control, sNPFR1.mnb (closed triangle) increased while sNPFR1.mnb-Ri (closed circle) decreased cumulative food
consumption. Data are presented as means 6 s.e.m. from three independent experiments. *P,0.05, **P,0.001 (One-way ANOVA analysis). (D-G)
Mnb/Dyrk1a Regulates Food Intake
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condition, the mnb overexpression and mnb inhibition flies did not
change the amount of food intake compared with the control flies
(sNPFR1-Gal4; tub-Gal80ts) (Figure S5A). However, in the restric-
tive condition, the mnb overexpression increased food intake
compared with the control and the mnb inhibition suppressed food
intake (Figure S5B). These results indicate that the food intake
phenotype of mnb mutants is not due to developmental effects.
sNPF-PKA-CREB-mnb Signaling in Drosophila Neuronal
To study how sNPFR1 regulates mnb expression, we treated
Drosophila central nervous system-derived BG2-c6 cells  with
synthetic sNPF peptide, which changed sNPF and sNPFR1
expression slightly (Figure S6A). Consistent with our initial
observations and with patterns in genetically manipulated flies,
sNPF treatment increased mnb mRNA more than 5-fold compared
to the control when measured by quantitative PCR (Figure 2A).
Then, we tested whether the induction of this mnb mRNA is
mediated by ERK, as we have previously observed for the
induction of Drosophila insulin like peptides (Dilps) by sNPF .
However, ERK inhibitor PD98059 treatment of the sNPF
peptide-treated cells did not suppress the mnb expression. On the
other hand, sNPFR1 is a G-protein coupled receptor (GPCR), and
the second messenger of GPCRs is cAMP or Ca++which
respectively activates PKA or PKC .Thus, we treated BG2-
c6 cells with the protein kinase A (PKA) inhibitor H89 or with
protein kinase C (PKC) inhibitor Chelerythrine Chloride (CC).
H89 decreased both basal and sNPF-induced mnb expression level
but the PKC inhibitor CC showed no effect (Figure 2A). sNPF
signaling appears to control mnb expression through PKA, not
through ERK or PKC. Consistent with this interpretation, BG2-c6
cells treated with sNPF showed increased levels of cAMP in a time-
dependent manner, peaking at 15 min (Figure S6B).
To find the Ga subunit of the sNPFR1 G-protein heterotrimer,
we examined Gas and Gai, both of which modulate cAMP .
When transfected into BG2-c6 cells Gas siRNA inhibited sNPF-
induced cAMP whereas transfection with Gai siRNA did not
(Figure 2C), suggesting that Gas is a Ga subunit of sNPFR1 that
can modulate the cAMP-PKA pathway in Drosophila neuronal
cells. Next, we examined the activation of the cAMP responding
element binding protein (CREB), which is a PKA down-stream
transcription factor . sNPF stimulated the phosphorylation of
CREB in control cells whereas Gas siRNA transfection suppressed
this sNPF dependent activation of CREB (Figure 2E). In addition,
Gas siRNA transfection completely blocked the induction of mnb
by sNPF, but Gai siRNA transfection did not (Figure 2G). These
data indicate that Gas is a key Ga subunit of the sNPFR1 G-
protein as it regulates mnb expression. Taken together, these
findings demonstrate that sNPF signaling effectively regulates mnb
expression through the Gas-cAMP-PKA-CREB pathway in
Drosophila neuronal cells.
NPY-PKA-CREB-DYRK1A Signaling in Mouse
Hypothalamic GT1-7 Cells
To compare the functional conservation of sNPF-sNPFR1-
PKA-CREB-mnb signaling with the signaling of mammalian NPY,
we conducted similar experiments with mouse GT1-7 hypotha-
lamic cells . NPY treatment increased Dyrk1a mRNA while the
PKA inhibitor H89 strongly suppressed NPY-induced Dyrk1a
expression (Figure 2B). NPY signaling activates Dyrk1a expression
through PKA, much like the PKA mediated mnb expression by
sNPF in fly neuronal cells. Next, we measured the cAMP level in
the NPY treated GT1-7 cells. As expected, cAMP level increased
time-dependently and peaked at 15 min (Figure S6C). Five NPY
receptors (NPYR1, 2, 4, 5, and 6) mediate the NPY signal .
Among them, NPYR1, 2, and 5 receptors are broadly expressed in
the mouse nervous system and mediate NPY-induced food intake
. We treated GT1-7 cells with chemical inhibitors against
these receptors: BIBO3304 for NPYR1, BIIE0246 for NPYR2,
and CGP71683 for NPYR5. The NPYR1 inhibitor BIBO3304
substantially decreased the NPY-induced cAMP level; little effect
was seen for the inhibitors of NPYR2 and NPYR5 (Figure 2D).
Thus, NPY appears to activate the cAMP-PKA pathway mainly
through NPYR1 in GT1-7 hypothalamic cells. Next, we measured
the CREB activation. As expected, inhibiting PKA or NPYR1
suppressed the NPY-induced activation of CREB (Figure 2F),
confirming that NPY signal is mediated through NPYR1-cAMP-
PKA-CREB. In addition, the NPYR1 inhibitor strongly sup-
pressed NPY-induced Dyrk1a expression; this was not seen with the
inhibitors of NPYR2 and NPYR5 (Figure 2H). Taken together,
these findings indicate that NPY signaling regulates Dyrk1a
expression mainly through the NPYR1-cAMP-PKA-CREB path-
way in mouse hypothalamic cells. Importantly, this signal
transduction pathway is conserved between fly neuronal cells
and mammalian hypothalamic cells.
Genetic Interactions among sNPFR1, Gas, PKA, CREB, and
mnb Genes, and CREB ChIP Analysis
To study genetic interactions among sNPFR1, Gas, PKA, CREB,
and mnb genes, we suppressed Gas, PKA, CREB, and mnb by RNAi
and Dominant Negative (DN) forms in neurons that simulta-
neously overexpressed sNPFR1. Each of these suppression
genotypes reduced the level of mnb mRNA compared with
sNPFR1-Gal4 and UAS controls (Figure 3A and Figure S7A). In
contrast to the strong induction of mnb produced by sNPFR1
overexpression alone (sNPFR1.sNPFR1), mnb induction was
inhibited in genotypes where sNPFR1 overexpression occurred
with each of the suppression constructs (sNPFR1.sNPFR1+
DN, sNPFR1.sNPFR1+mnb-Ri) (Figure 3B). In sNPFR1 neurons
of flies, as in isolated cells, Gas, PKA, and CREB may work
downstream of sNPFR1 to regulate mnb expression. The conse-
quences of these interactions are also seen in terms of food intake.
Gas, PKA, CREB, and mnb suppression mutant flies have reduced
food intake compared to those of the sNPFR1-Gal4 and UAS
controls (Figure 3C and Figures S1C, S7B). Furthermore,
increased food intake of sNPFR1 overexpression was suppressed
by co-inhibition of Gas, PKA, and CREB, respectively (Figure 3D).
These results suggest that the sNPFR1 may regulate food intake
through Gas, PKA, CREB, and mnb.
Based on promoter analysis of mnb genes from twelve Drosophila
species, we found a conserved cAMP responding element (CRE)
site (Figure S8). Interestingly, the promoters of human Dyrk1a and
mouse Dyrk1a genes contain CRE . To test whether CREB
binds to the promoter of the mnb gene, we performed the
chromatin immunoprecipitation (ChIP)-PCR analysis with the
CREB antibody in sNPF treated Drosophila neuronal BG2-c6 cells.
Neurons of the Drosophila adult brain expressing Mnb protein (green) do not overlap with insulin producing cells marked with Dilp2.DsRed (red). (H-
M) Mnb protein expression neurons (H, K, red) and sNPFR1 protein expression neurons (I, L, green) were overlapped in the median neurons (J, dot
box; M, arrows). Scale bars are 100 mm (D, H) and 50 mm (G, K).
Mnb/Dyrk1a Regulates Food Intake
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Figure 2. sNPF/NPY-sNPFR1/NPYR1-PKA-CREB-mnb/Dyrk1a signaling in Drosophila neuronal BG2-c6 cells and mouse hypothalamic
GT1-7 cells. (A) mnb mRNA in Drosophila neuronal BG2-c6 cells increased in response to treatment with sNPF peptide, but not when co-treated with
H89 PKA inhibitor. The ERK inhibitor PD98059 and PKC inhibitor CC did not suppress sNPF-induced mnb expression. (B) Dyrk1a mRNA in mouse
hypothalamic GT1-7 cells increased in response to treatment with NPY peptide, but not when co-treated with the PKA inhibitor H89. (C) In Drosophila
BG2-c6 cells, sNPF peptide induced cAMP, while transfection of cells with Gas siRNA but not Gai siRNA repressed this effect. (D) In mouse GT1-7 cells,
NPY peptide induced cAMP, while co-treatment with NPYR1 inhibitor BIBO3304 but not NPYR2 and NPYR5 inhibitors strongly decreased this effect.
(E) Western blot to detect activated CREB (pCREB) in Drosophila BG2-c6 cells. sNPF peptide increasd pCREB but not in cells transfected with Gas siRNA.
Mnb/Dyrk1a Regulates Food Intake
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CREB binding was enriched at the sNPF treated promoter region
of the mnb gene by 3-fold compared to the Act5C and sNPF non-
treated controls (Figure 3E). Together these in vivo and in vitro
findings indicate that sNPF-sNPFR1-Gas-PKA-CREB pathway
controls expression of the mnb target gene and regulates food
intake in Drosophila.
Positive Regulation of sNPF/NPY by the Mnb/Dyrk1a-Sir2-
A possible avenue through which Mnb regulates food intake
could involve Sirt1/Sir2. Notably, Dyrk1a kinase phosphorylates
Sirt1 in HEK293T cells , and activated Sirt1 deacetylates
FoxO1 to modulate the activity of this transcription factor in the
rat hypothalamus . Accordingly we determined if these
interactions were present and associated in mouse hypothalamic
GT1-7 cells. In cells transfected with Dyrk1a or treated with NPY,
phosphorylation of Sirt1 was increased as detected by immuno-
precipitation with Sirt1 antibody, followed by immunobloting with
phospho-threonine (pThr) antibody. Sirt1 phosphorylation was
reduced by Dyrk1a siRNA or Dyrk1a siRNA with NPY (Figure 4A).
In addition, FoxO1 acetylation was reduced in cells transfected by
Dyrk1a or treated with NPY, while FoxO1 acetylation was
increased by Dyrk1a siRNA, Dyrk1a siRNA with NPY, or Dyrk1a
transfection coupled with the Sirt1 inhibitor EX527 (Figure 4C).
Importantly, NPY mRNA itself was increased in cells transfected
with Dyrk1a or treated with NPY peptide, and NPY mRNA was
decreased by Dyrk1a siRNA, Dyrk1a siRNA with NPY, or Dyrk1a
overexpression in the presence of Sirt1 inhibitor (Figure 4B). In
mouse hypothalamic GT1-7 cells, Dyrk1a phosphorylates Sirt1
and this activated Sirt1 appears to deacetylate FoxO1 which in
turn positively regulates expression of NPY.
To study genetic interactions among mnb, Sir2, and dFOXO in an
animal model, we manipulated Sir2 and dFOXO in the Drosophila
mnb overexpression genotype. When mnb, Sir2, and dFOXO
were overexpressed in sNPFR1-Gal4 neurons (sNPFR1.mnb,
sNPFR1.Sir2, sNPFR1.dFOXO) (Figure S9A), sNPF mRNA and
food intake were increased compared to sNPFR1-Gal4 and UAS
controls (Figure 4D and 4E, Figure S7B and S7C). Conversely,
when mnb, Sir2, and dFOXO were inhibited in sNPFR1 expressing
neurons (sNPFR1.mnb-Ri, sNPFR1.Sir2-Ri, sNPFR1.dFOXO-Ri)
(Figure S9B), the expression levels of sNPF and food intake were
decreased or similar to those of sNPFR1-Gal4 and UAS controls
(Figure 4D and 4E, Figure S7B and S7C). Finally the level of sNPF
mRNA and food intake were reduced in adults when Sir2 or
dFOXO were inhibited in sNPFR1 neurons that overexpressed mnb
(sNPFR1.mnb+Sir2-Ri, sNPFR1.mnb+dFOXO-Ri) compared with
flies only overexpressing mnb (sNPFR1.mnb). These data suggest
that mnb may regulate sNPF expression and food intake through
Sir2 and dFOXO.
Since fasting can stimulate food intake, we tested whether an
acute period of food deprivation affected the expression of mnb and
sNPF of adult flies. Levels of mnb and sNPF mRNA increased 2-fold
after 12 h starvation (Figure 4F). We propose that dFOXO
contributes to this expression of sNPF in starved flies. We identified
a common dFOXO consensus binding site (RWWAACA) in the
sNPF promoter from twelve Drosophila species (Figure S10) and
performed a chromatin immunoprecipitation (ChIP)-tiled gene
array analysis with dFOXO antibody in fed and starved adult flies.
dFOXO binding was enriched at the promoter region of sNPF
gene more than 3-fold in the starved flies compared to the Act5c
and fed controls (Figure 4G). These results suggest that the
dFOXO transcriptional factor regulates sNPF mRNA expression
by direct binding to its promoter in Drosophila, as seen for FoxO1
regulation of NPY expression in mice .
Overall, these results from mouse hypothalamic GT1-7 cells
and Drosophila indicate that the Mnb/Dyrk1a-Sir2-FOXO path-
way positively regulates sNPF/NPY expression and food intake.
Negative Regulation of sNPF/NPY by Insulin Signaling
The positive feedback regulation of sNPF signaling we have
described to this point must occur alongside a system to negatively
regulate sNPF signaling. Insulin, one of several anorexigenic
hormones, inhibits food intake through AKT-mediated FoxO1
inactivation in the hypothalamus . In Drosophila, neuronal
overexpression of Dilps negatively regulates larval food intake .
To understand the inhibitory mechanism of insulin on food intake,
we analyzed the phosphorylation of FOXO and the expression of
NPY and sNPF. In the mouse hypothalamic GT1-7 cells, insulin
treatment increased FoxO1 phosphorylation and decreased NPY
mRNA while insulin combined with AKT inhibitor co-treatment
slightly decreased FoxO1 phosphorylation and increased NPY
expression (Figure 5A, 5B). Likewise, in fly neuronal BG2-c6 cells,
insulin with AKT inhibitor co-treatment increased sNPF mRNA
(Figure 5C). Thus, in both models AKT-mediated insulin signaling
increased FOXO phosphorylation and suppressed NPY or sNPF
We extended these results with analyses of Drosophila with insulin
and insulin receptor transgenes. Compared to Dilp2-Gal4 and
sNPFR1-Gal4 controls, sNPF mRNA and food intake were
decreased when Dilp2 was overexpressed in insulin producing
cells (Dilp2.Dilp2) and when insulin receptor (InR) was overexpressed
in sNPFR1 expressing neurons (sNPFR1.InRWT) (Figure 5D, 5E).
On the other hand, sNPF expression and food intake were
increased when InR was suppressed by a dominant negative
(Figure 5D, 5E). Fasting may contribute to sNPF expression and
the propensity for food intake because fasting in the adult reduces
the expression of several Dilps (Figure 5F), as previously observed
to occur in Drosophila larvae .
Taken together, the results from mouse and Drosophila neuronal
cells and from adult flies indicate that the insulin signaling
negatively regulates sNPF/NPY expression and food intake.
Dyrk1a TG Mice Regulate Food Intake through the FOXO-
To evaluate these Mnb/Dyrk1a-Sir2-FOXO-NPY interactions
and consequences in a mammalian animal model, we examined
FoxO1 acetylation and NPY expression in the hypothalamus of
transgenic mice containing the human Dyrk1a BAC clone (hDyrk1a
TG). As expected, in the Western blot, Dyrk1a in the hypothal-
amus was increased in hDyrk1a TG mice compared to controls
(Figure 6A). On the other hand, FoxO1 in the hypothalamus was
less acetylated in hDyrk1a TG mice compared to controls
(Figure 6C). Hypothalamic NPY mRNA as well as serum NPY
(F) Western blot to detect activated CREB (pCREB) in mouse GT1-7 cells. NPY peptide increased pCREB but not when cells are co-treated with PKA
inhibitor H89 or NPYR1 inhibitor BIBO3304. (G) In Drosophila BG2-c6 cells, sNPF peptide induced mnb mRNA, while transfection of cells with Gas siRNA
but not Gai siRNA repressed this effect. (H) In mouse GT1-7 cells, NPY peptide induced Dyrk1a mRNA, while co-treatment with NPYR1 inhibitor
BIBO3304 but not NPYR2 and NPYR5 inhibitors strongly decreased this effect. Data are presented as means 6 s.e.m. from three independent
experiments. *P,0.05 (One-way ANOVA analysis).
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levels were elevated in in hDyrk1a TG mice compared to controls
(Figure 6B). Thus, mammalian Dyrk1a appears to regulate FoxO1
acetylation and NPY expression in the mouse hypothalamus, as we
have observed for this system in Drosophila sNPFR1 neurons.
To assess whether mammalian Dyrk1a also regulates food intake
as seen for the homolog mnb of Drosophila, we monitored food
intake in seven-week-old hDyrk1a TG mice. Daily food consump-
tion was increased in the transgenic mice compared to littermate
controls (Figure 6D) and the average food intake of hDyrk1a
transgenic mice was elevated by 15% (Figure 6E). Correspond-
ingly, the hDyrk1a transgenic mice presented slightly increased
mass (Figure S11). Dyrk1a thus appears to regulate food intake
through the expression of NPY mediated by FOXO in a
molecular pathway that is evolutionarily conserved in Drosophila.
The production of sNPF and NPY in sNPFnergic and
hypothalamic neurons of flies and mammals respectively, is
increased during fasting. These neuropeptides are secreted to
produce paracrine and endocrine effects  but also feedback
upon their synthesizing neurons where they respectively induce
mnb and Dyrk1a gene expression through the PKA-CREB pathway
(Figure 6F). This Mnb/Dyrk1a kinase phosphorylates and
activates the Sir2/Sirt1 deacetylase, which in turn deacetylates
and activates the FOXO transcription factor. Among its many
potential targets, FOXO then increases sNPF/NPY mRNA
expression. Negative controls modulate the positive feedback of
sNPF/NPY. Feeding activates the insulin receptor-PI3K-AKT
pathway. FOXO becomes phosphorylated and transcriptionally
inactivated by translocation to the cytoplasm . In this state the
induction of sNPF/NPY by FOXO is decreased. Because sNPF
and NPY are orexogenic, their positive feedback during fasting
should reinforce the propensity for food intake whereas the
negative regulation of sNPF and NPY mRNA during feeding
condition would then contribute to satiety (Figure 6F).
FOXO family transcriptional factors are involved in metabo-
lism, longevity, and cell proliferation . FOXO is in part
regulated in these processes by post-transcriptional modifications
including phosphorylation and acetylation . In many model
systems, the ligand activated Insulin-PI3K-AKT pathway phos-
phorylates FOXO to inactivate this transcription factor by moving
it to the cytoplasm. The cytoplasmic localization of FOXO is
mediated by 14-3-3 chaperone proteins in Drosophila and mammals
[32,33]. FOXO may also be acetylated, as is FoxO1 of mice, by
the CREB-binding protein (CBP)/p300 acetylase and this inhibits
FOXO transcriptional function by suppressing its DNA-binding
affinity. Such FoxO1 acetylation can be reversed by SirT1 to help
activate the FoxO1 transcription factor . Here we describe for
Drosophila how dFOXO in sNPFR1 neurons regulates the
expression of sNPF and food intake (Figure 4D, 4E). This
mechanism parallels how hypothalamic FoxO1 regulates food
intake through its control of orexigenic NPY and Agrp in rodents
[12,27]. Post-transcriptional modification of FOXO is central to
these controls in both animals. sNPF and NPY expression is
increased when FOXO is deacetylated by Sir2/Sirt1, while sNPF
and NPY are decreased when FOXO is phosphorylated via the
Insulin-PI3K-AKT pathway. Post-transcriptional modifications of
FOXO proteins play a critical role for controlling food intake
through the sNPF and NPY expression in flies and rodents.
Mnb/Dyrk1a has been described to participate in olfactory
learning, circadian rhythm, and the development of the nervous
system and brain . Mnb and Dyrk1a proteins contain a nuclear
targeting signal sequence, a protein kinase domain, a PEST
domain, and a serine/threonine rich domain. The kinase domains
are evolutionary well-conserved from flies to humans . In
Down syndrome (DS), chromosome 21 trisomy gives patients three
copies of a critical region that includes the Mnb/Dyrk1a; trisomy of
this region is associated with anomalies of both the nervous and
endocrine systems . DS patients often show high Body Mass
Index due to the increased fat mass. Children with DS have
elevated serum leptin coupled with leptin resistance, both of which
contribute to the obesity risk common to DS patients [37,38]. We
now observe a novel function of Mnb/Dyrk1a that may underlay
this metabolic condition of DS patients. Mnb/Dyrk1a regulates
food intake in flies and mice. This is controlled by sNPF/NPY-
PKA-CREB up-stream signaling and thus produces down-stream
affects upon Sir2/Sirt1-FOXO-sNPF/NPY. Fasting not only
increases the expression of mnb, but also of sNPF, suggesting that
Mnb kinase activates a positive feedback loop where Sir2-dFOXO
induces sNPF gene expression. Notably, fasting increases Sirt1
deacetylase activity and localizes FoxO1 to the nucleus in the
orexogenic AgRP neurons of the mouse hypothalamus .
Increased dosage of Dyrk1a in DS patients may reinforce the
positive feedback by NPY and disrupt the balance between hunger
and satiety required to maintain a healthy body mass.
Insulin produced in the pancreas affects the hypothalamus to
regulate feeding in mammals . Insulin injected into the
intracerebroventrical of the hypothalamus reduces food intake
while inhibiting insulin receptors of the hypothalamic ARC
nucleus causes hyperphasia and obesity in rodent models
[39,40]. Here we saw a similar pattern for Drosophila where
overexpression of insulin-like peptide (Dilp2) at insulin producing
neurons decreased food intake while food intake was increased by
inhibiting the insulin receptor in sNPFR1 expressing neurons
(Figure 5E). Likewise, during fasting, serum insulin and leptin
levels are decreased in mammals , as is mRNA for insulin-like
peptides of Drosophila [29,41] (Figure 5F). Thus, the mechanism by
which insulin and insulin receptor signaling suppresses food intake
is conserved from fly to mammals in at least some important ways.
Previously, we reported how sNPF signaling regulates Dilp
expression through ERK in IPCs and controls growth in Drosophila
. Here, we show that sNPF signaling regulates mnb expression
through the PKA-CREB pathway in non-IPC neurons and
controls food intake (Figure 1B, 1D–1G). Since sNPF works
through the sNPFR1 receptor, sNPFR1 in IPCs and non-IPCs
neurons might transduce different signals and thereby modulate
different phenotypes. Four Dilps (Dilp1, 2, 3, and 5) are expressed
in the IPCs of the brain . Interestingly, levels of Dilp1 and 2
mRNA are reduced in the sNPF mutant, which has small body size
, but here we find only Dilp3 and 5 mRNA levels are reduced
upon 24 h fasting. Likewise, only Dilp5 is reduced when adult flies
are maintained on yeast-limited diets . In addition, Dilp1 and 2
Figure 3. Genetic interactions among sNPFR1, Gas, PKA, CREB, and mnb genes and CREB ChIP–PCR analysis. (A, C) mnb mRNA (A) and
feeding (C) were reduced by suppressing Gas, PKA, CREB, and mnb in sNPFR1 neurons relative to sNPFR1-Gal4 control. (B, D) mnb mRNA (B) and
feeding (D) were reduced by suppressing Gas, PKA, CREB, and mnb while overexpressing sNPFR1 in sNPFR1 neurons relative to overexpressing sNPFR1
alone (sNPFR1.sNPFR1). (E) In Drosophila BG2-c6 cells. CREB binding was enriched at the promoter region of the mnb gene by 3-fold compared to the
Act5c and sNPF peptide non-treated controls (ChIP-PCR). Data are presented as means 6 s.e.m. from three independent experiments. *P,0.05 (One-
way ANOVA analysis).
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null mutants show slight reduced body weights but Dilp3 and Dilp5
null mutants do not . These results suggest that Dilp1 and 2
behave like a mammalian insulin growth factor for size regulation
while Dilp3 and 5 act like a mammalian insulin for the regulation
of metabolism. However, in the long term starvation, Dilp2 and
Dilp5 mRNA levels are reduced and Dilp3 mRNA expression is
During fasting, sNPF but not sNPFR1 mRNA expression was
increased in samples prepared from fly heads (Figure 4F and
Figure S9C), which increases food intake. On the other hand, in
feeding, the high level of insulin signaling reduced sNPF but not
sNPFR1 mRNA expression and suppressed food intake (Figure 5D
and 5E, Figure S9D). Interestingly, in the antenna of starved flies,
sNPFR1 but not sNPF mRNA expression is increased and induces
presynaptic facilitation, which resulted in effective odor-driven
food search. However, high insulin signaling suppresses sNPFR1
mRNA expression and prevents presynaptic facilitation in DM1
glomerulus . These results indicate that starvation-mediated or
insulin signaling-mediated sNPF-sNPFR1 signaling plays a critical
role in Drosophila feeding behavior including food intake and food
search even though the fine tuning is different.
In this study, we present a molecular mechanism for how sNPF
and NPY regulate food intake in Drosophila and mice. We describe
a system of positive feedback regulation for sNPF and NPY
signaling that increases food intake and a mode of negative
regulation for sNPF and NPY by the insulin signaling that
suppresses food intake. Modifications of the FOXO protein play a
critical role for regulating sNPF and NPY expression, resulting in
the control of food intake.
Materials and Methods
Drosophila Culture and Stocks
Drosophila melanogaster were cultured and at 25uC on standard
cornmeal, yeast, sugar, agar diet. Wild-type Canton-S, w-, and UAS-
CREB-DN were obtained from the Bloomington stock center.
sNPFc00448was obtained from the Harvard stock center (Exelixis
stock collection). UAS-sNPF, UAS-2XsNPF, UAS-sNPF-Ri, UAS-
sNPFR1, UAS-sNPFR1-DN and sNPF-Gal4 transgenic flies were
described in our previous reports [2,3,16]. The sNPFR1-Gal4
construct was generated from a 2.5 kb genomic DNA fragment of
the 59-untranslated region of the sNPFR1 gene. The full-length
coding sequence of Drosophila minibrain-H (mnb, CG 42273) was
subcloned into the pUAS vector to generate the pUAS-mnb
construct. sNPFR1-Gal4 and UAS-mnb transgenic flies were
obtained by the P-element-mediated germ line transformation
. mnbG1767, an EP line for minibrain, was purchased from the
GenExel, Inc. (KAIST, Korea). UAS-sNPFR1-Ri (VDRC9379),
UAS-mnb-Ri (VDRC28628), UAS-Sir2-Ri (VDRC23201) and UAS-
FOXO-Ri (VDRC106097) were obtained from the Vienna
Drosophila RNAi Center (VDRC). Dilp2-Gal4, UAS-Gas-Ri, UAS-
PKA-DN (a dominant-negative form of PKA), UAS-Sir2 transgenic
flies were described previously [42,48,49,50,51]. To express these
UAS lines, UAS-Gal4 system was used . For minimizing the
genetic background effect among tested Drosophila lines, all stocks
were crossed with w- and then crossed to the second (w-; Bc, Elp/
CyO) or third (w-; D/TM3, Sb) chromosome balancers, respective-
ly. For making double mutants, w-; T(2:3) ApXa/CyO; TM3 was
crossed with the flies containing UAS-X transgene to produce w-;
UAS-X/CyO; +/TM3. Then, w-; +/CyO; UAS-Y/TM3 flies
generated by the similar way were crossed with w-; UAS-X/CyO;
+/TM3 to produce w-; UAS-X/CyO; UAS-Y/TM3.
Cell Culture, Stimulation, and Transfection
Drosophila BG2-c6 cells established by the single colony isolation
of primary cells derived from the third instar larval central nervous
system. This cell line synthesizes acetylcholine and expresses insect
neuron specific glycans and a RNA-binding protein Elav .
BG2-c6 cells purchased from the Drosophila Genomics Resource
Center (DGRC, Indiana University) were maintained at 26uC in
Schneider medium supplemented with 10% bovine calf serum.
Immortalized GT1-7 mouse hypothalamic neurons  were
cultured in 4.5 g/l glucose Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum, 2% of l-
glutamine, 100 mU/ml penicillin and 100 mg/ml streptomycin in
5% CO2at 37uC. The culture medium was changed every 2–3
days. Before peptide treatments, cells were starved for 8 h in the
serum-free medium containing 0.5% BSA and pretreated with a
chemical inhibitor or vehicle. PKA inhibitor H89 (10 mM,
Calbiochem), ERK-specific kinase MEK inhibitor PD98059
(10 mM, Calbiochem), PKC inhibitor Chelerythrine chloride
(1 mM, Sigma) were used. NPY1R inhibitor BIBO3304 (10 nM),
CGP71683 (1 mM) and Sirt1 inhibitor EX527 (10 mM) were
purchased from Tocris. Then, cells were treated with 100 nM
synthetic 19 amino acids sNPF2 or 100 nM human NPY 1–36
peptide (Sigma). For transfection, cells were cultured in the growth
medium without antibiotics and transfected with small interfering
RNA (siRNA) using Lipofectamine 2000 reagent (Invitrogen). Gas
and Gai siRNA constructs were designed by the BLOCK-iT RNAi
Designer and Dyrk1a siRNA was purchased from Invitrogen. The
sequences of siRNA are caggauauucuucggugccguguuu for Gas
and cggcgggauacuaucuaaauucgcu for Gai. The BLOCK-iT
Fluorescent Oligo, which is a fluorescent-labeled dsRNA oligomer,
was used as the non-targeting siRNA control. For the overexpres-
sion mouse Dyrk1a, a full-length mDyrk1a cDNA was cloned to
(50 nM),NPY5R inhibitor
Drosophila Food Intake Assay
We measured food intake of Drosophila in two ways. The CAFE
assay  was performed with 3 day-old adult male flies. Twelve
hours before the assay, ten flies were placed in the CAFE device
 containing 5% sucrose solution in calibrated glass micropi-
pettes (VWR, West Chester, PA). At time zero, the micropipettes
with 5% sucrose solution were replaced and the amount of liquid
consumed was measured every 6 h. A colorimetric food intake
Figure 4. Positive regulation of sNPF/NPY by the Mnb/Dyrk1a-Sir2-FOXO pathway. (A) Sirt1 phosphorylation was increased in mouse GT1-7
cells transfected with Dyrk1a or treated with NPY but reduced in cells transfected with Dyrk1a siRNA or Dyrk1a siRNA co-treated with NPY. (B) NPY
mRNA was increased in GT1-7 cells transfected with Dyrk1a or treated with NPY peptide, but reduced in cells transfected with Dyrk1a siRNA, Dyrk1a
siRNA co-treated with NPY, or Dyrk1a co-treated with Sirt1 inhibitor EX527. (C) FoxO1 acetylation was reduced in GT1-7 cells transfected with Dyrk1a
or treated with NPY peptide, but FoxO1 acetylation was increased in cells transfected with Dyrk1a siRNA, Dyrk1a siRNA co-treated with NPY, or Dyrk1a
co-treated with Sirt1 inhibitor EX527. (D, E) sNPF mRNA (D) and food intake (E) were reduced when Sir2 or dFOXO were inhibited while mnb was
overexpressed in sNPFR1 expressing neurons relative to levels observed for mnb overexpression alone (sNPFR1.mnb). (F) sNPF and mnb mRNA
increased in adults starved 12 h. (G) dFOXO binding to the promoter region of the sNPF gene in adult flies starved 12 h was elevated relative to the
Act5c and fed controls (ChIP-chip). Data are presented as means 6 s.e.m. from three independent experiments. *P,0.05, **P,0.001 (One-way ANOVA
Mnb/Dyrk1a Regulates Food Intake
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Figure 5. Negative regulation of sNPF/NPY by insulin signaling. (A, B) Insulin treatment increased FoxO1 phosphorylation and decreased NPY
expression in mouse hypothalamic GT1-7 cells while insulin with AKT inhibitor co-treatment slightly decreased FoxO1 phosphorylation and increased
NPY expression. (C) Insulin with AKT inhibitor co-treatment increased sNPF expression in fly neuronal BG2-c6 cells. (D, E) sNPF expression (D) and food
intake (E) were decreased in adult flies overexpressing Dilp2 in IPCs (Dilp2.Dilp2) and overexpressing insulin receptor (InR) in sNPFR1 neurons
(sNPFR1.InRWT), while sNPF mRNA and food intake increased when InR was suppressed in sNPFR1 neurons (sNPFR1.InRDN). (F) Fasting (at 24 h)
reduces Dilp3, and Dilp 5 mRNA but not Dilp2 mRNA. Data are presented as means 6 s.e.m. from three independent experiments. *P,0.05 (One-way
Mnb/Dyrk1a Regulates Food Intake
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Figure 6. hDyrk1a transgenic mice regulate food intake through the FOXO-NPY pathway. (A, C) In the hypothalamus of hDyrk1a
transgenic mice, Dyrk1a was increased and FoxO1 acetylation was reduced compared with those of the littermate control mice. (B) NPY mRNA from
hypothalamus and serum NPY were increased in hDyrk1a transgenic mice. (D) Daily food intake was increased in hDyrk1a transgenic mice compared
with the littermate controls. (E) Average food intake of hDyrk1a transgenic mice increased by 15%. Data are presented as means 6 s.e.m. from three
independent experiments. *P,0.05 (One-way ANOVA analysis). (F) The model of this study.
Mnb/Dyrk1a Regulates Food Intake
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assay was modified from published methods [2,53]. Since flies had
most fed color food in the crop during first 30 min and started to
excrete from 1 h (Figure S4C, S4D) , flies were starved in
PBS-containing vials for 2 h and fed for 30 min in vials containing
0.05% Bromophenol Blue dye and 10% sucrose in yeast paste.
Then, the flies were frozen, homogenized in PBS, and centrifuged
twice for 25 min each. The supernatant was measured at 625 nm.
Each experiment consisted of 20 flies, and the assay was repeated
at least three times.
Mouse Food Intake Assay
Dyrk1a transgenic mice expressed the human Dyrk1a BAC clone
in the C57BL/6 background . Seven weeks-old male Dyrk1a
TG and littermate control mice were used in the experiments
(n=7). The mice were housed individually in the standard plastic
rodent cages. They were maintained at 2262uC in a room with a
12-hour light/dark cycle and habituated to frequent handling.
Food intake and body weight were measured within 30 min before
the light turned on and off. Drinking water was available at all
times. Food intake data were corrected with body weight.
Animal care and all experiments were conducted according to
KRIBB Guidelines for the Care and Use of Laboratory Animals
and Inje University Council.
Twenty w- female flies were starved overnight and fed for 2 h
for the physiological synchronization. Then, starvations for the
experiment were started. The heads from starved flies were
collected for the Quantitative RT-PCR analysis. The experiments
were repeated three times.
Measurement of Drosophila Body Weight
Eggs laid by five female flies for 6 h at 25uC were cultured to
avoid over-crowding and lack of nutrition. For weight of individual
fly, over 50 three day-old adult male flies were measured with the
balancer (METTLER AJ100) and divided with the number of
flies. At least three experiments were performed in each assay.
Quantitative RT–PCR Analysis
Adult heads from 20 flies were collected for RNA preparation.
Total RNA was extracted using the easy-BLUE (TM) reagent
(iNtRON biotechnology). All RNA samples were treated with
RNase-free DNase (Promega). cDNA was synthesized using a
SuperScript III First-Strand Synthesis System (Invitrogen). For
quantitative RT-PCR analysis, ABI Prism 7900 Sequence
Detection System (Applied Biosystems) and SyberGreen PCR
Core reagents (Applied Biosystems) were used. mRNA levels were
expressed as the relative fold change against the normalized rp49
mRNA. The comparative cycle threshold (Ct) method (User
Bulletin 2, Applied Biosystems) was used to analyze the data. All
experiments were repeated at least three times. The statistical
significance was tested by Microsoft Excel-based application for
the student t-test statistical analysis. Primers used in the RT-PCR
analyses were listed in Table S2.
Generation of the Minibrain and sNPFR1 Antisera and
Immunostaining in the Adult Brain of Drosophila
Minibrain antiserum was generated by the immunization of
rabbits with the synthetic peptide (CQHRVRNWPTNGNQ)
corresponding to the N-terminal sequence (75–88) of the
Minibrain-H protein. Antiserum against sNPFR1 was generated
by the immunization of rat with the synthetic peptide (GEAI-
GAGGGAELGRRIN) corresponding to the C-terminal sequence
(585–600) of the sNPFR1 protein. For immunostaining, adult
brain from newly eclosed flies (3 day old) was dissected in PBS,
fixed in 4% paraformaldehyde, and blocked in 5% BSA and 5%
normal goat serum. Primary antibodies were incubated two days
at 4uC and secondary antibodies were incubated for 2 h at room
temperature. The tissues were mounted in the DABCO solution
(70% glycerol, 2.5% DABCO, Sigma, St Louis, MO) and
fluorescence images were acquired by FluoView confocal micro-
scope (Olympus). sNPF (1:200), sNPFR1 (1:200), and Minibrain
(1:200) primary antibodies, and anti-rat IgG Alexa 488, anti-rabbit
IgG Alexa 488, or Alexa 594 (1:200, Molecular Probes) and anti-
guinea pig Cy5 (1:200, Jackson ImmunoResearch) secondary
antibodies were used.
Western Blot Analysis
The cells were lysed by the Lysis buffer (Cell signaling)
containing NaF, PMSF and Na3VO4. Total cell lysates were
immunoprecipitated with Sirt1 antibody (Cell signaling) and
protein A-agarose (Pierce). The immunoprecipitates were washed
three times with Lysis buffer and solubilized in the SDS sample
buffer (63.5 mM Tris-HCl; pH 6.8, 2% w/v SDS, 10% glycerol,
50 mM DTT, 0.01% w/v bromphenol blue). Western blot
analyses were performed as described previously . Phospho-
CREB, phospho-Threonine, FoxO1 (1:1000, Cell signaling), Ac-
FKHR (1:1000, Santa Cruze), b-actin (1:3000, Abcam) primary
antibodies, and horseradish peroxidase-conjugated anti-rabbit IgG
(1:5000, Santa Cruze) and anti-mouse IgG secondary antibody
(1:5000, Sigma) were used.
Intracellular cAMP was measured with the cAMP Biotrak
Enzyme Immunoassay Kit (GE Healthcare) by the manufacturer’s
instruction. Briefly, samples were incubated with anti-cAMP
antibody, which was immobilized in the secondary antibody
coated micro-plates. Following enzyme substrate conversion, an
optical density was measured at 450 nm with microplate reader
(Fluostar Optima, BMG labtech). cAMP concentration was
expressed as the cAMP pM per mg of protein and converted to
the fold change relative to the basal control value.
ChIP-on-chip and ChIP–PCR Analysis
About 250 of 3-day-old W[DAH] female flies were collected
after 12 h starvation. Then, flies were homogenized and cross-
linked in 1X PBS containing 1% formaldehyde. The ChIP
protocol was performed as described in Teleman et al. .
Immunoprecipitation was performed using Dynal protean G
beads (Invitrogen) and anti-dFOXO antibody (a gift from Heather
Broihier). Purified DNA was amplified and labeled following
Affymetrix ChIP Assay Protocol. Drosophila Tiling 2.0R Array
was used to detect dFOXO binding enrichment. For ChIP-PCR
analysis, about 108of BG2-c6 cells were treated with sNPF2
peptide as described above. sNPF-treated and untreated cells were
cross-linked with 1% formaldehyde. After immunoprecipitation
with the CREB antibody (Cell signaling) and Protein A Sepharose
CL-4B (GE Healthcare), quantitative RT-PCR analysis was
performed using input DNA and immunoprecipitated DNA for
the CREB binding site in the mnb promoter region and the 3rd
axon of Actin5C.
Values in the paper are presented as means 6 s.e.m. Statistical
significant of all data were evaluated by the One-way ANOVA test
Mnb/Dyrk1a Regulates Food Intake
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(GraphPad Prism software). P,0.05 was accepted as statistically
UAS controls. (B) Relative food consumption of Figure 1B and
UAS controls. (C) Relative food consumption of Figure 3C,
Figure 4E, and UAS controls.
(A) mnb mRNA expression levels of Figure 1A and
Gal4.UAS-DsRed (sNPFR1.DsRed) in the fly adult brain. (A-D) In
the anterior focal planes, sNPFR1.DsRed was detected in the optic
lobes (OL, A), insulin producing cells (IPCs, C), mushroom body
(MB, B), and subesophageal ganglions (SOG, D). (E, F) In the
posterior focal planes, sNPFR1.DsRed was detected in the median
neurons above esophagus (E, dot box; F, asterisk). Scale bars are
100 mm (A, E), 50 mm (B, D, F) and 30 mm (C).
sNPFR1-Gal4 expression was detected by sNPFR1-
sNPFc00448mutant, sNPFR1-Gal4 control, sNPFR1 overexpression
(sNPFR1.sNPFR1), and sNPFR1 inhibition (sNPFR1.sNPFR1-Ri).
(B-F) Numbers of strong Mnb expression neurons (asterisks) are
similar in the sNPFR1-Gal4 control, sNPFR1 overexpression
(sNPFR1.sNPFR1), sNPFR1 inhibition (sNPFR1.sNPFR1-Ri),
and sNPFc00448mutant. Scale bars are 100 mm.
(A) Western blots with the Mnb antibody in the
represent exons, the triangle shows the p-element insertion site in
mnbG1767, and an arrow indicates the transcriptional initiation of
the mnb H isoform containing the longest coding sequences among
mnb isoforms. mnb deletion mutants (mnbd305and mnbd419) were
generated by imprecise excisions of the inserted p-element. (B) mnb
mRNA expression levels in the mnb overexpression (sNPFR1.mnb),
inhibition (sNPFR1.mnb-Ri), and mnbG1767mutant. (C) Western
blot with the Mnb antibody in the w- control and mnbG1767mutant.
(D) mnb overexpression (sNPFR1.mnb) increased the body weight
compared with the sNPFR1-Gal4 control whereas mnb suppression
(sNPFR1.mnb-Ri, mnbG1767) decreased the body weight. (E)
Amount of food intake by the normalized to body mass and to
the number of flies. Data are presented as means 6 s.e.m. from
three independent experiments. *P,0.05 (One-way ANOVA
(A) The mnb genomic organization. Open boxes
inducible system. (A) In the 22uC permissive temperature
condition in which tubGal80ts suppress sNPFR1-Gal4 expression,
Gal4+tubGal80ts.2Xmnb) and mnb inhibition (sNPFR1-Gal4+tub-
Gal80ts.mnb-Ri) flies did not change the amount of food intake
compared with the control flies (sNPFR1-Gal4;tub-Gal80ts). (B) In
the 30uC restrictive temperature in which tubGal80ts cannot
suppress sNPFR1-Gal4, the mnb overexpression increased food
intake compared with the control and the mnb inhibition
Adult specific food intake assay using the tubGal80ts
suppressed food intake. Data are presented as means 6 s.e.m.
from three independent experiments. *P,0.05 (One-way AN-
BG2-C6 cells after sNPF treatment. (B) cAMP level in Drosophila
BG2-c6 cells after sNPF treatment. (C) cAMP level in mouse GT1-
7 cells after NPY treatment.
(A) Expression levels of sNPF and sNPFR1 in Drosophila
consumption (B), and sNPF mRNA expression (C) in Gal4 and UAS
controls used in this study.
Levels of mnb mRNA expression (A), relative food
species reveals that the cAMP-response element (CRE), which is
TGACGTCA, was conserved in Drosophila species including D.
melanogaster (Adapted and modified from UCSC Genome Browser
Promoter analysis of mnb genes from twelve Drosophila
sNPFR1.mnb, sNPFR1.Sir2, and sNPF1.dFOXO overexpression
and in the sNPFR1.sNPFR1-Ri, sNPFR1.mnb-Ri, sNPFR1.Sir2-
Ri, and sNPFR1.dFOXO-Ri inhibition. (C) sNPFR1 expression
during fasting. (D) sNPFR1 mRNA expression was not changed in
Dilp2.Dilp2 compared to the Dilp2-Gal4 control and in
sNPFR1.InR and sNPFR1.InRDNcompared to the sNPFR1-
(A, B) RT-PCR analysis of sNPFR1, mnb, Sir2, and
mRNA in the
Drosophila species reveals that the dFOXO binding site, which is
RWWAACA, was conserved in five Drosophila species including D.
melanogaster (Adapted and modified from UCSC Genome Browser
Promoter analysis of sNPF genes from twelve
body weight. Data are presented as means 6 s.e.m. *P,0.05.
hDyrk1a transgenic mice showed slightly increased
mnb expression in the DNA microarray analysis.
PCR primer sequences in this study.
We thank J. S. Lee for helpful comments on the manuscript and K. H.
Jeong for technical support.
Conceived and designed the experiments: S-HH K-SL S-JK MT KY.
Performed the experiments: S-HH K-SL S-JK A-KK HB M-SJ. Analyzed
the data: S-HH K-SL S-JK O-YK W-JS KY. Wrote the paper: S-HH K-
SL MT KY.
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