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Neuropeptides
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Short communication
The effects of serotonin1A receptor on female mice body weight and food
intake are associated with the differential expression of hypothalamic
neuropeptides and the GABAA receptor q
Isma Butt a,1, Andrew Hong a,1, Jing Di a,1, Sonia Aracena c, Probal Banerjee b,c, Chang-Hui Shen a,b,
a
b
c
Department of Biology, College of Staten Island, City University of New York, Staten Island, NY 10314, USA
Institute for Macromolecular Assemblies, City University of New York, Staten Island, NY 10314, USA
Department of Chemistry, College of Staten Island, City University of New York, Staten Island, NY 10314, USA
a r t i c l e
i n f o
Article history:
Received 26 January 2014
Accepted 23 July 2014
Available online 2 August 2014
Keywords:
Serotonin
Neuropeptides
GABAA b subunits
Food intake
Body weight
Gene expression
qRT-PCR
a b s t r a c t
Both common eating disorders anorexia nervosa and bulimia nervosa are characteristically diseases of
women. To characterize the role of the 5-HT1A receptor (5-HT1A-R) in these eating disorders in females,
we investigated the effect of saline or 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) treatment
on feeding behavior and body weight in adult WT female mice and in adult 5-HT1A-R knockout (KO)
female mice. Our results showed that KO female mice have lower food intake and body weight than
WT female mice. Administration of 8-OH-DPAT decreased food intake but not body weight in WT
female mice. Furthermore, qRT-PCR was employed to analyze the expression levels of neuropeptides,
c-aminobutyric acid A receptor subunit b (GABAA b subunits) and glutamic acid decarboxylase in the
hypothalamic area. The results showed the difference in food intake between WT and KO mice was
accompanied by differential expression of POMC, CART and GABAA b2, and the difference in body weight
between WT and KO mice was associated with signicantly different expression levels of CART and
GABAA b2. As such, our data provide new insight into the role of 5-HT1A-R in both feeding behavior
and the associated expression of neuropeptides and the GABAA receptor.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
People suffering from eating disorders usually exhibit serious
disturbances in eating habits. These disturbances include an alteration of food intake as well as altered psychological perception
towards food, body weight, and self-image. Two common eating
disorders are anorexia nervosa (AN) and bulimia nervosa (BN).
AN is characterized by chronic food refusal, excessive weight loss,
fear of weight gain and a distorted self-image (Attia, 2010;
Weiselberg et al., 2011). On the other hand, BN is characterized
by frequent episodes of binge eating. Self-vomiting, abuse of laxatives and excessive exercise are common compensatory purging
mechanisms used in patients with BN (American Psychiatric
Association, 1994). The gender difference in both AN and BN is
q
Source of support: This work was supported by an NSF Grant (MCB-0919218) to
C.-H. S.
Corresponding author at: Department of Biology, College of Staten Island, City
University of New York, 2800 Victory Blvd, Staten Island, NY 10314, USA. Tel.: +1
(718) 982 3998; fax: +1 (718) 982 3852.
E-mail address: ChangHui.Shen@csi.cuny.edu (C.-H. Shen).
1
Contribute equally to this work.
http://dx.doi.org/10.1016/j.npep.2014.07.003
0143-4179/ 2014 Elsevier Ltd. All rights reserved.
314
Table 1
Oligonucleotides used in qRT-PCR reaction.
Oligonucleotides
Sequence
GAPDH ORFa
Forward primer
Reverse primer
50 -ACAGGGTGGTGGACCTCATG-30
50 -GTTGGGATAGGGCCTCTCTTG-30
GABAA b1 ORF
Forward primer
Reverse primer
50 -CTGCATCCTGATGGAACTGTTC-30
50 -CTCATCCAGAGGGTATCTTCGAA-30
GABAA b2 ORF
Forward primer
Reverse primer
50 -GTGGGCACGAGGGTTAGAAC-30
50 -GATCCACCACAGCAGCCATT-30
GABAA b3 ORF
Forward primer
Reverse primer
50 -CCACGGAGTGACAGTGAAAA-30
50 -CACGCTGCTGTCGTAGTGAT-30
GAD65 ORF
Forward primer
Reverse primer
50 -GGCTCTGGCTTTTGGTCCTTC-30
50 -TGCCAATTCCCAATTATACTCTTGA-3
NPY ORF
Forward primer
Reverse primer
50 -AGAGATCCAGCCCTGAGACA-30
50 -TTTCATTTCCCATCACCACA-30
AgRP ORF
Forward primer
Reverse primer
50 -GGCACAAGAGACCAGGACAT-30
50 -ACTTCTTCTGCTCGGTCTGC-30
POMC ORF
Forward primer
Reverse primer
50 -GAACAGCCCCTGACTGAAAA-30
50 -AACGTTGGGGTACACCTTCA-30
CART ORF
Forward primer
Reverse primer
50 -GAGGTCCAGAACCATGGAGA-30
50 -ACTTCTTGCAACGCTTCGAT-30
GAL ORF
Forward primer
Reverse primer
50 -AGCCTTGATCCTGCACTGAC-30
50 -AGGGTCACAACCAACAGGAG-30
GALP ORF
Forward primer
Reverse primer
50 -TGGTCCTCTTCCTCACCATC-30
50 -AGGACCCAGGAGGTAACCAG-30
PMCH ORF
Forward primer
Reverse primer
50 -TGCTGAGTCCACACAGGAAA-30
50 -GCCAACATGGTCGGTAGACT-30
315
study. Our results showed that the body weight of WT mice was
maintained at 38.31 0.31 g, but the body weight of KO mice
was only 24.43 0.71 g (Fig. 1A). Furthermore, the daily food
intake of KO mice was signicantly lower than WT mice
(Fig. 1B). As such, these results suggested that WT and KO mice
showed differences in body weight and daily food intake, and these
differences were subject to the presence of 5-HT1A-R.
We then examined how the 5-HT1A-R agonist 8-OH-DPAT
affects the body weight and food intake. Our results showed that
the administration of 8-OH-DPAT in WT mice caused the signicant decrease of food intake, but the food intake of WT mice was
still signicantly higher than the food intake of KO mice (Fig. 1B).
Intriguingly, we did not observe a change in body weight during
the 3 weeks period. Furthermore, the administration of 8-OH-DPAT
on KO mice did not have any inuence on body weight and daily
food intake. A similar pattern for both saline and 8-OH-DPAT treatment in KO mice was observed.
3.2. Effect of 5-HT1A-R on the expression of hypothalamic
neuropeptides
Since both body weight and food intake can be inuenced by 5HT1A-R, we examined the associated neuropeptides expression levels. We used quantitative real-time PCR to examine mRNA levels of
neuropeptides in the hypothalamus of WT and KO mice. These
neuropeptides included neuropeptide Y (NPY), agouti-gene related
protein (AgRP), pro-opiomelanocortin (POMC), cocaine- and
amphetamine-related transcript (CART), galanin (GAL), galaninlike peptide (GALP) and pro-melanin-concentrating hormone
(PMCH).
In the absence of the 8-OH-DPAT, the expression levels of
POMC, AgRP and CART were signicantly lower in KO mice than
in WT mice (Table 2). The levels of GAL, on the other hand, were
signicantly higher in KO mice. Regression analysis further showed
that the difference of body weight between WT and KO mice was
accompanied by signicantly different expression levels of CART
(Supplementary Table 1), and that the difference of food intake
between WT and KO mice was associated with signicantly different levels of POMC and CART (Supplementary Tables 2 and 3).
In the presence of the 8-OH-DPAT, expression levels of all neuropeptides decreased signicantly in WT mice. For KO mice, only
the expression of GAL and PMCH dropped signicantly. Again
p=0.02092
50
p=0.00013
30
20
10
p=0.00654
p=0.00597
40
p=0.02214
0
Saline
8-OH-DPAT
WT
Saline 8-OH-DPAT
KO
Saline
8-OH-DPAT Saline
WT
8-OH-DPAT
KO
Fig. 1. The role of 5-HT1A-R in food intake and body weight. (A) The body weight of WT mice and KO mice after 3 weeks of drug delivery. (B) The daily average food intake
under freely feeding scheme for both WT and KO mice during the 3 weeks period of drug delivery. p < 0.05, Students t test.
316
Table 2
The mRNA levels of hypothalamic neuropeptides.
WT
POMC
CART
AgRP
NPY
GAL
GALP
PMCH
KO
PBS
8OH-DPAT
PBS
8OH-DPAT
0.1675 0.0062
0.2355 0.0240
0.0115 0.0001
1.9884 1.4556
7.1020 2.2543
0.0055 0.0036
191.8407 75.8636
0.0014 0.0006
0.0353 0.0024
0.0002 0.0001
0.0769 0.0128
0.1872 0.1173
0.00004 0.00002
0.0332 0.0126
0.0035 0.0002
0.0592 0.0164
0.0004 0.0001
3.9509 1.0919
16.7854 4.6389
0.0078 0.0071
124.2015 34.3247
0.0031 0.0001
0.0562 0.0034
0.0024 0.0009
3.2804 1.2674
0.1022 0.0395
0.0121 0.0109
0.0012 0.0004
Summary of the real-time PCR analysis of neuropeptides mRNA relative to GAPDH mRNA. All real-time PCR reactions were done in triplicate.
we examined the effects of a 5-HT1A-R/5-HT7-R agonist, 8-OHDPAT, in food intake, body weight and neuropeptide expression
of female WT and KO mice. We demonstrated that the body weight
and food intake of KO mice were signicantly lower than the WT
mice (Fig. 1). Further analysis showed that such weight loss and
reduced food intake were associated with altered expression levels
of hypothalamic POMC, CART and GABAA b2 (Tables 2 and 3).
Finally, the body weight of KO mice was still signicantly lower
than that of the WT mice in the presence of 8-OH-DPAT, but only
the expression levels of hypothalamic GABAA b3 was associated
with this observation (Tables 2 and 3). As such, we have demonstrated how expression levels of neuropeptides and neurotransmitters are associated with food intake and body weight through
the presence of 5-HT1A-R.
Our results showed that the daily food intake and the body
weight of KO mice were signicantly lower than the daily food
intake and the body weight of WT mice (Fig. 1). This strongly suggested that 5-HT1AR might play an important role in food intake
and body weight. Previously, it has been demonstrated that KO
mice have an increased tendency to avoid a novel and fearful environment and to escape a stressful situation. As such, KO mice
showed behaviors consistent with an increased anxiety and stress
response (Parks et al., 1998). Furthermore, KO mice were impaired
in hippocampal-dependent spatial learning and memory tasks
such as the hidden platform version of Morris water maze and
the delayed version of the Y maze (Sarnyai et al., 2000). Clearly,
5-HT1A receptors are required for maintaining normal hippocampal
functions and this implicates a role for the 5-HT1A receptor in hippocampal-related symptoms, such as cognitive disturbances, in
stress-related disorders. Therefore, these abnormal behaviors
might affect KO mices food intake and subsequent change in body
weight.
We observed that administration of 8-OH-DPAT caused a significant decrease of food intake for WT female mice, which suggested
a satiety effect from 8-OH-DPAT in WT female mice (Fig. 1B). It has
been shown that the administration of 5-HT1A-R agonists increased
food intake in non-deprived or satiated rats (Dourish et al., 1985;
Gilbert and Dourish, 1987; Fletcher and Davis, 1990; Ebenezer,
1992; Ebenezer and Tite, 1994). Furthermore, a recent study
showed that the administration of 8-OH-DPAT to non-deprived
C57BL6 male mice increases food intake (Ebenezer and
Surujbally, 2007). Here, C57BL6 female mice were used in the test,
and our observations are not completely consistent with previous
ndings, suggesting that the discrepancy might arise from the
sex difference. Indeed, similar observations were also reported
for the female adult Wistar rats (Steffens et al., 2008). As such,
the gender difference indeed plays a role in 5-HT1A-R action.
Since female mice were used in this study, one might ask the
inuence of estrous cycle in the pharmacokinetic action of 8-OHDPAT. In general, the estrous cycle for a mouse is about 4.5 days
(Sullivan et al., 2002). Recent studies have demonstrated that the
pharmacokinetic action of 8-OH-DPAT is not inuenced by the
317
GABAA b1
GABAA b2
GABAA b3
GAD65
KO
PBS
8OH-DPAT
PBS
8OH-DPAT
345.13 75.42
2022.01 339.78
750.31 700.16
3791.44 540.60
155.35 46.65
482.51 92.85
24.12 21.03
283.55 144.24
360.96 265.32
64.95 17.95
1884.33 520.76
321.87 88.95
4.67 1.81
647.56 77.06
6198.68 1718.16
413.70 159.84
Summary of the real-time PCR analysis of neurotransmitters mRNA relative to GAPDH mRNA. All real-time PCR reactions were done in triplicate.
GABAA receptor agonists can reduce food intake and cause substantial weight loss (Turenius et al., 2009a,b). Here, we further
examined the transcripts levels of each subtype. Our results indicated that low food intake and low body weight are associated
with low GABAA b2 and high GABAA b3 (Fig. 1; Table 3). This is
the rst report to examine how different GABAA b subtypes affect
food intake and body weight. As such, we have provided a new
possible view to understand the relationship between GABAergic
system and food intake.
5. Conclusions
In this report, we examined the effects of a 5-HT1A-R/5-HT7-R
agonist, 8-OH-DPAT, in eating behavior at the molecular level.
Our ndings suggest that 5-HT1A-R plays an important role in
female mice feeding behavior and body weight. Our observations
have also demonstrated the effect of 5-HT1A-R on the expression
levels of neuropeptide and the peptide neurotransmitters. It is possible that 5-HT1A-R regulates eating behavior through its effects on
the levels of those peptides. A better understanding of the molecular mechanism underlying the unique features of 5-HT1A-R in
the expression of neuropeptide and GABAA receptor can facilitate
a better understanding of the neural regulation of feeding
behavior.
Acknowledgments
We are grateful to Dr. Lisa Manne for statistical analysis and to
Michelle Esposito for helpful discussion and comments on the
manuscript and to our laboratory colleagues for technical assistance. This work was supported by an National Science Foundation
Grant (MCB-0919218) to C.-H. S.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.npep.2014.07.003.
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