Nutrigenomic effects of edible bird's nest on insulin signaling in ovariectomized rats

Abstract

Estrogen deficiency alters quality of life during menopause. Hormone replacement therapy has been used to improve quality of life and prevent complications, but side effects limit its use. In this study, we evaluated the use of edible bird's nest (EBN) for prevention of cardiometabolic problems in rats with ovariectomy-induced menopause. Ovariectomized female rats were fed for 12 weeks with normal rat chow, EBN, or estrogen and compared with normal non-ovariectomized rats. Metabolic indices (insulin, estrogen, superoxide dismutase, malondialdehyde, oral glucose tolerance test, and lipid profile) were measured at the end of the experiment from serum and liver tissue homogenate, and transcriptional levels of hepatic insulin signaling genes were measured. The results showed that ovariectomy worsened metabolic indices and disrupted the normal transcriptional pattern of hepatic insulin signaling genes. EBN improved the metabolic indices and also produced transcriptional changes in hepatic insulin signaling genes that tended toward enhanced insulin sensitivity, and glucose and lipid homeostasis, even better than estrogen. The data suggest that EBN could meliorate estrogen deficiency-associated increase in risk of cardiometabolic disease in rats, and may in fact be useful as a functional food for the prevention of such a problem in humans. The clinical validity of these findings is worth studying further.

Full text

© 2015 Hou et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0)
License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further
permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on
how to request permission may be found at: http://www.dovepress.com/permissions.php
Drug Design, Development and Therapy 2015:9 4115–4125
Drug Design, Development and erapy Dovepress
submit your manuscript | www.dovepress.com
Dovepress 4115
ORIGINAL RESEARCH
open access to scientific and medical research
Open Access Full Text Article
http://dx.doi.org/10.2147/DDDT.S80743
Nutrigenomic effects of edible bird’s nest
on insulin signaling in ovariectomized rats
Zhiping Hou1,2
Mustapha Umar Imam1
Maznah Ismail1,3
Der Jiun Ooi1
Aini Ideris4
Rozi Mahmud5
1Laboratory of Molecular Biomedicine,
Institute of Bioscience, Universiti
Putra Malaysia, Serdang, Malaysia;
2Department of Pathology, Chengde
Medical University, Chengde, People’s
Republic of China; 3Department of
Nutrition and Dietetics, Universiti
Putra Malaysia, Serdang, Malaysia;
4Department of Veterinary Clinical
Studies, Faculty of Veterinary
Medicine, Universiti Putra Malaysia,
Serdang, Malaysia; 5Department
of Imaging, Faculty of Medicine
and Health Sciences, Universiti
Putra Malaysia, Serdang, Malaysia
Abstract: Estrogen deficiency alters quality of life during menopause. Hormone replacement
therapy has been used to improve quality of life and prevent complications, but side effects
limit its use. In this study, we evaluated the use of edible bird’s nest (EBN) for prevention
of cardiometabolic problems in rats with ovariectomy-induced menopause. Ovariectomized
female rats were fed for 12 weeks with normal rat chow, EBN, or estrogen and compared with
normal non-ovariectomized rats. Metabolic indices (insulin, estrogen, superoxide dismutase,
malondialdehyde, oral glucose tolerance test, and lipid profile) were measured at the end of
the experiment from serum and liver tissue homogenate, and transcriptional levels of hepatic
insulin signaling genes were measured. The results showed that ovariectomy worsened meta-
bolic indices and disrupted the normal transcriptional pattern of hepatic insulin signaling genes.
EBN improved the metabolic indices and also produced transcriptional changes in hepatic
insulin signaling genes that tended toward enhanced insulin sensitivity, and glucose and lipid
homeostasis, even better than estrogen. The data suggest that EBN could meliorate estrogen
deficiency-associated increase in risk of cardiometabolic disease in rats, and may in fact be use-
ful as a functional food for the prevention of such a problem in humans. The clinical validity
of these findings is worth studying further.
Keywords: ovariectomy, lipid metabolism, insulin resistance, antioxidant, aging
Introduction
Menopause is characterized by depletion of the ovarian follicles and decreasing levels
of estrogen and related hormones. As menopause sets in, the ovaries progressively fail
to produce estrogen, resulting in symptoms that may be severe and disabling and have
an impact on quality of life.1 Estrogen has modulatory effects on metabolic processes
including the maintenance of optimal glucose and lipid homeostasis, which it does
through regulation of key hormones and genes involved in cardiometabolic health.2
During menopause, fluctuations and/or relative or absolute deficiency of estrogen and
other sex hormones will result in loss of the balance maintained by these hormones,
thereby leading to a disturbed internal milieu and heightened risk of diseases including
insulin resistance and associated problems.3,4
The symptoms and diseases that are a consequence of menopause have received
considerable attention, due to their distressing nature and impact on quality of life. It is
increasingly becoming a concern for women because of increasing life expectancy. Hor-
mone replacement therapy has been used to restore premenopausal hormonal levels in
order to reverse menopausal problems, but associated side effects including cancers and
cardiovascular disease have necessitated the search for alternatives.5,6 Phytoestrogens from
plants have also been associated with side effects similar to those of hormone replacement
therapy.7 Alternatives like Remifemin® have not offered significant improvements in
Correspondence: Maznah Ismail
Laboratory of Molecular Biomedicine,
Institute of Bioscience, Universiti
Putra Malaysia, 43400, Serdang,
Selangor, Malaysia
Fax +60 3 8947 2116
Email maznahis@upm.edu.my
Journal name: Drug Design, Development and Therapy
Article Designation: Original Research
Year: 2015
Volume: 9
Running head verso: Hou et al
Running head recto: Edible bird’s nest prevents insulin resistance
DOI: http://dx.doi.org/10.2147/DDDT.S80743
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
14 August 2015
Number of times this article has been viewed

Drug Design, Development and Therapy 2015:9
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
4116
Hou et al
long-term sequelae of menopause like cancer and cardiometa-
bolic disease risk, and side effects further limit their use.8
The burden of cardiometabolic disease has grown consid-
erably over the years, and in women the risk of these diseases
increases significantly after menopause.3,4 Edible bird’s nest
(EBN) has been used as a traditional supplement mostly by
Asians to improve wellbeing.9 Recent evidence indicates
that EBN may have anti-inflammatory and antioxidative
properties,10–12 which can be beneficial in cardiometabolic
disease.13,14 In this study, we determined the effects of EBN on
cardiometabolic indices in ovariectomized rats, and a possible
mechanistic basis for such effects. We hypothesized that the
outcome of the study could provide the evidence needed for
use of EBN as a functional food in preventing cardiometabolic
disease in menopause, where the risk of such is increased due
to loss of protection from estrogen and related hormones.
Materials and methods
Materials
A rat insulin enzyme-linked immunosorbent assay kit was
purchased from Millipore (Billerica, MA, USA), while kits
for superoxide dismutase (SOD) and estrogen were from
Cell Biolabs (Cell Biolabs Inc, San Diego, CA, USA) and
Cusabio Biotech Co Ltd (Wuhan, People’s Republic of
China), respectively. The GenomeLab™ GeXP start kit
was from Beckman Coulter Inc (Miami, FL, USA) and the
RNA extraction kit was from RBC Bioscience Corp (Taipei,
Taiwan). MgCl2 and DNA Taq polymerase were purchased
from Thermo Fisher Scientific (Pittsburgh, PA, USA), while
RCL2 solution was purchased from Alphelys (Toulouse,
France). Glucometer strips were from Roche Diagnostics
(Indianapolis, IN, USA) and lipid profile kits (low-density
lipoprotein cholesterol, high-density lipoprotein cholesterol,
total cholesterol, and triglycerides) were purchased from
Randox Laboratories Ltd (Crumlin, County Antrim, UK). Rat
chow was obtained from Specialty Feeds (Glen Forrest, WA,
Australia). Ketamine/xylazine was from Sigma Chemical Co
(St Louis, MO, USA) and other solvents of analytical grade
were purchased from Merck (Darmstadt, Germany).
EBN samples
Ready-to-use EBN was supplied by Niah Bird’s Nest Trading
Company (Sarawak, Malaysia), and was incorporated into
standard rat chow for animal feeding. For determination of
nutritional composition, EBN was dried at 50°C for 3 days,
ground into powder, and used. Nutritional composition
(protein, carbohydrate, fat, ash, and moisture; Table 1) was
determined as reported previously.15
Animal handling and feeding
Approval for use of animals was granted by the Animal Care
and Use Committee of the Faculty of Medicine and Health
Sciences, Universiti Putra Malaysia (approval UPM/IACUC/
AUP-R012/2014), and the animals were handled as stipulated
by the guidelines for the use of animals. Thirty-six Sprague-
Dawley rats (3 months old, female, 160–180 g) were housed
under controlled conditions (12-hour light/12-hour dark
cycle, 20°C–22°C, 40%–50% humidity) with free access to
water and food for 2 weeks prior to the experiments. After
acclimatization, rats were ovariectomized under anesthesia
using 10 mg/60 mg/kg xylazine/ketamine (intraperitoneally),
except for the normal controls. The rats were then observed for
4 weeks and randomly assigned to one of five groups (n=6):
a sham group, maintained on standard rat chow; an ovariectomy
(OVX) control group maintained on standard rat chow; an OVX +
estrogen group maintained on standard rat chow and daily
estrogen (0.2 mg/kg body weight); an OVX + 3% EBN group
maintained on standard rat chow containing 3% EBN; and an
OVX + 1.5% EBN group maintained on standard rat chow con-
taining 1.5% EBN. The normal group was not ovariectomized
and was maintained on standard rat chow. Treatments lasted
for 12 weeks, and food intake in each group was adjusted to the
average intake each day according to observation of the OVX
group the day before. Weights were measured weekly, and the
total amount of feed (g) given was reviewed weekly based on
the weekly weights of the rats. At the end of the experiment, all
animals were exsanguinated after anesthesia (10 mg/60 mg/kg
xylazine/ketamine, intraperitoneally). The livers were collected
into RCL2 solution for gene expression studies.
OGTT and lipid prole
At the end of the intervention, an oral glucose tolerance
test (OGTT) was performed on each animal after 12 hours
of an overnight fast, and measurements were taken with a
glucometer. Serum samples from blood collected on the day
of euthanasia were used to analyze the rat lipid profiles (total
Table 1 Nutritional values for edible bird’s nest
Nutritional value*Crude protein Carbohydrate Fat Ash Moisture
Edible bird’s nest 67.72%±1.9% 31.07%±1.5% 1%±0.002% 0.06%±0.002% 0.15%±0.004%
Notes: *Nutritional values are expressed as percent dry weight. All the values are shown as the mean ± standard deviation (n=3).

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
4117
Edible bird’s nest prevents insulin resistance
cholesterol, high-density lipoprotein cholesterol, low-density
lipoprotein cholesterol, and triglycerides) using a Dimension
Xpand Plus Integrated chemistry system (Siemens, Germany)
with commercially available kits.
Determination of serum estrogen
and insulin
Serum samples were used for measurements of estrogen and
insulin using the respective enzyme-linked immunosorbent
assay kits according to the manufacturers’ instructions.
Absorbance was read on a microplate reader (BioTek
Synergy H1 Hybrid Reader, BioTek Instruments Inc,
Winooski, VT, USA) and results were calculated from the
respective standard curves: estrogen (y = -30.12x +113.73,
R2=1), insulin (y =0.762x -0.143, R2=0.966). Addition-
ally, homeostatic model assessment of insulin resistance
(HOMA-IR), a measure of insulin sensitivity, was com-
puted from the fasting plasma glucose and insulin levels
as reported previously.16
Hepatic antioxidative markers
Superoxide dismutase enzyme
Liver homogenates were used for SOD quantification using
the enzyme-linked immunosorbent assay kit according to
the manufacturer’s instructions. Absorbance were read on
a microplate reader (BioTek Synergy H1 Hybrid Reader)
and results were calculated from the standard curve
(y =0.143x -0.017, R²=1).
Thiobarbituric acid reactive substances assay
Thiobarbituric acid reactive substances were determined
using a modified method based on the protocol of Chan
et al.17 Briefly, homogenized liver tissues (50 mg/100 μL
phosphate-buffered saline) were added to 0.25 N HCl,
15% trichloroacetic acid, and 0.375% thiobarbituric acid
separately, then incubated at 100°C for 10 minutes, and
centrifuged at 3,000 rpm for 15 minutes. Finally, absorbance
of the supernatant were read at 540 nm using the Synergy
H1 Hybrid Multi-Mode microplate reader. Malondialdehyde
(MDA) was used as the standard (y =0.1982x -0.1898,
R2=0.9947).
RNA extraction, reverse transcription,
and multiplex polymerase chain reaction
analyses
RNA was extracted from rat livers using the Total RNA
isolation kit according to the manufacturer’s instructions.
Primer sequences were designed on the National Center for
Biotechnology Information website, except for the internal
control (KanR), which was supplied by Beckman Coulter.
The primers (Table 2) were supplied by Integrated DNA
Technologies (Singapore), and reconstituted in 1× TE buffer
according to the protocol outlined in the GenomeLab GeXP
kit. Reverse transcription and polymerase chain reaction were
performed according to the GenomeLab GeXP kit protocol
in an XP Thermal Cycler (Bioer Technology, Germany). The
polymerase chain reaction products were finally analyzed
with a GeXP genetic analysis system, and the results were
normalized using eXpress Profiler software based on the
manufacturer’s instructions.
Statistical analysis
Statistical analyses were done using one-way analysis of
variance with Tukey’s honest significant difference test.
The data are expressed as the mean ± standard error of
the mean. P,0.05 indicates statistical significance. All
values were statistically evaluated using Statistical Package
for the Social Sciences version 20.0 (SPSS Inc, Chicago,
IL, USA).
Results and discussion
Food intake and body weight
Menopause is characterized by a drop in estrogen levels,
which results in lipid and glucose metabolic perturbation.1,3,4
In this study, serum estrogen levels in the OVX group
were significantly lower than in the other groups (Table 3),
suggesting that removal of the ovaries produced a condition
similar to menopause in the rats. The higher levels in the
estrogen group could be attributed to exogenous estrogen
administration, while the high levels of the hormone in
the EBN group could have been due to increased extrago-
nadal synthesis of estrogen18 induced by EBN or possibly
estrogen-like compounds absorbed from EBN. Body weights
were similar for all groups at the start of the experiment
(Table 3), and calorie intake remained similar throughout
the intervention period. At the end, however, differences in
weight gain were observed. The OVX group had the highest
weight gain, indicating that loss of estrogen affected body fat
deposition and distribution. Moreover, estrogen is believed
to regulate weight in women, while menopause is associated
with increased weight gain, which is linked to increased
risk of cardiometabolic disease.2–4 Estrogen treatment and
EBN produced lower weight gains similar to the control
group, suggesting that both treatments had some weight-
modulating properties, possibly mediated through estrogen
and/or estrogen-mimetic effects.

Drug Design, Development and Therapy 2015:9
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
4118
Hou et al
Serum lipid prole
Table 4 shows the lipid profiles for the different groups.
Total cholesterol for the OVX group was highest although
no significant differences were observed between the groups.
Low-density lipoprotein cholesterol was also highest in
the OVX group, while the others were significantly lower,
except for the 1.5% EBN group. Triglyceride levels were
significantly lower in the EBN groups in comparison with
the OVX group, while high-density lipoprotein cholesterol
levels were not significantly different between the groups.
As expected, estrogen improved the lipid profiles. Estrogen
modulates cholesterol metabolism, and in estrogen-deficient
states similar to OVX, there is dysregulation of cholesterol
metabolism in favor of higher cholesterol levels, with an
increased risk of cardiometabolic disease.4 EBN on the
other hand was able to ameliorate lipid profile and cho-
lesterol ratios (Table 4) similar to estrogen. This effect of
EBN on the lipid profile may have been secondary to the
enhanced estrogen concentration induced by EBN, but other
EBN constituents may also have played a role since EBN
is known to be bioactive-rich.9 The improved lipid profiles
observed in this study suggest that EBN could lower the risk
of cardiometabolic disease without the associated risks of
estrogen administration.
OGTT, serum insulin, and HOMA-IR
The OGTT results are shown in Figure 1. The glycemic
response in the OVX group was impaired in comparison
with the other groups, which showed similar and significantly
(P,0.05) lower responses (Figure 1A). Moreover, the area
under the curve for glucose over 120 minutes (Figure 1B)
was significantly lower (P,0.05) for the EBN and estrogen
treatments in comparison with the OVX group. Additionally,
the insulin level was highest in the OVX group, which could
have been a counter-response to the loss of estrogen-regulated
insulin action in the rats (Figure 2). The estrogen group, on
the other hand, showed significantly lower insulin levels
(P,0.05), while the EBN groups were not significantly dif-
ferent from the OVX group. HOMA-IR results showed that
the OVX group had the highest tendency for insulin resis-
tance, while estrogen therapy significantly improved insulin
sensitivity (P,0.05). These effects of OVX and estrogen
therapy mirror what has been reported previously;4 OVX
tends to increase the risk of insulin resistance, which can
be prevented by estrogen therapy. EBN treatments showed
dose-dependent effects on insulin sensitivity, but only the 3%
EBN treatment showed significantly better results than those
seen in the OVX group. Although estrogen may prevent the
Table 2 Names, accession numbers, and primer sequences used in the study
Gene name Accession number Left sequence Right sequence
IRS2 NM_001168633 AGGTGACACTATAGAATAAGGCACTGGAGCCTTAC GTACGACTCACTATAGGGAGCAGCACTTTACTCTTTCAC
KCNJ11 NM_031358 AGGTGACACTATAGAATACTACTTCAGGCAAAACTCTG GTACGACTCACTATAGGGAGAACTTTCCAATATTTCTTTT
Insr NM_017071 AGGTGACACTATAGAATAAGCTGGAGGAGTCTTCAT GTACGACTCACTATAGGGAAAGGGATCTTCGCTTT
GCK NM_001270849 AGGTGACACTATAGAATAATCTTTTGCAACACTCAGC GTACGACTCACTATAGGGATTGTTGGTGCCCAGA
PKLR NM_012624 AGGTGACACTATAGAATATCGGAGGTGGAAATTG GTACGACTCACTATAGGGACTCTGGGCCGATTTT
B2M* NM_012512 AGGTGACACTATAGAATAATGCTTGCAGAGTTAAACA GTACGACTCACTATAGGGATGCATAAAATATTTAAGGTAAGA
HPRT1*,# NM_012583 AGGTGACACTATAGAATATCCTCATGGACTGATTATG GTACGACTCACTATAGGGACTGGTCATTACAGTAGCTCTT
MAPK1 NM_053842 AGGTGACACTATAGAATACATTTTTGAAGAGACTGCTC GTACGACTCACTATAGGGAAACTCTCTGGACTGAAGAAT
IKBKB NM_053355 AGGTGACACTATAGAATACTTGAACTTAAAGCTGGTTC GTACGACTCACTATAGGGAACATTTTACTGTTGTCAAAGAG
Kan (r)**
GLUT4 NM_012751 AGGTGACACTATAGAATACTCTGAAGATGGGGAAC GTACGACTCACTATAGGGAAGCTCTGTTCAATCACTTTC
ACTB* NM_031144 AGGTGACACTATAGAATAAACTACATTCAATTCCATCA GTACGACTCACTATAGGGATAAAACGCAGCTCAGTAAC
Pik3ca NM_133399 AGGTGACACTATAGAATACAAGGATCTGACTTATTTCC GTACGACTCACTATAGGGACTAACCATGCTGTTACCAA
Notes: *Housekeeping gene. #Normalization gene. Underlined sequences are left and right universal left and right sequences (tags). **Internal control supplied by Beckman Coulter Inc, as part of the GeXP kit. Reverse transcription
conditions were: 48°C for 1 minute; 37°C for 5 minutes; 42°C for 60 minutes; 95°C for 5 minutes, then hold at 4°C. Polymerase chain reaction conditions were initial denaturation at 95°C for 10 minutes, followed by two-step cycles of
94°C for 30 seconds and 55°C for 30 seconds, ending in a single extension cycle of 68°C for 1 minute.

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
4119
Edible bird’s nest prevents insulin resistance
OVX-induced risk of insulin resistance as demonstrated in
the present study (lower HOMA-IR in the estrogen group),
EBN may be preferred since its consumption is not associ-
ated with side effects as with estrogen therapy, except when
it is adulterated.9
Hepatic antioxidant capacity of EBN
Oxidative stress is increased while antioxidants are decreased
in estrogen-deficient states. This imbalance is thought to
underlie some problems related to menopause.19 Thus, in
this study, MDA levels, indicative of oxidative stress, were
potentiated in the OVX group while SOD, an antioxidant
enzyme, was suppressed (Figure 3), in keeping with a report
by Muthusami et al.20 Further, estrogen treatment reduced
MDA and improved SOD levels but not as well as EBN.
Moreover, the side effects of estrogen therapy may be asso-
ciated with the pro-oxidant effects of exogenous estrogen.21
EBN significantly ameliorated oxidative stress status and
improved antioxidant abilities, in agreement with reports on
its antioxidant properties.11,12 The results therefore suggest
that improved antioxidant enzymes may partly form the basis
for improved metabolic indices in ovariectomized rats fed
EBN, since improved antioxidant status has been linked with
better metabolic outcomes.22
mRNA levels of hepatic insulin signaling
genes
The results thus far indicate that EBN could lower the risk
of estrogen deficiency-induced perturbations in glucose
and lipid homeostasis. In an effort to understand some
of the mechanistic bases for the observed effects, we
evaluated mRNA levels for hepatic insulin signaling genes
(Figures 4–6). Figure 4 shows the effects of the interven-
tions on mRNA levels of the hepatic insulin receptor, insulin
receptor substrate 2 (IRS2), and phosphoinositide-3-kinase
(PI3K). Insulin exerts its effects after binding to its receptor
and activating a cascade of events mediated by IRS2,23 in
which PI3K plays a prominent role through enhanced insulin
signaling in the cell.24 Disruption of this pathway promotes
insulin resistance. In this study, OVX downregulated IRS2
and PI3K, suggesting that estrogen deficiency-induced inter-
ruption of cellular insulin signaling contributed to worsening
of glucose and lipid homeostasis in the rats. Interestingly,
EBN increased expression of the insulin receptor, IRS2, and
PI3K even better than estrogen therapy, indicating that its
ability to prevent estrogen deficiency-induced worsening
of glucose and lipid homeostasis in ovariectomized rats
was partly through transcriptional upregulation of insulin
signaling.
Furthermore, OVX upregulated mitogen-activated protein
kinase (MAPK) and inhibitor of kappa light polypeptide gene
enhancer in B-cells, kinase beta (IKBKB, Figure 5), which
have both been implicated in impaired insulin signaling.25,26
These genes have been reported to influence the activity of
IRS2, and the OVX-induced low transcriptional levels of
IRS2 observed in this study may have been due to upregula-
tion of MAPK1 and IKBKB.25,26 The EBN groups had lower
mRNA levels of these genes in comparison with other groups,
further suggesting that the effects of EBN on transcriptional
regulation of IRS2 may have been through MAPK1 and
IKBKB. Glucose transporter type 4 (GLUT4) transports
glucose into cells for metabolism and is downregulated in
insulin-resistant states.27 Although the exact contribution of
hepatic expression of GLUT4 to glucose transport remains
a matter of debate, there are indications that its dysregula-
tion is linked to insulin resistance.28 In this study, OVX
downregulated GLUT4, while EBN treatments upregulated
the gene in a dose-dependent manner. Moreover, lower lev-
els of MAPK, with consequently higher PI3K levels, have
been reported to enhance glucose uptake through increased
GLUT4 expression.27 In keeping with findings of decreased
MAPK and increased PI3K and GLUT4 in this study.
Table 3 Body weight, food intake, and serum estrogen levels in ovariectomized rats after 12 weeks of intervention
Rat groups Body weight
before intervention (g)
Body weight at end of
intervention (g)
Total food intake
(kcal/kg/day)
Serum estrogen
(pg/mL)
Normal control 221.5±33.5a227.5±6.0a134±4.8a151.1±8a
Sham control 217.8±44.0a224.6±6.8a134±4.8a156.7±13a
OVX control 230±29.7a283.1±23.1b134±4.8a35.6±0.9b
Estrogen-treated 236±33.3a239±3.0a134±4.8a169.8±11.4a
3% EBN 228.1±53.9a242.8±14.6a134.97±4.99a147.8±8.7a
1.5% EBN 216±31.5a242.6±26.7a,b 134.81±4.45a143.3±13.4a
Notes: Values are shown as the mean ± standard deviation, n=6. All rat groups received standard rat chow for 12 weeks, and in addition, the estrogen-treated groups
received 0.2 mg/kg/day of estrogen, while EBN groups received 3% or 1.5% EBN in their rat chow. Columns with different letters signify statistical difference (P,0.05).
Abbreviations: OVX, ovariectomy; EBN, edible bird’s nest.

Drug Design, Development and Therapy 2015:9
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
4120
Hou et al
Transcriptional levels of other genes that contribute
to enhanced cellular glucose sensing and homeostasis
(glucokinase, pyruvate kinase-liver isoform, and potas-
sium inwardly rectifying channel, subfamily J, member 11
[KCNJ11] genes) were downregulated in the OVX group
(Figure 6). Conversely, the EBN group showed increased
expression of these genes, except KCNJ11, even better
than estrogen therapy. The implications of these effects
could be increased hepatic glucose storage and utilization
(glucokinase and pyruvate kinase-liver isoform),29 which
may then enhance cellular adenosine triphosphate levels
and subsequent upregulation of KCNJ11 with improved
glucose homeostasis.30
Based on the effects of EBN observed in this study, we
propose that EBN transcriptionally regulates multiple targets
in the insulin signaling pathway (Figure 7) and that this is
the basis for the improved insulin signaling and glucose and
lipid homeostasis.
Overall, the data show that OVX worsened glucose
and lipid homeostasis and oxidative stress (low insulin
and SOD levels; high body weight, HOMA-IR, MDA, and
lipid profile levels; and impaired insulin signaling) in rats.
Estrogen therapy, on the other hand, was able to improve
the lipid profile and OGTT, SOD, and MDA levels, and
was even associated with some transcriptional changes in
hepatic insulin signaling genes that tended toward better
glucose and lipid homeostasis. However, EBN improved
glucose and lipid homeostasis, lowered weight and oxidative
stress, and enhanced estrogen deficiency-induced impaired
signaling of insulin better than estrogen. Simple linear
regression analyses indicate that estrogen levels significantly
correlated with changes in HOMA-IR (r= -0.82, P=0.046),
SOD (r=0.82, P=0.046), MDA (r= -0.87, P=0.023), and
PI3K-mediated insulin signaling (r=0.86, P=0.027). Addi-
tionally, SOD levels (r= -0.86, P=0.028) and PI3K-mediated
insulin signaling (r= -0.85, P=0.033), but not MDA levels
(r=0.53, P=0.28) correlated with HOMA-IR in this study.
Multiple linear regression analyses indicate further that
changes in estrogen were overall strong determinants of
changes in SOD, MDA, HOMA-IR, and PI3K expression
changes (R=1, R2=0.995), and that estrogen, SOD, MDA, and
PI3K expression was a significant predictor of HOMA-IR
(R=0.987, R2=0.873). The combined effects of EBN on these
markers of cardiometabolic disease, coupled with the known
side effects of estrogen therapy, suggest that EBN may be
better than hormone replacement therapy at preventing
menopause-associated cardiometabolic disease, especially
impaired glucose and lipid homeostasis.
Table 4 Serum lipid proles after 12 weeks of intervention
Rat groups TC (mg/dL) LDL (mg/dL) HDL (mg/dL) LDL/HDL (ratio) VLDL* (mg/dL) TC/HDL (ratio) TG (mg/dL)
Normal control 1.858±0.240a,b 0.194±0.035a1.268±0.127a,b 0.153±0.027a0.396±0.078a,d 1.465±0.189a,b 0.473±0.033a,c
Sham 1.690±0.234a0.198±0.035a1.092±0.120a0.181±0.032a0.400±0.079a,d 1.548±0.214a,b 0.587±0.090a,b,d
OVX control 2.220±0.248b0.374±0.046b1.360±0.157a,b 0.275±0.034b0.486±0.045a1.632±0.183a0.657±0.067b
Estrogen 1.885±0.105a,b 0.286±0.038c1.375±0.077b0.208±0.028c0.224±0.011b1.371±0.076b0.460±0.026c
3% EBN 1.683±0.301a,b 0.298±0.021c1.316±0.135a,b 0.226±0.016b,c 0.068±0.014c1.278±0.229a,b 0.455±0.021c
1.5% EBN 1.897±0.254a,b 0.320±0.020b,c 1.260±0.132a,b 0.254±0.016b,c 0.317±0.010d1.505±0.201a,b 0.527±0.006d
Notes: Values are shown as the mean ± standard deviation. Columns with different letters indicate statistical difference (P,0.05). *Total cholesterol – (HDL + LDL). All rat groups received standard rat chow for 12 weeks, and in addition,
the estrogen treated groups received 0.2 mg/kg/day of estrogen, while EBN groups received 3% or 1.5% EBN in their rat chow.
Abbreviations: TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein; HDL, high-density lipoprotein; VLDL, very low-density lipoprotein; EBN, edible bird’s nest; OVX, ovariectomy.

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
4121
Edible bird’s nest prevents insulin resistance
$








DE









DE D
EEE
&RQWURO 6KDP 29; (VWURJHQ (%1KLJK (%1ORZ
  
7LPHPLQXWHV
5DWJURXSV
%ORRGJOXFRVHPPRO/
$8&RYHU
PLQVPPRO/
%
*URXSV
1RUPDO
6KDP
29;FRQWURO
(VWURJHQ
(%1KLJK
(%1ORZ
Figure 1 Effects of 12 weeks of supplementation with EBN on (A) oral glucose tolerance test and (B) AUC for glucose in ovariectomized rats.
Notes: EBN high, 3% EBN; EBN low, 1.5% EBN. Different letters for bars in (B) indicate a statistically signicant difference (P,0.05). All rat groups received standard rat
chow for 12 weeks, and in addition, the estrogen treated groups received 0.2 mg/kg/day of estrogen, while EBN groups received 3% or 1.5% EBN in their rat chow.
Abbreviations: AUC, area under the curve; EBN, edible bird’s nest; OVX, ovariectomy.
a,b
A
B
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
8
7
6
5
4
3
2
1
0
a,b a,b
a,b
a
a
a
a
a
a
b
b
Sham
Sham
Rat groups
Insulin (ng/mL)HOMA-IR
Rat groups
OVX Estrogen
Estrogen
EBN high
EBN high
EBN low
EBN lowOVX
Normal control
Normal control
Figure 2 Effects of 12 weeks of supplementation with EBN on (A) serum insulin and (B) HOMA-IR in ovariectomized rats.
Notes: EBN high, 3% EBN; EBN low, 1.5% EBN. Different letters for bars in each panel indicate a statistically signicant difference (P,0.05). All rat groups received standard
rat chow for 12 weeks, and in addition, the estrogen treated groups received 0.2 mg/kg/day of estrogen, while EBN groups received 3% or 1.5% EBN in their rat chow.
Abbreviations: EBN, edible bird’s nest; OVX, ovariectomy; HOMA-IR, homeostatic model assessment of insulin resistance.

Drug Design, Development and Therapy 2015:9
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
4122
Hou et al
Control
Control
Sham
Sham
Estrogen
Estrogen
EBN high
EBN high
EBN low
EBN low
OVX
OVX
Rat groups
MDA content
(nmol/L) SOD (U/mg protein)
Rat groups
a
aad
a
a
12
A
B
10
8
6
4
2
0
3
2.5
2
1.5
1
0.5
0
cc
c
bb
b
Figure 3 Effects of 12 weeks of supplementation with EBN on serum (A) SOD levels and (B) MDA levels in ovariectomized rats.
Notes: Different letters for bars in each panel indicate a statistically signicant difference (P,0.05). All rat groups received standard rat chow for 12 weeks, and in addition,
the estrogen treated groups received 0.2 mg/kg/day of estrogen, while EBN groups received 3% or 1.5% EBN in their rat chow.
Abbreviations: EBN, edible bird’s nest; MDA, malondialdehyde; SOD, superoxide dismutase; OVX, ovariectomy.
AB
C
a,b
a
a
a
a
4
3.5
3
2.5
2
1.5
1
0.5
0
aa
c
cb,c
b,c
b,c
a,c
a,c
1.2
1
0.8
0.6
0.4
0.2
0
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Normal Normal
Normal
bb
b
b
Sham Sham
Sham
OVX
control
OVX
control
OVX
control
Estrogen Estrogen
Estrogen
EBN
high
EBN
high
EBN
high
EBN
low
EBN
low
EBN
low
Rat groupsRat groups
Relative expression
Relative expression
Relative expression
PI3K
Insr IRS2
Rat groups
Figure 4 Hepatic mRNA levels of the (A) Insr, (B) IRS2, and (C) PI3K in ovariectomized rats fed with EBN for 12 weeks.
Notes: Different letters for bars in each panel indicate a statistically signicant difference (P,0.05). All rat groups received standard rat chow for 12 weeks, and in addition,
the estrogen treated groups received 0.2 mg/kg/day of estrogen, while EBN groups received 3% or 1.5% EBN in their rat chow.
Abbreviations: EBN, edible bird’s nest; PI3K, phosphoinositide-3-kinase; IRS2, insulin receptor substrate 2; OVX, ovariectomy; Insr, insulin receptor.

Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
4123
Edible bird’s nest prevents insulin resistance
AGLUT4
14
aaa
c
b
Normal Sham Estrogen EBN
high
EBN
low
OVX
control
a,c
12
10
8
6
4
2
0
Rat groups
Relative expression
a
c
bb
NormalSham Estrogen EBN
high
EBN
low
OVX
control
a,b
a,b
2.5
2
1.5
1
0.5
0
Relative expression
Rat groups
MAPK1
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
acc
b
a,b,c
NormalSham Estrogen EBN
high
EBN
low
OVX
control
a,c
Relative expression
Rat groups
IKBKB
C
B
Figure 5 Hepatic mRNA levels of (A) GLUT4, (B) IKBKB, and (C) MAPK1 in ovariectomized rats fed with EBN for 12 weeks.
Notes: Different letters for bars in each panel indicate a statistically signicant difference (P,0.05). All rat groups received standard rat chow for 12 weeks, and in addition,
the estrogen treated groups received 0.2 mg/kg/day of estrogen, while EBN groups received 3% or 1.5% EBN in their rat chow.
Abbreviations: EBN, edible bird’s nest; GLUT4, glucose transporter type 4; IKBKB, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta; MAPK1, mitogen-activated
protein kinase 1; OVX, ovariectomy.
NormalNormalSham ShamEstrogen EstrogenEBN
high
EBN
high
EBN
low
EBN
low
OVX
control
OVX
control
Rat groups Rat groups
Relative expression
Relative expression
GCK
AB
C
KCNJ11
a
2.5 25
20
15
10
5
0
2
1.5
1
0.5
0
a
a,c
cc
ccc
NormalSham Estrogen EBN high EBN lowOVX control
LPK
Relative expression
Rat groups
a
aa
a
4
3.5
3
2.5
2
1.5
1
0.5
0
c
b
b
b
b
b
Figure 6 Hepatic mRNA levels of (A) GCK, (B) KCNJ11, and (C) LPK in ovariectomized rats fed with EBN for 12 weeks.
Notes: Different letters for bars in each panel indicate a statistically signicant difference (P,0.05). All rat groups received standard rat chow for 12 weeks, and in addition,
the estrogen treated groups received 0.2 mg/kg/day of estrogen, while EBN groups received 3% or 1.5% EBN in their rat chow.
Abbreviations: EBN, edible bird’s nest; GCK, glucokinase; KCNJ11, potassium inwardly rectifying channel, subfamily J, member 11; OVX, ovariectomy; LPK, pyruvate kinase-liver
isoform.

Drug Design, Development and Therapy 2015:9
submit your manuscript | www.dovepress.com
Dovepress
Dovepress
4124
Hou et al
.&1- $733./%
*OXFRVH
KRPHRVWDVLV
*OXFRVH
OLSLG
KRPHRVWDVLV
*&.
,.%.%
,56
0$3.
*/87
YHVLFOH
8SUHJXODWHGE\(%1 'RZQUHJXODWHGE\(%1
3LNFD
*/87
*/87
,165
Figure 7 Proposed schematic showing targets of EBN action in the insulin signaling pathway. EBN prevents insulin resistance in ovariectomized rats by inuencing the
transcriptional regulation of multiple genes.
Abbreviations: EBN, edible bird’s nest; GLUT, glucose transporter; MAPK, mitogen-activated protein kinase; GCK, glucokinase; IKBKB, inhibitor of kappa light polypeptide
gene enhancer in B-cells, kinase beta; IRS, insulin receptor substrate; KCNJ11, potassium inwardly rectifying channel, subfamily J, member 11; INSR, insulin receptor; LPK,
pyruvate kinase-liver isoform; Pik3ca, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha.
Conclusion
OVX promotes metabolic perturbations that may give
rise to cardiometabolic disease, and although the use of
estrogen may offer some protection against some of these
cardiometabolic problems, as suggested by the present data,
use of estrogen therapy is limited by side effects. EBN has
demonstrated an ability to improve cardiometabolic indices
and enhance transcriptional regulation of insulin signaling
genes even better than estrogen. These results demonstrate for
the first time the potential for EBN to be used as a functional
food for prevention of cardiometabolic disease associated
with estrogen deficiency. These findings are worth studying
further in humans to ascertain the clinical validity of EBN
in preventing these diseases.
Acknowledgments
The authors acknowledge the financial support of the
Ministry of Science, Technology and Innovation, the
e-ScienceFund, Malaysia (vote 5450666); and the staff of
the Laboratory of Molecular Biomedicine for their assistance
during the study.
Disclosure
The authors report no conflicts of interest in this work.
References
1. Hess R, Thurston RC, Hays RD, et al. The impact of menopause on
health-related quality of life: results from the STRIDE longitudinal
study. Qual Life Res. 2012;21(3):535–544.
2. Louet JF, Cedric LM, Franck MJ. Antidiabetic actions of estrogen:
insight from human and genetic mouse models. Curr Atheroscler Rep.
2004;6(3):180–185.
3. Szmuilowicz ED, Stuenkel CA, Seely EW. Influence of menopause on
diabetes and diabetes risk. Nat Rev Endocrinol. 2009;5(10):553–558.
4. Gaspard UJ, Gottal JM, van den Brûle FA. Postmenopausal changes of
lipid and glucose metabolism: a review of their main aspects. Maturitas.
1995;21(3):171–178.
5. Beral V, Banks E, Reeves G. Evidence from randomised trials on
the long-term effects of hormone replacement therapy. Lancet. 2002;
360(9337):942–944.
6. Nelson HD, Humphrey LL, Nygren P, Teutsch SM, Allan JD. Post-
menopausal hormone replacement therapy: scientific review. JAMA.
2002;288(7):872–881.
7. Tempfer CB, Froese G, Heinze G, Bentz E-K, Hefler L-A, Huber JC.
Side effects of phytoestrogens: a meta-analysis of randomized trials.
Am J Med. 2009;122(10):939–946.
8. Nedrow A, Miller J, Walker M, Nygren P, Huffman LH, Nelson HD.
Complementary and alternative therapies for the management of
menopause-related symptoms: a systematic evidence review. Arch
Intern Med. 2006;166(16):1453–1465.
9. Marcone MF. Characterization of the edible bird’s nest the “Caviar of
the East”. Food Res Int. 2005;38(10):1125–1134.
10. Vimala B, Hussain H, Nazaimoon WW. Effects of edible bird’s nest
on tumour necrosis factor-alpha secretion, nitric oxide production
and cell viability of lipopolysaccharide-stimulated RAW 264.7 mac-
rophages. Food Agric Immunol. 2012;23(4):303–314.
11. Yew MY, Koh RY, Chye SM, Othman I, Ng KY. Edible bird’s nest
ameliorates oxidative stress-induced apoptosis in SH-SY5Y human
neuroblastoma cells. BMC Complement Altern Med. 2014;14(1):391.

Drug Design, Development and erapy
Publish your work in this journal
Submit your manuscript here: http://www.dovepress.com/drug-design-development-and-therapy-journal
Drug Design, Development and Therapy is an international, peer-
reviewed open-access journal that spans the spectrum of drug design
and development through to clinical applications. Clinical outcomes,
patient safety, and programs for the development and effective, safe,
and sustained use of medicines are a feature of the journal, which
has also been accepted for indexing on PubMed Central. The manu-
script management system is completely online and includes a very
quick and fair peer-review system, which is all easy to use. Visit
http://www.dovepress.com/testimonials.php to read real quotes from
published authors.
Drug Design, Development and Therapy 2015:9 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
Dovepress
4125
Edible bird’s nest prevents insulin resistance
12. Yida Z, Imam MU, Ismail M. In vitro bioaccessibility and antioxidant
properties of edible bird’s nest following simulated human gastro-
intestinal digestion. BMC Complement Altern Med. 2014;14(1):468.
13. Roberts CK, Sindhu KK. Oxidative stress and metabolic syndrome. Life
Sci. 2009;84(21):705–712.
14. Hotamisligil GS. Inflammation and metabolic disorders. Nature.
2006;444(7121):860–867.
15. Imam MU, Ishaka A, Der Juin O, et al. Germinated brown rice regu-
lates hepatic cholesterol metabolism and cardiovascular disease risk in
hypercholesterolemic rats. J Funct Foods. 2014;8:193–203.
16. Cacho J, Sevillano J, de Castro J, Herrera E, Ramos MP. Validation of
simple indexes to assess insulin sensitivity during pregnancy in Wistar
and Sprague-Dawley rats. Am J Physiol Endocrinol Metab. 2008;295(5):
1269–1276.
17. Chan KW, Khong NM, Iqbal S, Ch’ng SE, Babji AS. Preparation of
clove buds deodorized aqueous extract (CDAE) and evaluation of its
potential to improve oxidative stability of chicken meatballs in com-
parison to synthetic and natural food antioxidants. J Food Qual. 2012;
35(3):190–199.
18. Ye L, Chan MY, Leung LK. The soy isoflavone genistein induces
estrogen synthesis in an extragonadal pathway. Mol Cell Endocrinol.
2009;302(1):73–80.
19. Sánchez-Rodríguez MA, Zacarías-Flores M, Arronte-Rosales A,
Correa-Muñoz E, Mendoza-Núñez VM. Menopause as risk factor for
oxidative stress. Menopause. 2012;19(3):361–367.
20. Muthusami S, Ramachandran I, Muthusamy B. Ovariectomy induces
oxidative stress and impairs bone antioxidant system in adult rats. Clin
Chim Acta. 2005;360(1):81–86.
21. Bhat HK, Calaf G, Hei TK, Loya T, Vadgama JV. Critical role of
oxidative stress in estrogen-induced carcinogenesis. Proc Natl Acad
Sci U S A. 2003;100(7):3913–3918.
22. Rahimi R, Nikfar S, Larijani B, Abdollahi M. A review on the role
of antioxidants in the management of diabetes and its complications.
Biomed Pharmacother. 2005;59(7):365–373.
23. Brady MJ. IRS2 takes center stage in the development of type 2 diabetes.
J Clin Invest. 2004;114(7):886–888.
24. Hirsch E, Costa C, Ciraolo E. Phosphoinositide 3-kinases as a com-
mon platform for multi-hormone signaling. J Endocrinol. 2007;194(2):
243–256.
25. Igarashi M, Wakasaki H, Takahara N, et al. Glucose or diabetes activates
p38 mitogen-activated protein kinase via different pathways. J Clin
Invest. 1999;103(2):185–195.
26. Arkan MC, Hevener AL, Greten FR, et al. IKK links inflammation to
obesity-induced insulin resistance. Nat Med. 2005;11(2):191–198.
27. Carlson CJ, Koterski S, Sciotti RJ, Poccard GB, Rondinone CM.
Enhanced basal activation of mitogen-activated protein kinases in adi-
pocytes from type 2 diabetes potential role of p38 in the downregulation
of GLUT4 expression. Diabetes. 2003;52(3):634–641.
28. Karim S, Adams DH, Lalor PF. Hepatic expression and cellular distri-
bution of the glucose transporter family. World J Gastroenterol. 2012;
18(46):6771–6781.
29. Lenzen S. A fresh view of glycolysis and glucokinase regulation: history
and current status. J Biol Chem. 2014;289(18):12189–12194.
30. Proks P, Girard C, Ashcroft FM. Functional effects of KCNJ11 muta-
tions causing neonatal diabetes: enhanced activation by Mg ATP. Hum
Mol Genet. 2005;14(18):2717–2726.