Edible Bird’s Nest Attenuates Procoagulation Effects of High-Fat Diet in Rats

7/26/2019 Edible Birds Nest Attenuates Procoagulation Effects of High-Fat Diet in Rats

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Research ArticleEdible Birds Nest Prevents High Fat Diet-InducedInsulin Resistance in Rats

Zhang Yida,1,2 Mustapha Umar Imam,1 Maznah Ismail,1,3

Der-Jiun Ooi,1 Nadarajan Sarega,1 Nur Hanisah Azmi,1 Norsharina Ismail,1

Kim Wei Chan,1 Zhiping Hou,1 and Norhayati Binti Yusuf1

Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Cardiology Department, Affiliated Hospital of Chengde Medical University, Chengde, Hebei , ChinaDepartment of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Correspondence should be addressed to Maznah Ismail; [email protected]

Received January ; Revised March ; Accepted March

Academic Editor: Joseph Fomusi Ndisang

Copyright Zhang Yida et al. Tis is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Edible birds nest (EBN) is used traditionally in many parts o Asia to improve wellbeing, but there are limited studies on itsefficacy. We explored the potential use o EBN or prevention o high at diet- (HFD-) induced insulin resistance in rats. HFD

was given to rats with or without simvastatin or EBN or weeks. During the intervention period, weight measurements wererecorded weekly. Blood samples were collected at the end o the intervention and oral glucose tolerance test conducted, aferwhich the rats were sacriced and their liver and adipose tissues collected or urther studies. Serum adiponectin, leptin, F-isoprostane, insulin, and lipid prole were estimated, and homeostatic model assessment o insulin resistance computed. Effectso the different interventions on transcriptional regulation o insulin signaling genes were also evaluated. Te results showed thatHFD worsened metabolic indices and induced insulin resistance partly through transcriptional regulation o the insulin signalinggenes. Additionally, simvastatin was able to prevent hypercholesterolemia but promoted insulin resistance similar to HFD. EBN, onthe other hand, prevented the worsening o metabolic indices and transcriptional changes in insulin signaling genes due to HFD.Te results suggest that EBN may be used as unctional ood to prevent insulin resistance.

1. Introduction

Te growing burden o cardiometabolic diseases, even in

the ace o increasing advances in medical sciences, is thedriving actor behind the heightened interest in alternativetherapies in the management o these diseases and associatedproblems [,]. Additionally, rising obesity rates globally dueto unhealthy liestyle actors promote these rising diseasetrends; obesity promotes insulin resistance and eventuallycardiometabolic diseases []. Inact, it isestimated thati per-sons at risk o insulin resistance and cardiometabolic diseasesare accurately determined using sensitive diagnostic tech-niques, the numbers o those needing interventions to man-age their conditions would be much higher than establishedgures []. Tere are different theories used to hypothesizethe underlying mechanisms involved in the progression rom

obesity to insulin resistance and cardiometabolic diseases.Popularly, excess calories are thought to promote depositiono visceral at around organs, with consequent changes in

the adipose tissue metabolism in the body, and ultimatelyincrease in insulin resistance especially in liver, as a resulto glucolipotoxicity []. Te ensuing insulin resistance causesdisruption in the propagation o insulin signals on insulin-responsive cells. In act, the perceived role o this phe-nomenon is the reason why therapeutic approaches to themanagement o insulin resistance and other associated car-diometabolic diseases involve the use o agents that promoteinsulin signaling.

Edible birds nest (EBN) is traditionally consumed amongAsians or its nutritional value. It is believed to enhanceenergy levels, prevent aging, and improve overall well-being.Furthermore, there are scientic reports o its antioxidative,

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2015, Article ID 760535, 11 pageshttp://dx.doi.org/10.1155/2015/760535
http://dx.doi.org/10.1155/2015/760535http://dx.doi.org/10.1155/2015/760535


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Journal o Diabetes Research

: Food composition and animal groups.

Animal group Normal pellet Cholesterol/cholic acid Palm oil Starch Others

Normal %

High at diet %

High at diet + simvastatin % Simvastatin ( mg/kg)

High at diet + % EBN % % EBNHigh at diet + .% EBN .% .% EBN

EBN: edible birds nest.

anti-inammatory, and bone-strengthening effects [].However, its effects on insulin resistance and cardiometabolicindices have not been documented. In view o the largepatronage o EBN by Asians, especially o Chinese origin[], we decided to evaluate the effects o EBN consumptionon cardiometabolic indices in high at diet- (HFD-) ed rats.Based on the anti-inammatory and antioxidant effects oEBN, we assumed it would have avorable effects on car-diometabolic indices, since both effects have been reportedto avor insulin sensitivity. As the rst study o its kind,we hypothesized that the results could provide the evidenceor continued use o EBN as a supplement and may evenpaveway or evidence-based development o unctional oodsand nutraceuticals using EBN or managing cardiometabolicdiseases.

2. Materials and Methods

.. Materials. Leptin, F-isoprostane, and insulin ELISA kitswere purchased rom Elabscience Biotechnology Co., Ltd(Wuhan, China), while adiponectin ELISA kit was rom Mil-

lipore (Billerica, MA, USA). Lipid prole kits were purchasedrom Randox Laboratories Ltd (Crumlin, County Antrim,UK). GenomeLab GeXP Start Kit was rom Beckman CoulterInc (Miami, FL, USA), and RNA extraction kit was romRBC Bioscience Corp. (aipei, aiwan). Simvastatin wasrom Pzer (New York, NY, USA) and RCL Solution romAlphelys (oulouse, France). Analytical grade ethanol waspurchased rom Merck (Darmstadt, Germany). Cholesteroland cholic acid were purchased rom Amresco (Solon, OH,USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA),respectively. Standard rat pellet was rom Specialty eeds(Glen Forrest, WA, USA), while palm oil was supplied by YeeLee Edible oils Sdn. Bhd. (Perak, Malaysia). EBN, oAero-

dramus fuciphagus (white nest swiflet) origin, supplied byBlossom View Sdn. Bhd (errengganu, Malaysia) was cleanedunder tap water or mins, dried at room temperature, andground into powder manually using mortar and pestle beoreincorporating it into rat pellet.

.. Bioactive and Proximate Analyses. Te proximate analy-sis o EBN was done as reported in our previous publication[], based on the official methods o Association o OfficialAnalytical Chemists. Briey, nitrogen content was deter-mined using micro-Kjeldahl apparatus (Kjeltech AutoDistillation Unit, FOSS ecator, Hoganas, Sweden), and thenprotein content was determined as N .. Furthermore,

the ashing process was done by incinerating the sample ina urnace (Furnace , Barnstead/Termolyne, Dubuque,IA, USA) set at C, while the at content was determinedas the dried ether extract o EBN. Ten, carbohydrate contentwas determined using the ollowing ormula: (% proteincontent moisture content ash content crude at content).All results were expressed as percentage o dry weight. Teamounts o major bioactives in EBN (sialic acid [SA], lacto-errin [LF], and ovotranserrin [OVF]) were analyzed usingELISA-based techniques (LF and OVF) and HPLC-DAD(SA). Briey, EBN was ground to powder and dissolvedin water at C or h on a shaking incubator (LSI-,DaihanLab tech Co.Ltd, Korea) andnally ltered. Te waterextract was then used to detect LF and OVF concentrationsusing Chicken Lactoerrin and Ovotranserrin Elisa Kits,Biosource (San Diego, Caliornia, USA), according to manu-acturers instructions. Additionally, water extract o EBN wasalso analysed or SA content using HPLC-DAD as reportedpreviously [].

.. Animal Study. Te Animal Care and Use Committee

(ACUC) o the Faculty o Medicine and Health Sciences,Universiti Putra Malaysia, approved the use o animals inthis study (Project approval number UPM/IACUC/AUP-R/), and animals were handled as stipulated by theguidelines or the use o animals. Sprague Dawley rats (-week old, g, = 30) werehousedat the animal house(2 5 2C, / h light/dark cycle) and allowed to acclimatizeor weeks with ree access to normal pellet and water.Afer acclimatization, rats were ed HFD containing .%cholesterol and .% cholic acid with or without treatmentusing simvastatin or EBN (able ), except the normal group( = 6). Intervention lasted or another weeks, afer whichrats were sacriced and their organs harvested or urtherstudies. Additionally, blood samples were collected at the endo the intervention or biochemical analyses.

.. Food Intake and Weight. Food intake was calculatedby subtracting the lefover ood rom what was added theprevious day. Weight was recorded afer acclimatization andweekly thereafer until sacrice.

.. Biochemical Analyses. Lipid prole analyses were per-ormed using serum rom blood collected at the beginningand end o the study by cardiac puncture afer an overnightast. Samples were analyzed using Randox analytical kitsaccording to manuacturers instructions using a Selectra XL


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instrument (Vita Scientic, Dieren, Te Netherlands). Bloodglucose was measured using glucometer (Roche Diagnostics,Indianapolis, IN, USA), and homeostatic model assessmento insulin resistance (HOMA-IR), a measure o insulin sen-sitivity, was computed rom the asting plasma glucose andinsulin levels using the ormula, HOMA-IR = (asting glucose

level [mg/dL]/asting plasma insulin [uU/mL])/ [].

.. Serum Adiponectin, Leptin, F-Isoprostane, and Insulin.Serum rom blood collected in plain tubes was used or mea-surements o adiponectin, leptin, F-isoprostane, and insulinusing the respective ELISA kits according to the manuac-turers instructions. Absorbance was read on BioeK SynergyH Hybrid Reader (Bioek Instruments Inc., Winooski, V,USA) at the appropriate wavelengths ( nm or insulin,leptin, and F-isoproatane and and or adiponectin).Te results were analyzed on http://www.myassays.com/

using our parametric test curve: adiponectin (2 = 0.9914),insulin (2 = 1), leptin (2 = 0.9996), and F-isoprostane

(2

= 1).

.. Gene Expression

... Primer Design. Rattus norvegicusgene sequences romthe National Center or Biotechnology Inormation web-site (http://www.ncbi.nlm.nih.gov/nucleotide/) were used todesign primers (able ) on GenomeLab eXpress Prolersofware. In addition to the genes o interest, primers werealso designed or housekeeping genes, while the internal con-trol (Kanr) was supplied by Beckman Coulter Inc. Primerswere tagged with an -nucleotide universal orward and -nucleotide universal reverse sequence, respectively. Primers

were supplied by Integrated DNA echnologies (Singapore)and reconstituted in RNAse ree water.

... RNA Extraction, Reverse ranscription, and PCR. RNAwas extracted rom liver and adipose tissues using thetotal RNA isolation kit (RBC Biotech Corp., aipei, ai-wan) according to the manuacturers instructions. Reversetranscription ( ng) and PCR were done according to theGenomeLab GeXP Start Kit protocol (Beckman Coulter,USA), using the conditions shown inable .

... GeXP Genetic Analysis System and Multiplex DataAnalysis. PCR products ( uL) were mixed with . L

sample loading solution and . L DNA size standard (GenomeLab GeXP Start Kit; Beckman Coulter, Inc, USA) ona -well sample plate and loaded on the GeXP genomelabgenetic analysis system (Beckman Coulter, Inc, Miami, FL,USA), which separates PCR products based on size bycapillary gel electrophoresis.Figure shows a representativeelectropherogram. Results were analyzed with the FragmentAnalysis module o the GeXP system sofware and normal-ized on the eXpress Proler sofware.

.. Data Analysis. Te meansstandard deviations ( = 6)o the groups were used or the analyses. One-way analysis o

variance (ANOVA) was perormed using SPSS . sofware

F : Representative electropherogram ollowing gene expres-sion analysis on GenomeLab GeXP genetic analysis system (Beck-man Coulter Inc., USA). Te genes and their expected sizes were

Irs-; Slca-; Kcnj-; Insr-; Glut-; Irs-; Gck-; Mapk-; Pklr-; Prkcd-; Bm-; Hprt-; Mapk-; Socs-; Rpla-; Prkcz-; Ikbkb-; Kan(r)-;Mtor-; Pdx-; Pikcd-; Actb-; Pikr-; Pikca-;Hk-.

(SPSS Inc., Chicago, IL, USA) to assess the level o signi-cance o differences between means with a cutoff o < 0.05.

3. Results and Discussions

.. Proximate andBioactiveAnalyses. Te proximate analysiso EBN showed that it contained mostly protein and carbo-

hydrates (able ), in agreement with previous ndings [].Additionally, it contained a signicant amount o SA (%) asbioactive, with lesser amounts o LF (%) and OVF (.%).Previous reports have indicated that EBN is bioactive-rich[], and it is likely that ood synergy plays role in its overalleffects []. Te presence o any one bioactive compound maynot explain the bioactivity o EBN, but the concentration othe leading bioactive compounds like SA may have an inu-ence to a great extent, albeit with the contribution o otherbioactives. Moreover, SA, LF, and OVF have all been reportedto have varying unctional effects [, ], andtheir synergismmay even produce better. Tis is similar to the concept obioactive-rich raction we have advocated or recently, in

which a lead bioactive compound in an extract produces bet-ter bioactivity in the presence o other bioactive compounds[]. Tereore, in view o recent advocacy or the study ooods but not their individual constituents as the unctionalunit o nutrition [], we decided to study the bioactivity oEBN as a whole.

.. Weight Changes. Figure shows the changes in bodyweights o rats over weeks o intervention. No statisticallysignicant changes were observed but the changes in HFD-ed (untreated control) group (% increase) were higher,in comparison with normal (%), simvastatin (%), .%EBN (%), and % EBN (%) groups. Interestingly, as


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T:Names,accession

number,andprimersequencesusedinth

estudy.

Accessionnumber

Lefsequence

Right

sequence

Irs

NM

AGGTGACACTATAGAATAAGGC

ACTGGAGCCTTAC

GTAC

GACTCACTATAGGGAGCAGCACTTTACTCTTTCAC

Kcnj

NM

AGGTGACACTATAGAATACTACTTCAGGCAAAACTCTG

GTAC

GACTCACTATAGGGAGAACTTTCCA

ATATTTCTTTT

Insr

NM

AGGTGACACTATAGAATAAGCTGGAGGAGTCTTCAT

GTAC

GACTCACTATAGGGAAAGGGATCTT

CGCTTT

Gck

NM

AGGTGACACTATAGAATAATCTTTTGCAACACTCAGC

GTAC

GACTCACTATAGGGATTGTTGGTGC

CCAGA

Pklr

NM

AGGTGACACTATAGAATATCGG

AGGTGGAAATTG

GTAC

GACTCACTATAGGGACTCTGGGCCG

ATTTT

Prkcd

NM

AGGTGACACTATAGAATATCAAGAACCACGAGTTCA

GTAC

GACTCACTATAGGGATCTTTCTGGAAGATGGTG

Bm

NM

AGGTGACACTATAGAATAATGCTTGCAGAGTTAAACA

GTAC

GACTCACTATAGGGATGCATAAAATATTTAAGGTAAGA

Hprt,#

NM

AGGTGACACTATAGAATATCCTCATGGACTGATTATG

GTAC

GACTCACTATAGGGACTGGTCATTACAGTAGCTCTT

Mapk

NM

AGGTGACACTATAGAATACATTTTTGAAGAGACTGCTC

GTAC

GACTCACTATAGGGAAACTCTCTGGACTGAAGAAT

Prkcz

NM

AGGTGACACTATAGAATACTTTAACAGGAGAGCGTACT

GTAC

GACTCACTATAGGGATATTGTCATGT

TTCCGAGAT

Ikbkb

NM

AGGTGACACTATAGAATACTTGAACTTAAAGCTGGTTC

GTAC

GACTCACTATAGGGAACATTTTACTG

TTGTCAAAGAG

Kan(r)

Mtor

NM

AGGTGACACTATAGAATATGGA

ACTTCGAGAGATGAG

GTAC

GACTCACTATAGGGATCACTTCAAACTCCACATAC

Actb

NM

AGGTGACACTATAGAATAAACTACATTCAATTCCATCA

GTAC

GACTCACTATAGGGATAAAACGCAG

CTCAGTAAC

Pikr

NM

AGGTGACACTATAGAATACATCAGTATTGGCTTACG

GTAC

GACTCACTATAGGGATCATTTACTTC

TTCCCTTGA

Housekeepinggenes.

#Normalizationgene.Underlinedsequencesarelefandrightunivers

allefandrightsequences(tags).

InternalcontrolsuppliedbyBeckmanCoulterInc(Miami,

FL,

USA)aspartothe

GeXPkit.

RTconditionswereCormin;Cormin;Cormin;Cormin

andthenholdatC.

PCRconditionswereinitialdenaturationatCormin,

ollowedby

two-stepcyclesoC

orsecandCorsec,endinginasingleextensioncycleoCormin.


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: Proximate analyses and lactoerrin, ovotranserrin, andsialic acid concentrations o edible birds nest (EBN).

Bioactive/nutrient EBN

Lactoerrin .. g/mg

Ovotranserrin .. g/mg

Sialic acid .. g/mg

Crude at ..%

Ash ..%

Moisture ..%

Carbohydrate ..%

Crude protein ..%

0

50

100

150

200

250

300

350

400

450

500

Normal SIM EBNL EBNH

Weig

ht(g)

Rat groups

Baseline

Week1

Week2

Week3Week4

Week5

Week6

Week7Week8

Week9

Week10

Week11

Week12

Untreatedcontrol

F : Effects o edible birds nest (EBN) on body weight changesin high at diet- (HFD-) ed rats over weeks. Te normal groupreceived standard rat chow, while the other groups received HFDcontaining .% cholesterol and .% cholic acid (untreated controlgroup), HFD containing .% cholesterol and .% cholic acid + mg/kg/day simvastatin (SIM), HFD containing .% cholesteroland .% cholic acid + .% EBN (EBNL, EBN low), or HFD con-taining .% cholesterol and .% cholic acid + % EBN (EBNH,EBN high).

shown inable , calorie intake or the different groups wassimilar over the intervention period. Te results indicatedthereore that EBN had some weight-modulating properties,

although the weight gain was lowest or simvastatin-treatedgroup. Moreover, simvastatin is reported to have some weightreducing properties [].

.. OG, Insulin, HOMA-IR, and Lipid Prole. Seruminsulin levels at the end o intervention were not remarkablydifferent between the groups except or the .% EBN group,which was signicantly lower ( < 0.05) than others(able ). However, absolute insulin levels may not reect thestate o the underlying insulin responsiveness since insulinresistance ofen starts with high insulin levels and ends upwith lower levels. Tereore, we computed the HOMA-IR asa marker o insulin resistance that combines insulin levels

0

2

4

6

8

10

12

Baseline 30 60 120Bloo

dg

lucose

(mmo

l/L)

Time (min)

Normal

Untreated controlSIM

EBNL

EBNH

F : Effects o edible birds nest(EBN) on oralglucosetolerancetest in ed high at diet- (HFD-) ed rats. Groupings are similar toFigure . indicates signicant difference ( < 0.05) in comparisonwith untreated control.

and asting glucose levels. Te data showed that untreated

control and simvastatin groups had a tendency to causeinsulin resistance. Tis mirrors earlier ndings on the effectso HFD eeding [] and simvastatin [] on development oinsulin resistance. EBN groups had lower HOMA-IR values incomparison with other groups, although not signicantly di-erent rom normal (both EBN groups) and untreated control(% EBN group) groups.

Te cholesterol levels in the untreated control groupwere signicantly increased in comparison with the normalgroup (able ). Moreover, worseningo lipid prole hasbeenassociated with insulin resistance []. Te total cholesterolwas signicantly reduced by simvastatin and % EBN group( < 0.05). As seen rom other cholesterol indices in the table,

simvastatin, which is used to manage hypercholesterolaemiawas able to improve lipid prole but not as well as % EBNtreatment. Furthermore, Figure shows the OG resultsor the intervention groups. Te glycemic response or thediabetic untreated group was higher than other groups (

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Edible Bird’s Nest Attenuates Procoagulation Effects of High-Fat Diet in Rats