Effects
of
high
fat
diet
on
salivary
a-amylase,
serum
parameters
and
food
consumption
in
rats
Le´nia
Rodrigues
a,
Raquel
Mouta
b,
Ana
Rodrigues
Costa
c,
Alfredo
Pereira
d,
Fernando
Capela
e
Silva
e,
Francisco
Amado
f,
Ce´lia
M.
Antunes
g,
Elsa
Lamy
h,*
aInstitutode CieˆnciasAgra´riase AmbientaisMediterraˆnicas (ICAAM),Universidade de E´vora,7002-554 E´vora,
Portugal
bICAAM,UniversidadedeE´vora,7002-554E´vora,Portugal
c
ICAAMe Departamento deQuı´mica, Escolade Cieˆnciase Tecnologia, Universidadede E´vora, 7002-554E´vora,
Portugal
dICAAMe DepartamentodeZootecnia, EscoladeCieˆnciase Tecnologia,UniversidadedeE´vora,7002-554E´vora,
Portugal
eICAAMe Departamento de Biologia,Escola de Cieˆnciase Tecnologia, Universidade de E´vora,7002-554 E´vora,
Portugal
f
Quı´micaOrgaˆnica,ProdutosNaturaiseAgro-Alimentares(QOPNA)eDepartamentodeQuı´mica,Universidadede
Aveiro,Aveiro,Portugal
gCentrodeNeurocieˆnciaseBiologiaCelular,UniversidadedeCoimbra,Coimbra,Portugal
hICAAM,UniversidadedeE´vora,702-554,E´vora,Portugal
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Accepted20February2015 Keywords: Weightgain High-fatdiet Leptin Rats Salivaryamylasea
b
s
t
r
a
c
t
Salivarya-amylase,amajorproteininsaliva,hasbeendescribedasamarkerfor
sympa-theticnervoussystem activity,henceformetabolicenergy balance.Inthiscontext,its
expressioninoverweight andobesityis ofinterest.Ratsfedwith adietenriched with
sunfloweroildifferentiallygainedweightyieldingtwosubgroupsaccordingtotheir
sus-ceptibility(OP)orresistance(OR)toobesity.ElevatedplasmaticlevelsofleptinintheOP
subgroupandalteredplasmaticlipidprofiles(lowertriglyceridesandhighertotal
choles-terol/HDLratiocomparedtocontrols) inORsubgroupwereobserved.AnimalsfromOP
subgroup presentedhighera-amylase expressionandactivityevenpriortothedietary
treatment, suggestingthatthis salivary proteinmay constitutea putative indicator of
susceptibilityforfattissueaccumulation.After18weeks ofhigh-fatdietconsumption,
salivarya-amylaselevelsdidnotsignificantlychangedinOPsubgroup,butincreased3-fold
inORsubgroup.Theraiseofa-amylaseforthelattermightrepresentanadaptationtolower
starchintake.Theseresultssuggestthatsalivarya-amylasesecretionmightbeusefulto
predictsusceptibilityforweightgaininducedbyhigh-fatdietconsumption.
#2015ElsevierLtd.Allrightsreserved.
*Correspondingauthorat:ICAAM,UniversidadedeE´vora,702-554,E´vora,Portugal.Fax:+351266760841.
E-mail addresses: lrodrigues-88@hotmail.com (L. Rodrigues), rakymouta@hotmail.com (R. Mouta), acrc@uevora.pt (A.R. Costa),
apereira@uevora.pt(A.Pereira),fcs@uevora.pt(F.CapelaeSilva),famado@ua.pt(F.Amado),cmma@uevora.pt(C.M.Antunes),
ecsl@uevora.pt(E.Lamy).
Available
online
at
www.sciencedirect.com
ScienceDirect
journalhomepage:http://www.elsevier.com/locate/aob
http://dx.doi.org/10.1016/j.archoralbio.2015.02.015
1.
Introduction
Duringthepastdecades,obesityprevalencehasenormously
increased worldwide, reaching epidemic proportions in
Europe.1 Animbalance between energyintake and output,
manytimesinvolvingtheconsumptionofhighamountsoffat,
isthemainreason.
Sunflower oil is one of the most consumed liquid fats
worldwide,mainlyusedforfryingfoodsandinsalads,being
preferredbymanypeopleforitsneutraltaste.2Sunfloweroilis
richinunsaturatedfattyacids,suchaslinoleicacid(n-6PUFA)
andoleicacid(n-3-PUFA).3Despitesomecontroversy,oilswith
highcontentsofunsaturatedfattyacidsmayinduceweight
gainwithoutmostofthedeleteriouseffectsassociatedwith
saturatedfat. Some studies reported that diets rich inn-6
PUFA decrease plasmatic levels of triacylglyceroland total
cholesterol,aswellasliverfat,improvingmetabolicstatus,4
insulinsensitivity5,6andcardiovascularfunction,7
compara-tivelytosaturatedfattyacids.
Salivahasavarietyofproteinswithimportantrolesinoral
health and food perception, some of which have been
suggestedtoinfluenceingestivebehaviour.8,9Theinterestin
the studyofthis fluid increasedinthe last decade due to
methodologicaldevelopments,whichrevealeditspotentialas
a source of biomarkers of pathological and physiological
conditions,helpingintheunderstandingofbiological
mecha-nisms.10Nonetheless,relativelyfewstudieswerefoundabout
salivaryproteincompositioninobesity.11
The levels of salivary a-amylase, a protein present in
considerableamountsinmixedsalivaandresponsibleforthe
hydrolysisof starch, aregenerally referred as amarker of
stress and an indicator of sympathetic nervous system
activity,12whichinturnplaysanessentialroleinmetabolic
homeostasis.Severalstudieshavefoundelevatedlevelsofthis
salivary proteinconcomitant to overall changes in
norepi-nephrineinresponsetostress.13Automomicnervoussystem
influencestheregulationofsalivarysecretion,with
sympa-theticnervoussystemactivationdecreasingsalivaryflowand
increasingsalivaryproteinoutput.Thestimulationof
beta-adrenergicreceptorsfromsalivaryparotid glandsincreases
alpha-amylasesecretion.14
The relation between obesity and sympathetic nervous
systemactivityhasbeensomehowcontroversial.Lowactivity
ofthis branch ofthe autonomic nervous system hasbeen
suggested to be a risk factor for weight gain and obesity
development.15 However, some evidences point that the
sympathetic neural transmitter neuropeptide Y (NPY) is
involved in stress-induced augmentation of diet-induced
obesity.16 From our knowledge it has not been addressed
whether the expression and enzymatic activity of salivary
alpha-amylasediffersinobesity.
Similarly to humans, in some rodent strains there are
subjectsproneandsubjectsresistant toobesityinduced by
high-fatdietconsumption.17Althoughthefactorsunderlying
this different propensity are yet poorly understood, these
animalsconstituteagoodmodeltostudyobesity.Thisstudy
hadthreemainobjectives:toinvestigatehowsalivary
alpha-amylaselevelsrelatetoratssusceptibilityforweightgain;to
evaluatetheeffectsoftheconsumptionofahigh-fatdietin
salivary alpha-amylase secretion; and to assess the
indivi-dual’sresponsestohigh-fatdiet,byevaluatingbiochemical
markersofmetabolicstatus.
2.
Materials
and
methods
2.1. Treatmentofanimals
Twenty outbredfemale Wistarrats4weeksold (160–223g)
weremaintainedona12h/12hlight/darkcycleataconstant
temperature(228C)andhumidity(65%)withadlibitumaccess
tofoodandwater.Theanimalsweresinglecagedinstandard
ratcagesandmaintainedonastandarddiet(SAFEA04)for4
weeksacclimation. Afterthattimeanimalswererandomly
dividedintwodietarygroups(controlN=6andsunfloweroil
treated N=14), with the control group receiving the same
standarddietofthepre-trialperiodandthetreatmentgroup
receivingahighfatdietrichinsunfloweroilduring18weeks,
bothadlibitum.
Theexperimentalhigh-fatdiet waspreparedbysoaking
thestandardfeedinsunfloweroilduring4days,followedby
dryinginanoven(temperaturebelow408C)andthenstored,
protectedfromlightandoxygen.Sunfloweroilusedtoprepare
thehigh-fatdietwasobtainedfromacommercialtrademark
availableforhumanconsumption.Fattyacidcompositionwas
measuredbygaschromatography(SupplementaryTableS1).
Analyses of the macronutrient content ofthe usedrodent
dietswereperformedintheLaboratoryofAnimalNutritionof
the ICAAM,UniversityofE´vora:sunfloweroilenricheddiet
containing 14.5% total protein, 48.2% carbohydrates, 14.7%
totalfat,withanenergeticcontentof3.8kcal/gandstandard
dietcontaining17.4%totalprotein,53.7%carbohydrates,2.7%
totalfat,withanenergeticcontentof3.1kcal/g.
Supplementary material related to this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
archoralbio.2015.02.015.
Duringboththeacclimationandexperimentalperiodsthe
animalswereweighedweeklyandfoodconsumption
moni-tored,byweighingtheamountdistributedandtherefusals.At
theendoftheexperimenttheanimalswereeuthanizedby
exsanguination under anaesthesia and blood collected for
furtheranalysis.Allanimalprocedureswereapprovedbythe
scientific committee, and were supervised by a scientist
trained by the Federation of European Laboratory Animal
ScienceAssociations(FELASA),andconformedtoPortuguese
law,18 which followed European Union Laboratory Animal
ExperimentationRegulations.
2.2. Oralglucosetolerancetest
Oral glucose tolerance test (OGTT) was performed at the
beginning, in the middle and at the end of dietary
treatment.Afterovernight(16-h)fasting,bloodsampleswere
collected from the tail vein followed by oral
adminis-trationofglucose(75g/kgofbodyweight).Bloodglycaemia
wasmeasuredinfastandafter30,60,90and120minafter
glucosebolusadministration,usingahand-heldglucometer
(Accu-Check, Roche). Whole blood glucose levels were
2.3. Serumbiochemicalparameters
Bloodobtainedbyheartpuncture,after18weeksexperiment,
was collected in EDTA coated tubes and centrifuged at
3000rpmandroomtemperaturefor15min.
Plasmaconcentrationsofinsulinandleptinweremeasured
byenzyme-linkedimmunosorbentassay(ELISA).Forinsulina
competitive ELISA was used [Mouse anti-ratinsulinserum
(ref. BT53-3033-39 Linco Research, USA) and rat-insulin
standards (ref.813K,Linco, Research, USA)] whereasleptin
determinationwasperformedusingacommercialkit
accord-ingtothemanufacturer’s instructions(EZRL-83KMillipore).
PlasmaconcentrationsoftotalandHDLcholesterol,aswellas
triglyceridesweremeasuredusingenzymatictests(Sentinel
Diagnostics, Milano, Italy). Precision and reproducibility of
measurementsweremonitored byusingamulti-parameter
control for quantitative clinical chemistry determinations
(Clin Chem Control 1, and Clin Chem Control 2, Sentinel
Diagnostics,Milan,Italy).
2.4. Salivasamplecollectionandproteinquantification
Whole mouth saliva was collected from each individual
animalunderparasympatheticstimulation,toincreaseflow,
facilitating saliva collection. Animals were injected with
pilocarpine (7mg/kgbody weight s.c.)and saliva aspirated
fromthemouthusingamicropipetteandcollectedtoatube
maintainedonice.Althoughpilocarpineadministrationcan
leadtosmallincreasesinamylaseactivity,thedoseusedwas
thesame forall theanimals, toassurethatanychange in
protein levels would only reflect experimental treatment.
Collections were performed on each individual in two
differentdaysbeforethebeginningofthedietarytreatment
andattheendofthe18weeksexperimentalperiod.Thetwo
individual different collections from the beginning were
pooled.Aftersalivacollection,sampleswerecentrifugedat
13,000g,for5mintoremovefoodandcelldebris,
superna-tantaliquotedand storedat 808Cuntilanalysis. Bradford
method (BioRad Protein Assay) was used for total protein
quantification.
2.5. Westernblot
ForWesternblot,SDSPAGEseparationof3mgproteinfrom
eachsamplewasperformedusing12%gelsrunataconstant
voltageof100V.Theseparatedproteinsweretransferredtoa
PDVFmembranebyelectroblottingusingaTris-glycinebuffer
system,andtheproteinpatternswerevisualizedbystaining
withPonceauSdye(0.3%w/vin10%aceticacid)for10min.
Theproteinprofilesweredevelopedbywashingawayexcess
dyewithdistilledwater.ForWesternblotthemembranewas
incubated withanti-a-amylase primaryantibody(Sc-46657,
SantaCruzBiotechnology;1:200dilution).Asolution
consist-ing in5% non-fatdry milk inTBS-Tween20 was used for
blocking,for2h,withagitationatroomtemperaturefollowed
by incubation with primary antibody overnight at 48C.
Salivary amylase bands were detected with an alkaline
phosphatase-linkedsecondaryantibodyagainstmouseIgG
(anti-mouse,GEHealthcare,1:10,000dilution)usinga
chemi-fluorescent substrate(ECL Plus Western Blotting Detection
Reagents,GE,Healthcare).Membraneswererevealedusinga
transiluminator(Bio-RadGel-docsystem)anda
semi-quanti-tativeanalysisofsalivaryamylaseexpressionwascarriedout
byusingQuantityOnesoftware(BioRad).
2.6. Salivarya-amylaseenzymaticactivity
Dinitrosalicylic acid assay19 was used for measuring the
starch-hydrolyzing activityofsalivarya-amylase.The
reac-tionmixtureconsisted100mLof1%starchsolution,100mLof
20mMphosphatebuffer(pH7.0)and50mLsaliva(dilutedtoa
proteinconcentrationinenzymaticassayof1.5–2.5mg/mlin
the same phosphate buffer). After incubation at 378C for
20min,inathermostablewaterbath constant stirring,the
reactionwasstoppedbytheadditionof100mLDNSreagent
(1%dinitrosalicylicacid,0.2%phenol,0.05%sodiumsulfite,1%
sodium hydroxide). Samples werethen placed in aboiling
waterbathfor5min,andsubsequentlycooleddowntoroom
temperature. Further, 200mL of sodium and potassium
tartrate (40%)and 900mLofbidistilledwaterwereaddedto
samples.Absorbancewasmeasuredusinga
spectrophotome-ter(HitachiU200)at540nmagainstablankpreparedusingthe
identical method, except the enzyme solution, which was
addedtothemixtureaftertheadditionoftheDNSsolution.
The absorbance values were then converted to glucose
equivalentusingastandardcurve(0.2–2.0mg/mL).Theresults
were expressedasmmol glucose/min/mgproteinand asa
percentageofcontrol(groupreceivingstandarddiet)
consid-eredas100%.
2.7. Statisticalanalysis
Considering theobjectives ofthestudy,differentstatistical
approacheswerecarriedout.Aftertheendofexperiment,the
measured and calculated variables were analyzed with
descriptive statistics. Subsequent analysis was carried out
usingamediananalysisandestablishingarankingaccording
tothepercentiledistributionofthevariables:weightatthe
endofthetrial,weightgainthroughoutthetestandweight
gainrelativetotheinitialweight.Theanimalsfromsunflower
oiltreatmentweredividedintotwosubgroupsbasedonthe
resultsofthepreviousanalysis.Thenormalityand
homosce-dasticity were tested respectively by Kolmogorov–Smirnov
and Levene methodologies.Variables such asbody weight,
totalfoodconsumptionandtotalenergyconsumptionwere
testedaccordingANCOVA,withtheinitialbodyweightasa
covariant.Serumbiochemicalparametersweretested
accord-ing ANOVA-GLM. When significantdifferences werefound,
posthocpoolingwasperformedbyTukey’smethod.Pearson
correlations between different variables were carried out,
takingintoaccountthelevelofsignificanceandthevalueofR.
AllstatisticalanalyseswereperformedbySPSS20software.
3.
Results
3.1. Bodyweightandfoodintake
At the end of acclimation time (week 0), animals were
(N=6)andhigh-fattreated(N=14)],whichreceiveddietary
treatmentfor18weeks.Atthistimemeanbodyweightsfrom
controlandfromthegrouptreatedwiththehighfatdietdid
notpresentsignificantdifferences(Fig.1A).
Afterthe 18 weeks all animals, including controls, had
gainedweight.However,atthistime,fortheanimalsfromthe
high-fat dietarygroup, there was a largedispersion inthe
values, suggesting the existence of animals with different
propensitytoweightgain.Theratiobetweentheweightgain
and initial body weightwas usedtoallocateanimals from
high-fatdietarygroupintwosubgroups.Sincequitedifferent
ratios were observed, medians were used to create more
homogeneousgroupsaccordingtotherespectivepercentiles.
AnalysisofvariancewasoneANCOVAtocorrectthepossible
effectofthedifferentinitialbodyweights.Thetwodistinct
subgroups,fromthehigh-fattreatmentgroupwere
constitut-edby:(i)individualswhosebodyweightgainsandfinalbody
weightsremainsimilartocontrolgroup(obeseresistant,OR;
N=7);(ii)individualsforwhichbodyweightgainsandfinal
bodyweightswereatleast10%higherthanincontrolgroup
(obeseprone,OP;N=6)(Fig.1andTable1),accordingtothe
criteriareportedbyothers.20Oneratfromthehigh-fatdiet
treatmentgroupwasexcludedfromtheanalysisbecauseit
wasidentifiedasoutlier.Thiswasduetoitsabnormalweight
gainrelativelytoitsfinalbody weight,asevidencedbythe
statisticalanalysis(abnormalvalueinthebox-plotafterusing
the Z-score procedure afterwards confirmed by the Dixon
test).
Food intake differed among the groups. The animals
maintainedonthehigh-fatdietingestedaloweramountof
food compared to controls (2.600.06kg control vs
2.380.08kg for OP and 2.120.039kg for OR groups
treatment;valuesforthetotalamountconsumedinthe18
weeksofexperiment).Consideringtotalenergyconsumption,
Fig.1–Initialbodyweights(A)andgrowthrate(B)ofcontrol(Cont;N=6)andtreatment(HFD;N=13)groups.OR=obese
resistant(N=7);OP=obeseprone(N=6).WeekS3representsthetimeatwhichtheanimalswereallocatedtoindividual
cagesforacclimation,whereasweek0representsthestartofhigh-fatdietadministrationtorandomlyselectedanimals.
DataarepresentedasmeansWSEM.(*significantforP<0.05)(ANCOVAanalysisusingtheinitialbodyweightasa
covariant).
Table1–Bodyweightandfoodconsumption.
Control OP OR
Bodyweight(week 3)(g) 127.511.0 156.86.0 133.48.6
Bodyweight(week0)(g) 182.19.5a 211.54.4a 187.19.2a
Finalbodyweight(g) 268.74.0a 314.25.0b 267.38.4a
Totalfoodconsumption(kg) 2.600.06a 2.38
0.08b 2.12
0.04b
Totalenergyconsumption(Kcal)# 8.07
0.19a 9.06
0.30b 8.06
0.15a
OR=obeseresistant;OP=obeseprone.ValuesaremeansSEM.
a,bForeachparameter,differentlettersrepresentsignificantdifferencesamongmeans.MeancomparisonswereperformedusingtheANCOVA
analysiswiththeinitialbodyweightascovariantandP<0.05.
# Calculatedvaluesaccordingtotheenergycontentafternutritionalanalysis(3.8kcal/gforthehighfatdietand3.1kcal/gforthestandard
however, the caloric intake was 12.3% higher in the OP
subgroupcompared toboth the controland OR subgroups
(Table1).
3.2. Bloodbiochemicalparameters
Allanimalspresentednormaloralglucosetolerancetestsboth
atthebeginning andattheendoftheexperiment.Inboth
timesthemeanglycaemiclevels,fromallthegroups,ranged
from 68 to 90mg/dL glucose following overnight fast,
increasedto100–115mg/dL30minaftertheadministration
oftheoralbolusofglucoseandreturnedtofastingvaluesat
60min (results not shown), indicating that no significant
differencesinglucosetolerancewereinducedbythe
sunflow-er oil enriched diet. Accordingly, fasting plasmatic insulin
levels,attheendofthedietarytreatment,weresimilaramong
the groups (Table 2). Concerning fasting plasmatic leptin
levels,differenceswereobserved,withOPsubgroup
present-ingvaluesalmosttwicehigherthantheORsubgroup(Table2).
Beingleptinahormonethatpromotessatiety,therelation
betweenleptinplasmaticlevelsandenergyconsumptionwas
investigated.Astrongpositivecorrelationwasobservedfor
theanimalsfedthehighfatdiet(r=0.71;P=0.003),i.e.,higher
plasmatic leptin levels wereassociated withhigher energy
consumption(Fig.2B),indicatinghigheringestion.
The consumption of the sunflower oil enriched diet
induceddifferentialchangesinlipidprofilesamongORand
OP subgroups. A 40% decrease in the plasmatic levels of
triglycerideswasobservedinORsubgroupwhileOPpresented
values similar to the onesfrom controls. Concerning total
cholesterolnodifferenceswereobservedbetweenthegroups.
However, high-density lipoprotein (HDL) cholesterol was
approximately30%lowerintheORsubgroupand,accordingly,
totalcholesteroltoHDLratiowassignificantlyincreasedin
thissubgroup(Table2).
3.3. Salivarya-amylaseexpressionlevelsandenzymatic
activity
Themainaimofthepresentexperimentwastostudysalivary
a-amylasesecretioninindividualswithdifferent
susceptibili-tiestoobesity.Differencesinthissalivaryproteinexpression
levels and enzymatic activity were observed among the
experimental groups. Beforethe beginningofdietary
treat-ment, whereallanimalswerefedstandardrodentdiet,OP
subgroup presented higher expression levels of a-amylase
whencomparedtoboththecontrolandORgroups,beingthese
lasttwogroupssimilar(Fig.3A).
Afterthechronicconsumptionofsunfloweroilenriched
diet, OR subgroup presented an increased expression of
salivarya-amylasecomparativelytotheotherexperimental
groups(Fig.3B).Itisnoteworthythatwhencomparedtothe
expressionlevelspriortothetreatment,a-amylaseexpression
wassignificantlyincreasedintheORsubgroup,whereasitwas
maintainedinthecontrolandOPsubgroup(Fig.3B).
In concordance withprotein expression,the salivary
a-amylase enzymatic activity from whole saliva collected at
thebeginningofthetrial(whenanimalswereallmaintained
on astandard rodentchowdiet) was1.6 foldhigherinOP
individuals(Fig.4).
At this time point, a-amylase enzymatic activity was
stronglycorrelatedwithfutureindividualbodyweightgains
(r=0.70;P=0.032),i.e.,individualswhichgainedmoreweight
duringthe18weekshighfatdiettreatmentpresentedhigher
salivary a-amylase activities (Fig. 5A). Interestingly, this
relation (Pearson correlation coefficient) is even higher
considering only the animals that fed the high fat diet
(r=0.76;P=0.042)(Fig.5B).
Inthecontrary,attheendofthedietarytreatmentthe
a-amylaseactivityinOPsubgroupwassimilartothecontrols
while in the OR subgroup a-amylase activity was 3-fold
higher.Noticethat,comparedtothebeginningofthetrial,
salivarya-amylaseenzymatic activityinORsubgroupwas
increased3times(Fig.4).
Table2–Serumbiochemicalparametersmeasuredafter18weeksofahighfatorcontroldiet.
Control OP OR Refvalues#
Insulin(ng/mL) 0.750.02a 0.750.07a 0.850.05a 0.60–0.9021,22 Leptin(ng/mL) 1.690.17a,b 2.390.41b 1.340.17a 1.37–2.2923 TG(mg/dL) 79.16.8a 61.07.3a 45.32.8b 40.0–230.024,25 Totalcholesterol(mg/dL) 127.09.4a 129.6 7.4a 119.3 8.8a 70.0–125.025 HDL(mg/dL) 60.33.6a 52.7 2.5a 43.5 5.2b 38.0–63.04 Totalcholesterol/HDL 2.120.14a 2.480.14a 2.860.27b
OR=obeseresistant;OP=obeseprone.DataarepresentedasmeanSEM.
# NormalmeanvaluesforfemaleWistaradultrats.
a,bForeachparameter,differentlettersrepresentsignificantdifferencesamongmeans.MeancomparisonswereperformedusingANOVA
analysisforP<0.05.
Fig.2–Correlationbetweenplasmaticleptinlevelsand
4.
Discussion
Theexistenceofindividualswithpropensityandindividuals
with resistance to weight gain has been referred for both
animalsandhumans.InWistarratsthisdifferentialresponse
in body weight changes has been already referred for
individuals submitted to high saturated fat diets26,27 and
hasbeenattributedtodifferentcausessuchas
overconsump-tion,duetothesedietshighpalatability,ortothemodulation
ofgene expression linked to lipogenesis.28 Studies are not
consensual in terms of the potential of diets rich in
polyunsaturatedfatforobesitydevelopment.Someauthors
refersimilar29andotherslower30bodyweightgains,induced
byPUFAcomparativelytotheonesinducedbysaturatedfat.
In the present study, a differential individual response in
the rateofweightgain wasobservedforratsconsuminga
high-fat-enriched with sunflower oildiet.Despite beingan
unsaturatedfat,thesunfloweroilusedinthisstudycontainsa
considerableamountofn-6PUFAovern-3PUFA,theopposite
ofotheroilssuchasoliveorrapeseedoils,3andakeyfactorin
high-fatinducedobesity.31Ourresultssupportthefactthata
high-fat diet rich in n-6 PUFA induce weight gain and
eventually obesity in animals with genetic susceptibility,
suchastheOPsubgroup.
Despiteobesity developmentinsusceptibleanimals, the
effectsofthetesteddietappearednottobeasdeleteriousas
theonesproducedbysaturatedfat.Highamountsofsaturated
dietary fats lead to insulin resistance in rats32 what can
Fig.3–RepresentativeWesternblotanalysisofa-amylase
inmixedsalivasamplesfromcontrol(C),obese-prone(OP)
andobeseresistant(OR)animals.A:beforedietary
treatment;B:after18weeksdietarytreatment;Lower
panelsshowa-amylaseexpressionquantifiedby
densitometricanalysisofthisproteinimmunoreactive
band.DataarepresentedasmeanWSEM.(*differences
betweenhighfatdietsubgroupsandcontrolare
significantforP<0.05).
Fig.4–Salivaryamylaseactivityofmixedsalivasamples
beforeandafterdietarytreatment.OP=obeseprone;
OR=obeseresistant.DataarepresentedasmeanWSEM.
P<0.05.
Fig.5–Correlationbetweenbodyweightgainsanda-amylaseenzymaticactivityfromsalivacollectedbeforehigh-fat
ultimatelyresultinalteredglucosetoleranceanddiabetes.In
thepresentstudy,nochangesinoralglucosetolerancetestsor
fastinginsulinemiawereobservedasaconsequence ofthe
consumptionofthishighfatdietconsumptionfor18weeks.
Thefattyacidcompositionmaybeapossibleexplanationfor
theseresults,sincesunfloweroilisrichinpolyunsaturatedfat,
therefore not inducing insulin resistance, as suggested by
others.33
Accordingly with fasting leptin levels, it is possible to
hypothesize that the weightgain observedin someofthe
animals(OPanimals)canbetheresultofadegreeofleptin
resistanceby these. Leptinis mainly produced by adipose
tissueandishighlyinvolved inthecontrolofingestion by
inducing satiety. Despite the increased levels ofplasmatic
leptininOPanimals,higherenergyintakewasobservedinthis
subgroupsuggestingareducedresponsetothishormoneand
aconsequentlackofthefallinsatiety,afactoralreadypointed
outasresponsiblefordietinducedobesity.34
Lipid profiles were accessed with the aim of adding
informationonthemetaboliceffectsofthefatused.These
effectswereobservedtobedependentonthepropensityfor
obesity. In OR subgroup the dietary treatment induced a
decreaseintriacylglycerolandHDLlevels.Themaintenanceof
totalcholesterolresultedinanincreaseoftotalcholesterol/
HDLratiointhissubgroup.Similarresultswerereportedby
other authors for sunflower oil enriched diets, for both
triacylglycerol levels35 and total cholesterol/HDL ratio.36
Cholesterol is precursor of corticosteroids and androgens,
which increased levels are ultimately related to insulin
resistance.37,38Althoughinsulinemiasandglucosetolerance
curvesweresimilartotheonesfromcontrolindividuals,as
discussedabove,thehypothesisofORindividualsbecameless
sensitive to insulin and/or present metabolic changes, if
maintainedforalonger period inahigh fatdiet, isnotto
discard.Bythecontrary,lipidprofilesfromOPindividualsdid
notdifferfromcontrolanimals.Besidesconcludingthatthe
polyunsaturatedhighfatdietdidnotinducesevere
dyslipi-daemias in either OP or OR subgroups, for the period
considered inthis study, it is worthwhileto note thatthe
effectsofpolyunsaturatedfatarecomplexandshouldnotbe
generalized,butinsteadshouldhaveintoconsiderationthe
metaboliccharacteristicsofeachindividual,wherethegenetic
backgroundiscertainlyplayingacentralrole.
In the present study differences in the expression and
enzymaticactivityofsalivarya-amylase,bothbeforeandafter
thehighfatdiettrialwereobservedamongthegroups.The
a-amylaselevelsmeasuredbeforeanimalsenteredthetrialwas
higher in individuals which were further allocated to OP
subgroupandwerestronglycorrelatedwiththerateofbody
weightgainachievedduringtheexperiment.Consequently,
thelevelsofthisproteinseemtobehaveasapredictorofthe
susceptibilityindividualshave tobecomeobese.Themajor
roleofthissalivaryproteinintheoralcavityistohydrolyse
starchto maltose and maltotriose.It has been stated that
considerablestarchhydrolysisoccursintheoralcavity39and
cancontinueafterswallowing,duetothepresenceofpartially
digestedstarch,whichappearstoprotectsalivarya-amylase
from acid inactivation.40 Therefore, higher levels of this
salivaryenzyme could leadtoahigherefficiency instarch
digestion,whatcanbehypothesizedtoincreasecarbohydrate
absorption,contributingtothegreaterincreaseinbodyweight
observedinOPanimals.
Salivary a-amylase has been proposed as a marker of
sympatheticnervoussystemactivity.12Stimulationof
beta-adrenergic receptors results in increases in salivary total
proteinanda-amylaseoutput.14Basedonthat,wecanassume
thatOPanimalspresentedahigheractivityofthisbranchof
autonomic nervous system, at an early age. Sympathetic
nervous system activity is related to energy homeostasis,
increasing energy expenditure by stimulating adrenergic
thermogenesis, which in turn results in expending energy
as heat.41 However,a chronic sympatheticstimulationhas
been suggested to result in desensitization of the
beta-adrenergicsignallingpathway,whichhasbeenconsidereda
possible explanation for weight gain increases with age.42
Moreover,fromourobservations,attheendofthe18weeks
experiment OP animals did not present a-amylase levels
augmented,evenpresentinghigherbodyweightsandhigher
plasmaticleptinlevels.Thisalsogoesinaccordancewiththe
beta-adrenergicdesensitizationhypothesis.
Also,itwasobservedthatwhiletheORanimalsadjusted
foodintaketofulfiltheircaloricneedstheOPanimalsdidnot.
Indeed the OP subgroup presented a significantly higher
caloricintakecomparedtobothORandcontrolsubgroups,
hence contributing to their augmented weight gain. The
reasonsforfood consumptionabove theirneedsin theOP
subgroupareunknownbutanincreasedpalatability,widely
reportedasadrivetoconsumption43duetothehighersalivary
a-amylaselevelsmightbeonecontributingfactor.
Anotherimportantfindingwasthata-amylasesecretionis
3-foldincreasedafter18weeksconsumingthehighfatdietin
the subgroup resistant to weight gain. Since OR animals
consumedloweramountsofstarchthantheotherindividuals,
the increase in a-amylasesecretion might be an adaptive
responsemechanismtoincreasedigestiveefficiency.
More-over,sympatheticnervoussystemactivityincreaseswiththe
consumptionofhighfatdiet17and,inthesamelineofthought
describedabove,sinceORanimalscontinuestobesensibleto
adrenergicstimulation,itisnotsurprisingariseina-amylase
secretion.
Nonetheless,theuse ofsalivarya-amylaseasmarkerof
sympathetic activity has some limitations44 and further
studiesareimportanttoelucidatethemeaningofincreases
inthissalivaryproteinexpressioninducedbyhighfatdiets
onlyinanimalsresistanttoweightgain.
Finally, from our knowledge, although saliva has been
extensively exploredas a sourceofbiomarkers for diverse
pathologicalconditions,thereisalackofstudiescomparing
salivaryproteincompositionaccordingtoindividual
propen-sity forobesity.Ourresultssuggestthatsalivarya-amylase
secretion differ between individuals with and without
propensitytoweightgainandthatitmaybemeaningfulin
increasingunderstandingaboutobesitydevelopment.
More-over,arecentstudydemonstratedthatsalivaryamylasegene
(AMY1) copy number variations predisposes to obesity.45
Although,nosalivaryproteinexpressionwasaccessedinthis
study,itemphasisestheimportanceofthissalivaryprotein
relatedtoweightgainandBMI.Furtherstudieselucidatingthe
mechanismswhichresultinthisobservedhighersecretionof
othersalivaryproteinsinapre-obesitystate,canultimately
allowearly dietaryand behaviouralinterventions,avoiding
thepathologicalstate.
5.
Conclusion
In summary, chronic consumption of high amount of
sunfloweroildifferentiallyinducedobesityinrats,according
to individual propensity, without producing the negative
effects usually attributed to saturated fat, namely insulin
resistanceand/ordyslipidaemia.Itisconcluded that saliva
maybeusefultoevaluatesusceptibilityforobesity.Salivary
a-amylaselevels in young animalsarerelated tothe rate of
futureweightgain.Althoughthemechanismsresponsiblefor
thedifferentialexpressionofsalivarya-amylaseaccordingto
obesitysusceptibilityneedtobeexplored,theobtainedresults
showthatsalivacompositionrelatestotheamountofweight
gain.Moreover,theconsumptionforseveralweeksofahigh
fatdiet evokedanincrease in salivarya-amylase levels in
individualsresistanttoobesity,possiblyasanadaptationto
optimizethe usageofapoorer starchdiet.Takingtogether
these results reinforce the notion that salivary a-amylase
levelsaredependenton dietcharacteristicsandemphasize
thepotentialofsalivainstudiesaboutsusceptibilityforweight
gainandobesitydevelopment.
Funding
ThispaperisfundedbyFEDERFundsthroughtheOperational
ProgrammeforCompetitivenessFactors-COMPETEandNational
Funds through FCT-Foundation for Science and Technology
under the Strategic Projects PEst-C/AGR/UI01, PEst-OE/AGR/
UI0115/2014,15/2011,PEst-C/SAU/LA0001/2011andPEst-C/QUI/
UI0062/2011.Authors acknowledgealso thefinancial support
fromthePortugueseScienceFoundation(FCT)intheformof
Post-Doctoralgrant(SFRH/BPD/63240/2009)ofElsaLamy.The
Portu-gueseScienceFoundation(FCT)playednoroleinthe
develop-mentofthepresentworkoruponitssubmissionforpublication.
Conflict
of
interest
statement
Theauthorshavenopotentialconflictsofinterest.
Ethical
approval
All animal procedures were conformed to Portuguese law
(PORTARIA1005/92),whichfollowedEuropeanUnion
Labora-tory Animal Experimentation Regulations and were
super-visedby a scientist trained by the Federationof European
LaboratoryAnimalScienceAssociations(FELASA).
Acknowledgments
Theauthors thanks toDoutoraRaquelGarcia and Doutora
MariaIsabelFerrazdeOliveira,fromICAAM,forsunfloweroil
fattyacidcompositiondeterminationanddietmacronutrient
analysis,respectively.
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