Hyperleptinemia in obese adolescents deregulates neuropeptides during weight loss

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ContentslistsavailableatScienceDirect

Peptides

jo u rn al h om epa g e:w w w . e l s e v i e r . c o m / l o c a t e / p e p t i d e s

Hyperleptinemia

in

obese

adolescents

deregulates

neuropeptides

during

weight

loss

Ana

R. Dâmaso

a,b,∗

_,

_Aline

_de

_Piano

c

_,

_Priscila

_L.

_Sanches

c

_,

_Flávia

_Corgosinho

c

_,

_Lian

_Tock

c

_,

Lila

M. Oyama

d

_,

_Luciana

_Tock

e

_,

_Claudia

_M.

_Oller

_do

_Nascimento

d

_,

_Sérgio

_Tufik

f

_,

Marco

Túlio

de

Mello

f

a_Biosciences_Department,_Federal_University_of_São_Paulo_{– Paulista}_Medicine_School_–_UNIFESP-EPM,_São_Paulo,_Brazil

b_Post_Graduate_Program_of_{Interdisiciplinary}_Health_Sciences,_Federal_University_of_São_Paulo_–_Paulista_Medicine_School_–_UNIFESP-EPM,_São_Paulo,_Brazil c_Post_Graduate_Program_of_Nutrition,_Federal_University_of_São_Paulo_–_Paulista_Medicine_School_–_UNIFESP-EPM,_São_Paulo,_Brazil

d_Physiology_Department,_Federal_University_of_São_Paulo_–_Paulista_Medicine_School_–_UNIFESP-EPM,_São_Paulo,_Brazil e_{Endocrinology}_Department,_Federal_University_of_São_Paulo_–_Paulista_Medicine_School_–_UNIFESP-EPM,_São_Paulo,_Brazil f_{Psychobiology}_Department,_Federal_University_of_São_Paulo_{– Paulista}_Medicine_School_{– UNIFESP-EPM,}_São_Paulo,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received15March2011

Receivedinrevisedform28April2011 Accepted28April2011

Availableonline6May2011

Keywords: Leptin Obesity Neuropeptides Adolescent

a

b

s

t

r

a

c

t

Leptinhasemergedoverthepastdecadeasakeyhormonenotonlyinenergybalanceregulationbutalso inneuroendocrineandinflammatoryprocesses.Theaimofthepresentstudywastoevaluatewhether hyperleptinemiaderegulatesneuropeptidesduringweightloss.Atotalof86post-pubertalobese adoles-cents(withorwithouthyperleptinemia)participatedinoneyearofinterdisciplinaryweightlosstherapy (clinical,nutritional,psychologicalandexercise-related).Adipokineandneuropeptideconcentrations weremeasuredbyELISA,visceralfatwasmeasuredbyultrasoundandbodycompositionwas mea-suredbypletismography.Thehyperleptinemicpatientspresentedaloweralpha-MSHconcentration andhigherNPY/AgRPratiowhiletheadiponectin/leptin(A/L)ratiowaslowercomparedwiththe non-hyperleptinemicgroup.Aftertherapy,significantimprovementsinBM,BMI,bodyfatmass,visceraland subcutaneousfat,HOMA-IR,QUICKI,totalcholesterolandtriglycerideswereobservedinbothgroups. Indeed,weobservedsignificantincreasesinadiponectinandA/Laswellasreductionsinleptinand NPY/AgRPratiointhehyperleptinemicgroup.Inthestepwisemultiplelinearregressionanalysiswith leptinconcentrationasthedependentvariable,␣-MSHandbodyfatmass(%)weretheindependent

predictorstoexplainleptinconcentration.Fortheentiregroup,wefoundpositivecorrelationsbetween leptinemiaandBMIandbodyfatmass(%)aswellasanegativecorrelationwithfreefatmass(%)and alpha-MSH.Finally,weverifiednegativecorrelationsbetweenadiponectin/leptinratiowithtotal choles-terolandLDL-c,onlyinhyperleptinemicpatients.Inconclusion,thehyperleptinemiainobeseadolescents deregulatesneuropeptidesduringweightloss.

1. Introduction

Leptin,anadipokinethatisprimarilyexpressedbyadipose tis-sue, is considered to beinvolved in neuroendocrine control of energybalance.However,inhumanobesitystatesof hyperleptine-mia,centralandperipheralleptininsensitivityissuggested.Indeed, recentstudiesshowedthatthehypothalamusisnotleptin resis-tantinhyperleptinemiaconditions.Leptindeficiencyresultsfrom decreasedleptintransportacrossthebloodbrainbarrier[18,27,29].

∗ _{Corresponding} _author_at: _Rua _Professor _Francisco _de _Castro, _no _93,_Vila Clementino–SãoPaulo/SP,PostalCode:04020-050,Brazil.Tel.:+551155720177; fax:+551155720177.

E-mailaddress:ana.damaso@unifesp.br(A.R.Dâmaso).

Inthecontextofobesityandco-morbidities,statesof

hyper-leptinemia began as an idea that the body’s biomolecular

milieu decreases overall sensitivity to leptin effects such that normal or, classically, increased levels produce an inadequate

response [18,27]. This concept is reinforced by the

observa-tion that most obese individuals, including adolescents have

increasedserumleptinconcentrations,causinghyperleptinemia,as recentlydemonstratedintheliteratureandbyourresearchgroup

[24,27,43].

Inapreviousstudy,itwasdemonstratedthattheprevalence ofhyperleptinemiawas25.92%amongobeseadolescents[11]and

that20%of postmenopausalwomen presentedhyperleptinemia

[2].Sinceitsdiscoverymorethanadecadeago,leptinhasbeen establishedasakeyregulatorofenergybalance[7,38];however, recentevidenceindicatesthatleptindeficiencyisapivotallinkin obesity-relateddiseases[3,14].

Open access under the Elsevier OA license.

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Asmentioned above,hyperleptinemiaiscommonlyobserved inobesehumansandanimals[4,42,45].Inversely, adecreasein adiponectinconcentrationwasdemonstratedbyseveral investi-gationsinobeseadolescentsand adults.However,thepotential

mechanismsfordiminishedadiponectinemiaandhyperleptinemia

asrelatedtoinflammationremaintobeinvestigatedinobese ado-lescents[9,37].Thus,theinterplayamongadipokines,leptinand adiponectinmaybeanimportantcontributorinthepathogenesis ofobesityandotherco-morbidities.

Inthecentralnervoussystem,NPY,AgRPand␣-MSHproduced

by neuronsin the hypothalamus actlocally toregulate energy

balance. NPYexerts a physiologically important role in energy homeostasis [25,41]. However, blood NPY levelswill reflect its secretionfromtheadrenalgland,sympatheticnervoussystemand adipocytes,whichmaycontributetoadiposityanditsmetabolic consequences[13,26].

␣-MSHexertsanimportantroleintheenergybalanceinobese

adolescents;changesinexpressionwerecorrelatedtoweight sta-tuschanges[24,31].However,previousauthorsdidnotinvestigate thisassociationwithchangesinleptinconcentration,asrelatedto hyperleptinemicstatus.Becausethemelanocortin(MC)systemlies downstreamofleptinsensitivity,itisimportanttounderstandthis interactionduringclinicalweightlossinterventiontooptimizethe clinicalapproachtoimproveenergybalanceasakeystrategyfor obesitycontrol.

Severalstudieshaveshownarelationshipbetweenleptin lev-elsandenergybalanceinobeseyoungsters;however,theresults areinconclusive,withleptinlevelsthateitherdecreaseorremain unchangedafterexerciseordietaryintervention[5].

Therefore,theroleofalong-terminterdisciplinaryweightloss programonpro-anti-inflammatorypathwaysandthecentral reg-ulationofenergybalancewereanalyzedinobeseadolescentswith orwithouthyperleptinemia.Theaimsofthepresentstudywereto investigatetheroleofhyperleptinemiaduringweightlosstherapy onenergybalanceinobeseadolescentsandtodeterminewhether astateofhyperleptinemiacouldadverselyderegulate neuropep-tides.

2. Materialsandmethods

2.1. Subjects

A total of 86 obese adolescents(39 boysand 47 girls) who

enteredtheInterdisciplinaryObesityProgramoftheFederal Uni-versityofSãoPaulo–PaulistaMedicalSchoolwereassignedtotwo

sub-groups:hyperleptinemic(H) or non-hyperleptinemic(n-H).

Thosewhowereconsideredhyperleptinemicpresentedbaseline

valuesabove20ng/mlforboysand24ng/mlforgirls,asbasedon referencevaluescitedbyGutinetal.[12]andWhatmoreetal.[44]. Thesepatientsweresubmittedtoweightlosstherapy.The evalu-ationswereperformedatbaseline,after6monthsandafter1year ofaninterdisciplinaryapproach.

The ages of the participants ranged from 15 to 19 years

(16.6±1.67years). BMIwas37.03±3.78kg/m2_._All _participants wereconfirmedasmeetingtheinclusioncriteriaofpost-pubertal StageV[40](basedontheTannerstages ofobesity(BMI>95th percentileoftheCDCreferencegrowthcharts))[6].Exclusion cri-teriawereidentifiedgenetic,metabolicorendocrinediseaseand previousdrugutilization.Informedconsentwasobtainedfromall subjectsand/ortheirparents,includingagreementofthe adoles-centsandtheirfamiliestoparticipateasvolunteers.Thisstudywas performedinaccordancewiththeprinciplesoftheDeclarationof Helsinkiand wasformallyapprovedby theInstitutionalEthical

Committee(#0135/04).

2.2. Studyprotocolandmedicalscreening

Thesubjectsweremedicallyscreened;theirpubertalstagesand theiranthropometricmeasureswereassessed(height,weight,BMI andbodycomposition).Theendocrinologistcompletedaclinical interview,including questionstodetermineeligibilitybased on inclusionandexclusioncriteria.Abloodsamplewascollectedand analyzed,andultrasound(US)wasperformedtomeasurevisceral andsubcutaneousfat.Allsubjectsunderwentanergometrictest. Indeed,theprocedureswerescheduledforthesametimeofdayto removeanyinfluenceofdiurnalvariations.

2.3. Anthropometricmeasurementsandbodycomposition

Subjectswereweighedwearinglightclothingandnoshoeson aFilizolascaletothenearest0.1kg.Heightwasmeasuredtothe nearest0.5cmbyusingawall-mountedstadiometer(Sanny,model

ES2030).BMIwascalculated asbodyweightdividedbyheight

squared.Bodycompositionwasestimatedbyplethysmographyin

theBODPODbodycompositionsystem(version1.69,Life Measure-mentInstruments,Concord,CA)[10].

2.4. Serumanalysis

Bloodsampleswerecollectedintheoutpatientclinicaround 8h after an overnight fast. Insulin resistance was assessed by

the homeostasis model assessment-insulinesistance (HOMA-IR)

indexandthequantitativeinsulinsensitivitycheckindex(QUICKI).

HOMA-IRwas calculated using thefasting blood glucose (FBG)

and immunoreactive insulin (I): [FBG (mg/dL)×I (mU/L)]/405;

QUICKIwascalculatedas1/(logI+logFBG).Totalcholesterol,TG,

HDL,LDLandVLDLwereanalyzedusingacommercialkit(CELM,

Barueri,Brazil).TheHOMA-IRdatawereanalyzedaccordingto ref-erencevaluesreportedbySchwimmeretal.[35].Thea-MSH,NPY, AgRP,total ghrelin, total adiponectin and leptinconcentrations

were measured using a commercially available enzyme-linked

immunosorbentassay(ELISA)kitfromPhoenixPharmaceuticals,

Inc.(Belmont,CA,USA)accordingtothemanufacturer’s instruc-tions. The coefficient of variation (CV) for the adipokines and neuropeptideprocedurewascalculated:a-MSH(CV=6.48%),NPY (CV=11.91%),AgRP(CV=13.47%),ghrelin(CV=6.82%),adiponectin (CV=4.5%)andleptin(CV=4.07%).Forthisstudy,theleptindata wereanalyzedaccordingtoreferencevaluesdescribed byGutin etal.[12]andtheghrelinreferencevalueadoptedwas10–14ng/ml. accordingtoWhatmoreetal.[44].

2.5. Visceralandsubcutaneousadipositymeasurements

Allabdominalultrasonographicproceduresandmeasurements

of visceraland subcutaneous fat tissue were performedby the

samephysician,whowasblindedtosubjectassignmentgroups

atbaselineand afterintervention. Thisphysicianwasa special-istinimagingdiagnostics.A3.5-MHzmultifrequencytransducer (broadband)wasusedtoreducetheriskofmisclassification.The intra-examinationcoefficientofvariationforultrasound(US)was 0.8%.

USmeasurementsof intra-abdominal(visceral)and

subcuta-neous fat were obtained. US-determined subcutaneous fat was

definedasthedistancebetweentheskinandexternalfaceofthe rectusabdominismuscle,andvisceralfatwasdefinedasthe dis-tancebetweentheinternalfaceofthesamemuscleandtheanterior walloftheaorta.Cut-offpointstodefinevisceralobesityby

ultra-sonographicparameterswerebasedonpreviousmethodological

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2.6. Dietaryprogram

Energyintakewassetatthelevelsrecommendedbythedietary referenceintakeforsubjectswithlowlevelsofphysicalactivityof thesameageandgenderfollowingabalanceddiet[22].Nodrugs

or antioxidants were recommended. Once a week, adolescents

haddieteticlessons(providinginformationonthefoodpyramid, dietrecordassessment,weight-lossdietsand“miracle”diets,food labels,dietetics,fat-freeandlow-caloriefoods,fats(kinds,sources andsubstitutes), fast-foodcalories and nutritionalcomposition, goodnutritionalchoicesonspecialoccasions,healthysandwiches, shakesandproductstopromoteweightloss,functionalfoodsand decisionsonfoodchoices).Allpatientsreceivedindividual nutri-tionalconsultationduringtheinterventionprogram.

Atthebeginningofthestudyandat6monthsand12months intotheprogram,a3-daydietaryrecordwascollected.Portions weremeasuredintermsoffamiliarvolumesandsizes.The dieti-ciantaughttheparentsandtheadolescentshowtorecordfood consumption.Thesedietarydataweretransferredtoacomputerby thesamedietician,andthenutrientcompositionwasanalyzedbya softwareprogramdevelopedattheFederalUniversityofSãoPaulo –PaulistaMedicalSchool(Nutwinversion1.5forWindows,2002) thatuseddatafromWesternandlocalfoodtables.Inaddition,the parentswereencouragedbyadieticiantocalliftheyneededextra information.

2.7. Exerciseprogram

Duringthe1-yearinterdisciplinaryinterventionperiod, adoles-centsfollowedapersonalizedaerobictrainingprogramincluding

a60-minsession threetimesaweek(180min/week)underthe

supervisionofanexercisephysiologist.Eachprogramwas devel-opedaccordingtotheresultsofaninitialoxygenuptaketestfor aerobicexercises(cycle-ergometer and treadmill).Theintensity wassetataworkloadcorrespondingtoaventilatorythresholdof 1(50–70%ofoxygenuptaketest).Attheendof6months, aero-bictestswereperformedtoassessphysicalcapacity,andphysical trainingintensitywasadjustedforeachindividual.Duringthe aero-bicsessions,adolescentsweresubmittedtoheart-ratemonitoring.

Theexerciseprogramwasbasedonthe2001recommendations

providedbytheAmericanCollegeofSportsMedicine[1].

2.8. Psychologicalintervention

Diagnosesofcommonpsychologicalproblemsassociatedwith

obesity,suchasdepression,disturbancesofbodyimage,anxiety anddecreasedself-esteem,wereestablishedbyvalidated question-naires.Duringtheinterdisciplinaryintervention,theadolescents hadweeklypsychologicalsupportgroupsessionswherethey dis-cussedbodyimageandalimentarydisorders,suchasbulimiaand anorexianervosa,bingeeating;theirsigns,symptomsandhealth consequences;therelationshipbetweentheirfeelingsandfood; problemsinthefamily,suchasalcoholism,andothertopics.

Indi-vidualpsychologicaltherapywasrecommendedwhenwefound

individualswithnutritionalandbehavioralproblems.

2.9. Statisticalanalysis

Alldata wereanalyzedusingSTATISTICA version 6for

Win-dows, with the significance level set at p<0.05. Data are

expressed as the mean±SD unless otherwise stated.

Distribu-tionalassumptionswereverifiedbytheKolmogorov–Smirnovtest,

andnon-parametricmethodswereperformedwhenappropriate.

Adipokinesandneuropeptideswereanalyzedwithnon-parametric

testsandexpressedasmedian,minimumandmaximumvalues.

Comparisonsbetweenmeasuresatbaselineandafterweight-loss Table

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Table 2 Adipokines and neuropeptides among obese adolescents with hyperleptinemia and without hyperleptinemia before and after weight loss intervention. Variables/time Hyperleptinemic patients Non-hyperleptinemic patients Baseline 6 months After intervention Baseline 6 months After intervention Adiponectin (ug/l) 5.46 (1.82–13.86) 5.89 (1.40–15.06) b 5.46 (2.48–15.99) 5.26 (2.71–10.65) 5.63 (2.65–16.13) b 6.77 (2.78–13.23) c Leptin (ng/ml) 43.50 (24.37–69.44) a 28.37 (1.29–55.57) a , b 31.24 (1.10–51.75) a , c , d 7.93 (1.23–23.77) 5.15 (1.24–49.94) 5.05 (1.26–38.41) c Adiponectin/leptin ratio 0.13 (0.03–0.53) a 0.22 (0.05–21.88) a , b 0.23 (0.06–10.24) a , c 0.72 (0.14–6.52) 1.46 (0.13–5.41) b 1.35 (0.09–4.90) Ghrelin (ng/ml) 1.19 (0.31–13.30) a 1.31 (0.18–13.30) 1.22 (0.52–23.47) a 3.12 (0.90–42.66) 1.97 (0.68–11.61) 3.89 (0.79–23.47) c NPY (ng/ml) 0.89 (0.21–6.71) a 1.17 (0.27–27.21) b 0.94 (0.17–19.67) a , d 1.17 (0.23–15.43) 1.17 (0.75–14.36) 1.27 (0.18–4.68) AgRP (ng/ml) 0.25 (0.11–1.02) 0.28 (0.13–0.76) a 0.34 (0.02–0.88) a , c , d 0.31 (0.13–0.78) 0.40 (0.21–0.85) 0.49 (0.23–15.43) c , d NPY/AgRP ratio 3.74 (0.33–30.49) a 3.01 (1.83–68.11) b 3.08 (0.18–19.05) c 2.88 (0.36–42.08) 5.30 (1.52–64.46) 2.55 (0.22–57.76) Alpha-MSH (ng/ml) 0.71 (0.24–14.40) a 0.64 (0.28–17.13) a 0.74 (0.30–13) 7.72 (0.64–28.36) 2.01 (0.60–15.41) b 1.41 (0.72–7.48) c Values expressed by median (minimum–maximum). Reference values: leptin (1–20 ng/ml for boys and 4, 9–24 ng/ml for girls) [12;44]. a Comparison of the hyperleptinemic group vs Non-hyperleptinemic group at the same study period, p ≤ 0.05. b Comparison of baseline vs 6 months, p ≤ 0.05. cComparison of baseline vs after 1 year of intervention, p ≤ 0.05. d Comparison of 6 months vs after 1 year of intervention.

intervention weremadeusing ananalysisof variance(ANOVA)

for repeated measures or the Wilcoxon signed rank test of

non-parametricvariables.Comparisonsbetweengroupswere

per-formed using a one-way ANOVA or the Mann–Whitney test

(non-parametricvariables).Pearson’scorrelationwasperformed totestthedirectionandstrengthoftherelationshipbetween lep-tinconcentrationandthevariablesofinterestandtoselectthose variablesthatdidnotpresentcollinearity,toselectthepredictorsin themultipleregression.Stepwisemultiplelinearregression anal-ysiswasperformedtoestimatetheassociationwithparameters knowntoinfluenceleptinconcentration.

3. Results

Atthebeginningoftherapy,86obeseadolescentswereenrolled intheprogram.Theresultsarepresentedforthewholepopulation studied:wedidnotfindsignificantgenderdifferencesinBMIat baseline.Indeed,thesubjectswerepairedaccordingtoBMI,then dividedandanalyzedaccordingtoleptinemicstate.

3.1. Normoleptinemicpatients

Afterweightlossintervention,weobservedsignificant

improve-ments in BM, BMI, body fat mass (% and kg), visceral and

subcutaneousfat,insulin concentration,HOMA-IR,QUICKI,total cholesterolandtriglycerides.Indeed,short-andlong-term inter-ventionsincreasedthefreefatmass(%)(Table1Table1).

Based on the adipokine and neuropeptide data, we verified

increasesinadiponectinconcentrationandadiponectin/leptinratio

witha concomitantreduction in alpha-MSHconcentration.The

leptinconcentrationdecreased,whiletheorexigenicfactors (ghre-linandAgRP)increasedafter1year.WhenweanalyzedtheAgRP from6monthsto1yearofintervention,asignificantincreasewas observed(Table2Table2).

3.2. Hyperleptinemicpatients

Afterweightlossintervention,weobservedsignificant

improve-ments in BM, BMI, body fat mass (% and kg), visceral and

subcutaneousfat,insulin concentration,HOMA-IR,QUICKI,total cholesterolandtriglycerides,similartothetrendobservedinthe normoleptinemicpatients.Onlylong-term(1year)treatmentwas abletopromoteasignificantincreaseinfreefatmass(%)(Table1). Thegroupwithhyperleptinemiaexhibitedasignificantincrease inadiponectin,NPYconcentrationandadiponectin/leptinratioas wellasareductioninleptinandNPY/AgRPratiowithshort-term intervention. After one year,this grouppresented a significant increase in adiponectin/leptin ratio and in AgRP concentration, withareductionintheNPY/AgRPratio(Table2).

3.3. Comparisonbetweenthegroups

Itisimportanttonotethathyperleptinemicpatientspresented loweradiponectin/leptinratio,alpha-MSHandghrelin concentra-tionatbaseline.Indeed,theNPY/AgRPratiowashighercompared withthatobservedinthenon-hyperleptinemicgroup.

Wefoundpositivecorrelationsbetweenleptinconcentrations and BMIand bodyfat mass(%)atbaseline, intheentire group (Fig.1aandb).Ontheotherhand,theleptinconcentrationswere negativelycorrelatedwithfreefatmass(%)andalpha-MSH(Fig.2a andb).Negativecorrelationsbetweenadiponectin/leptinratioand total cholesterol and LDL-c wereconfirmed atbaseline only in hyperleptinemicpatients(Fig.3aandb).

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depen-Fig.1. PositivecorrelationbetweenleptinconcentrationwithBMI(a)andbodyfat(%)(b)atbaselineconditionsintheentiregroup.

Fig.2.Negativecorrelationbetweenleptinconcentrationswithfreefatmass(%)(a)andalpha-MSH(b)atbaselineconditionsintheentiregroup.

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Table3

Multipleregressionanalysisfortheleptinconcentrationintheentiregroup.

Leptin

Regressioncoefficientˇ p

Age 0.08 0.29

Alpha-MSH(ng/ml) −0.47 0.001 Bodyfatmass(%) −0.33 0.001 Visceralfat(cm) −0.04 0.60 Subcutaneousfat(cm) 0.08 0.39

dentvariable.␣-MSHandbodyfatmass(%)weretheindependent predictorstoexplainleptinconcentrationinthepresentstudy.

4. Discussion

Obesityhasbeenshowntocauseresistanceorreduced sensi-tivitytoseveralhormones,includingleptinandadiponectin.Obese individuals appear to have higher sympathetic nervous system (SNS)activity;however,themetabolicresponsetoSNS stimula-tionappearsreducedinthispopulation.Thisfindingsuggeststhat, inobesity,anycompensatoryeffectoftheSNSonmetabolismto increaseenergyexpendituremaynotoccur,renderingweightloss moredifficult[8,33].

We verified that, after the therapy, obese adolescents have decreasedbodyweight,totalbodyfat,visceralfatandleptin

con-centration;however,someofthemremaininahyperleptinemic

state(Table1).

Studieshavereportedhyperleptinemiaininsulin-resistant indi-vidualsindependentlyofthelevelofobesity.Indeed,theyreported cross-sectionalassociationsbetweenhyperleptinemiaandinsulin resistanceindependentlyofbodymassindexinapopulation-based cohort.Thesestudiesindicatethatleptinandinsulinareinvolved inacomplexregulatoryloopandhighlightthepivotalroleofleptin inglucosehomeostasis,actingasaninsulinsensitizerwhenleptin levelsareatlowandnormallevelsandpossiblycontributingto insulinresistancewhenleptinischronicallyelevated[32,36].

Inaddition,inthenon-hyperleptinemicgroup,therewasa sig-nificantincreaseinfreefatmass(%)aftershort-termtherapy.In thehyperleptinemicpatients,thisincreaseoccurredafteronlyone yearof intervention.In fact, evidencederived fromanimal and humanstudiessuggeststhattheabilityofleptinandadiponectinto stimulatefatacid(FA)oxidationinmuscleisimpairedinobesity. Thus,leptindeficiencyandadiponectinresistancemaybe initiat-ingfactorsintheaccumulationofintramuscularlipids.Thisfinding maypartiallyexplainwhythefatfreemass(%)wassignificantly increasedonlyafterlong-terminterventioninthehyperleptinemic group[8].

Inthepresentstudy,hyperleptinemicpatientspresentedhigher valuesoforexigenicfactors.Thisfactsuggeststhattheleptinemic stateaffectstheneuroendocrineenergeticbalance,stimulatingthe orexigenicpathways,which make weightlossdifficultinobese adolescents.

Oneofthemostimportantfindingsinthepresentinvestigation

wastheloweralpha-MSHconcentrationatbaseline,whichwas

maintainedafterweightlossinthevolunteerswith

hyperleptine-mia(Table2).Wealsoshowedthatatbaseline,leptinconcentration

wasnegativelycorrelatedwithalpha-MSH,reinforcingtheconcept thatadisruptionbetweenthemechanismsinvolvedinenergy bal-anceoccursinobeseadolescents,renderingweightlossdifficult andultimatelypredisposingtheseindividualstoweightregain[33]. However,attheendoftherapy,alpha-MSHwassimilarinboth analyzedgroups.Inaddition,weverifiedthatthehyperleptinemia decreasedsignificantlyafterweightlossintervention,suggesting theimportantroleofthis typeoftherapy inprovidingsuperior neuroendocrineregulationofenergybalance.

Animalexperiments recentlyshowedthat thecomplexityof

melanocortin(MC)systemeffectsvarieswiththenutritionalstate andthatresponsivenesstotheeffectsofalpha-MSHmaybe main-tained even inleptin-resistant animals, suggestingthat theMC system(receptorsandpost-receptorsignaltransductionpathways) isoperantevenintheabsenceofleptininput[33].Similarly,in mul-tipleregressionanalyses,alpha-MSHwasanegativeindependent predictorofleptinconcentration(Table3).

Therefore,itisnecessarytoconfirmthesefindingsindifferent populationsbecauseage-relatedobesityinthelong-term regula-tionofbodyweightisknowntobeassociatedwithleptinresistance

[34,39]andalterationsinbodyweightandcomposition.These

find-ingsmaybe,atleastpartly,causedbychangesintheactivityof

anorexigenicandorexigenicneurohumoralsystems.Components

oftheMCsysteminthehypothalamusareconsideredtobemajor playersintheregulationofenergymetabolismandbodyweight

[28].

Inagreementwiththeliterature,weobservedthatin hyper-leptinemicstatus,theghrelinconcentrationwaslowerduringthe interventionincomparisonwiththenon-hyperleptinemicgroup. Anincrease inghrelinconcentrationat theendof therapy was observedonlyinthenon-hyperleptinemicpatients.Suchachange isconsideredasanadaptivefunctionofghrelininresponseto neg-ativeenergybalance[7].Thesedatareinforcetheconceptofleptin resistanceinleptinexcessstatus,asobservedinobesity,asitwas previouslydemonstratedthatleptininhibitsghrelineffluxfromthe stomachandreducedghrelin-inducedfeeding[15,21,23].

Important evidence in the present investigation is that the

NPY/AgRPratiowassignificantlyhigheratbaselineinthe

hyper-leptinemic group. This findingcould beexplained by impaired

leptinfunctioninmaintainingenergyhomeostasis,restrainingthe releaseofNPY,inthehyperleptinemiagroup[15].However,both groupspresentedareductionofthisratiointhecourseofweight losstherapy,showingsimilarvalues attheendofthe interven-tion.These datareinforcetherole of circulatinglevelsof these peptidesinenergyhomeostasisinobeseadolescents.Previously,it wasdemonstratedthatNPYandleptinformaloopsystem respon-sibleforprovidingfeedbacktothecentralnervoussystemonthe stateoftheperipheralenergystores.Thesuggestedmechanism includesnitricoxide-mediatedregulationofleptinandNPYduring foodintakeinmice[19,20].However,thesemechanismsneedto befullyinvestigatedinhumansinfutureresearchefforts.

RecentstudiesshowedthatelevatedcirculatingNPYlevelsand leptinwereobservedinpatientswithcardiovasculardiseases,such asacutemyocardialinfarction,anginapectoris,heartfailureand hypertensionwheresympatheticnerveactivityisincreased, indi-catingtheclinicalimportanceofNPYinregulatingvesselfunction

[16,26].Moreover,theinteractionsbetweenNPYandthereleaseof

inflammatorycytokines,suchasleptin,inanatheroscleroticmilieu mayplayamajorroleinthecardiovascularsystem[26].

Adiponectin levels improved significantly after short- and

long-termtherapiesinthenormoleptinemicgroup;however,the hyperleptinemicpatientsshowedanincreaseinthisvariableonly afterlong-termtherapy.Theslightchangein adiponectinlevels couldbelinkedtothetimeofinterventionandmaynotbesufficient fordetection.Inastudywithobesechildren,afterlifestyle inter-vention,adiponectinlevels,togetherwithseveralothermetabolic parameters,weresignificantlyimproved,potentiallyduetoweight loss,improvementofmetabolicstatus,orboth[5].

Leptin and adiponectin are involved in the regulation of

metabolichomeostasisandinflammatoryprocessinaconstellation ofchronicdiseases.Severalstudieshavereportedtheassociation ofadipokines,especiallyA/Lratio,withthepresenceofmetabolic syndrome[14,17,46].Inagreement,Jungetal.[14]showedinadults thattheA/Lratiowasdecreasedinthepresenceofmetabolic

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components.Ourstudycorroboratedthesefindings,revealing a negativecorrelationbetweentheA/Lratioandtotalcholesteroland LDLcholesterolinthehyperleptinemicgroup.

Thus,oneimportantfindingfromthepresentstudyisthatthe A/Lratiowassignificantlylowerthroughouttheintervention in

thosewithhyperleptinemiacomparedwithnon-hyperleptinemic

patients.However,weightlosstherapywaseffectiveinimproving thisratioinbothanalyzedgroups.

Ourstudypresentedsomelimitations,suchasareducednumber ofsubjects,andwemeasuredtotalghrelinratherthanacylghrelin, althoughtheacylationofthispeptideisnecessarytocrosstheblood brainbarriertoreleaseGHandexertothersendocrinefunctions.

However,wedemonstrate inobeseadolescents thattheA/L

ratio was negatively correlated with total cholesterol and LDL

cholesterol and higher values of NPY/AgRP in hyperleptinemic

patients.Alltogether,thesedatareinforcetheroleof hyperleptine-miainthederegulationofenergybalanceinobeseadolescents, suggestingthatthispivotalinterplayofleptininenergybalance andinflammationneedstobeconsideredinaclinicalintervention.

5. Conclusions

Inconclusion,ourstudyrevealsthatlong-terminterdisciplinary

therapy promotessignificantimprovement in the disruptionof

homeostaticcross-talkbetweentheafferenthormonalsignalsfrom

theperiphery and thehypothalamic network ofNPY, observed

mainly in hyperleptinemic obese patients. Finally, these data

canelucidate theinterplaybetweenhyperleptinemicstatus and increasedNPY/AgRPratiowithaconcomitantdecreasein alpha-MSH,factorsimplicatedinimpairedweightlosscontrol.

Grantsupport

AFIP, FAPESP 2008/53069-0 and 2006/00684-3, FAPESP

(CEPID/Sleep#9814303-3S.T)CNPq,CAPES,CENESP,FADA,and

UNIFESP-EPM,supportedtheCEPE-GEOInterdisciplinaryObesity

InterventionProgram.

Disclosurestatement

Thereisnoconflictinterest.

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