against Hepatic EncephalopathyWord文档格式.docx

against Hepatic EncephalopathyWord文档格式.docx

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∙Á

ngelaRojas,

∙Iné

sCamacho,

∙BlancaFigueruela,

∙JuanD.Bautista,

∙ManuelRomero-Gó

mez 

mail

Abstract

Aim

Toinvestigatetheinfluenceofmetforminuseonliverdysfunctionandhepaticencephalopathyinaretrospectivecohortofdiabeticcirrhoticpatients.Toanalyzetheimpactofmetforminonglutaminaseactivityandammoniaproduction 

invitro.

Methods

Eighty-twocirrhoticpatientswithtype2diabeteswereincluded.Forty-onepatientswereclassifiedasinsulinsensitizersexperienced（metformin）and41ascontrols（cirrhoticpatientswithtype2diabetesmellituswithoutmetformintreatment）.Baselineanalysisincluded:

insulin,glucose,glucagon,leptin,adiponectin,TNFr2,AST,ALT.HOMA-IRwascalculated.BaselineHEriskwascalculatedaccordingtominimalhepaticencephalopathy,oralglutaminechallengeandmutationsinglutaminasegene.Weperformedanexperimentalstudy 

invitro 

includinganenzymaticactivityassaywhereglutaminaseinhibitionwasmeasuredaccordingtodifferentmetforminconcentrations.InCaco2cells,glutaminaseactivityinhibitionwasevaluatedbyammoniaproductionat24,48and72hoursaftermetforminatreatment.

Results

Hepaticencephalopathywasdiagnosedduringfollow-upin23.2%（19/82）:

4.9%（2/41）inpatientsreceivingmetforminand41.5%（17/41）inpatientswithoutmetformintreatment（logRank9.81;

p=0.002）.Inmultivariateanalysis,metforminuse[H.R.11.4（95%CI:

1.2–108.8）;

p=0.034],ageatdiagnosis[H.R.1.12（95%CI:

1.04–1.2）;

p=0.002],femalesex[H.R.10.4（95%CI:

1.5–71.6）;

p=0.017]andHErisk[H.R.21.3（95%CI:

2.8–163.4）;

p=0.003]werefoundindependentlyassociatedwithhepaticencephalopathy.Intheenzymaticassay,glutaminaseactivityinhibitionreached68%withmetformin100mM.InCaco2cells,metformin（20mM）decreasedglutaminaseactivityupto24%at72hourspost-treatment（p<

0.05）.

Conclusions

Metforminwasfoundindependentlyrelatedtooverthepaticencephalopathyinpatientswithtype2diabetesmellitusandhighriskofhepaticencephalopathy.Metformininhibitsglutaminaseactivity 

invitro.Therefore,metforminuseseemstobeprotectiveagainsthepaticencephalopathyindiabeticcirrhoticpatients.

Figures

12

Citation:

AmpueroJ,RanchalI,Nuñ

ezD,Dí

az-HerreroMdM,MaraverM,etal.（2012）MetforminInhibitsGlutaminaseActivityandProtectsagainstHepaticEncephalopathy.PLoSONE7（11）:

e49279.doi:

10.1371/journal.pone.0049279

Editor:

CarlosM.Isales,Georgia 

HealthSciences 

University,UnitedStatesofAmerica

Received:

July25,2012;

Accepted:

October8,2012;

Published:

November15,2012

Copyright:

©

2012Ampueroetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.

Funding:

ProyectodeExcelencia（CTS-7991/2011）.Consejerí

adeEconomí

a.JuntadeAndalucí

a.GovermentofAndalusia,Spain.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.

Competinginterests:

Theauthorshavedeclaredthatnocompetinginterestsexist.

Introduction

Hepaticencephalopathy（HE）isoneofthemajorcomplicationsoflivercirrhosisaffectingonethirdofcirrhoticpatients 

[1].Ithasrelevantsocio-economicimpactsinceHEreducesquality-of-lifeandisassociatedwithhighermortalityrate 

[2].HEoccursasaresultofthecoexistenceofhyperammonemiaandinflammationinpatientswithliverdysfunctionand/orporto-systemicshunts 

[3].AmmoniaproductiontakesplacemainlyinthesmallintestinewhereglutaminasetypeKactivityiscrucialforthepathogenesisofHE 

[4].Type2diabetesmellitusandinsulinresistance（IR）arecharacterizedbythereleaseofpro-inflammatorycytokines,suchasTNFαandIL-6,resultinginaninflammatorystate 

[5].Diabeteshasbeenindependentlyrelatedtocontrolofactivevaricealbleeding 

[6] 

andisassociatedwithanincreasedriskofhepatocellularcarcinomadevelopment 

[7].Type2diabetesmellitushasalsobeenfoundassociatedwithhepaticencephalopathyinpatientswithHCV-relatedcirrhosis 

[8].Insulinsensitizers,likemetformin,decreaseinsulinsecretionandreducehyperinsulinemicstate.MetforminincreasesbetaoxidationandreducesthehepaticgluconeogenesisviaactivationofAMP-Kpathway;

decreasesintestinalglucoseabsorptionandincreasesglucoseuptakeinskeletalmuscle 

[9].Recently,ithasbeenfoundabletomodulatetheexpressionofcytokines,suchasTNFα 

[10].Thus,IRstatecouldinfluencehepaticencephalopathydevelopmentinpatientswithcirrhosis.Insulin-sensitizersseemtodecreaseHCCinpatientswithcirrhosisC 

[11].Therefore,theammoniaproduction,IRandthepro-inflammatorystateseemtotriggercirrhosisprogression,andmaybeinterestingastherapeutictargetsinthenearfuture,improvingtheprognosisofcirrhoticpatients.

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Table1. 

Comparisonofbaselinecharacteristicsbetweengroups.

doi:

10.1371/journal.pone.0049279.t001

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Table2. 

Univariateanalysisbetweenhepaticencephalopathyandoutcomes.

10.1371/journal.pone.0049279.t002

Theaimofthisstudywastodeterminewhetherthemetforminusewasassociatedwithdecreasedriskofhepaticencephalopathyindiabeticcirrhoticpatientsandtoanalyzetheabilityofmetformintoinhibitglutaminaseactivity 

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Figure1. 

KaplanMeiercurveshowingtheimpactofmetforminuseonhepaticencephalopathy（n=82;

logRank:

9.45;

p=0.002）.

10.1371/journal.pone.0049279.g001

Patients

Eighty-twoconsecutivediabeticcirrhoticpatientsfromtheUnitforClinicalManagementofDigestiveDiseases,UniversityHospitalofValme,wereincluded.ThestudystartedeitherwiththefirstvisittoHepatologyofficeorwiththefirsthospitaladmissionandoutcomestofinishweresurvivalandlivertransplantation.Exclusioncriteriawere:

age≤18years;

non-diabeticpatients;

patientswithtype1diabetesmellitus;

andpatientswithtreatmentongoingforcirrhosis.TheprotocolwasapprovedbytheCEICofUniversityHospitalofValme（Sevilla,Spain）andallpatientsprovidedwritteninformedconsenttoparticipateinthisstudy.ThestudywasconductedinaccordancewiththeethicalguidelinesoftheDeclarationofHelsinkiandInternationalConferenceonHarmonizationGuidelinesforGoodClinicalPractice.Atotalof41casesand41controlswereincluded.Theywereclassifiedaccordingtoinsulinsensitizersexperienced.Casesweredefinedaspatientswhounderwentmetformintreatment,whilecontrolsweredefinedascirrhoticpatientswithtype2diabetesmellituswithoutmetformintreatment.Metformin-experiencedaveragetimewas33.4±

26.7months.Type2diabetesmellituswasdiagnosedaccordingtothe 

AmericanDiabetesAssociation 

[12].

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Table3. 

MultivariateanalysisaccordingtoovertHE.

10.1371/journal.pone.0049279.t003

BiochemicalandClinicalParameters

Baselineanalysis,usingcommercialtests,included:

insulin,glucose,glucagon,TNFr2,leptin,adiponectin,ASTandALT.HOMA-IRwascalculated[glucose（mmol/L）*Insulin（IU/ml）/22,5].Cirrhosiswasdefinedandbasedonliverbiopsy,ultrasound,endoscopicanalysisandbiochemicalparameters.

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Figure2. 

Glutaminaseactivityinchemicalassay（%）,accordingtometforminconcentration.

Eachbarrepresentsthemean±

SD（allexperimentswereconductedbytriplicate）.

10.1371/journal.pone.0049279.g002

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originalimage（364KB）

Figure3. 

Effectofmetforminonglutaminaseactivity 

3A）Glutaminaseactivityinhibitionincellsassay（%）,accordingtometforminconcentration;

3B）Ammoniaconcentrationincellsassay,accordingtometforminconcentration.Eachbarrepresentsthemean±

SD（allexperimentswereconductedbytriplicate）.*p≤0.05vs.thecorrespondingcontrolsample.#p≤0.05vs.thesamegroupcollectedattheprevioustimepoint.

10.1371/journal.pone.0049279.g003

EncephalopathyManagement

Minimalhepaticencephalopathy（MHE）wasdiagnosedbasedonpsychometrichepaticencephalopathyscore（PHES）andcriticalflickerfrequency（CFF）（Hepatonorm™Analyzer（R&

RMedi-BusinessFreiburgGmbH,Freiburg,Germany））.Thisbatterycomprisesthedigitsymboltest（DST）,thenumberconnectiontestA（NCT-A）,thenumberconnectiontestB（NCT-B）,theserialdottingtest（SDT）,andthelinedrawingtest（LDT）.PatientswereclassifiedashavingMHEwhenthePHESscorewaslessthan−4pointsortheCFFvaluewasbelowthecut-off（38Hz） 

[13].Fororalglutaminechallenge（OGC）analysis,bloodsamplesweretakenatbaselineand60minutesfollowingglutamineload（10gglutaminedissolvedin100mlwater（L-Glutamine,SHSS.A.,Spain））.AmmoniawasmeasuredusingtheDaFonseca-Whollheimmethodinanauto-analyzer（Hitachi911;

RocheDiagnostics,Mannheim,Germany）.Apathologicalresponsecurveforglutaminetolerancewasdefinedasanammoniariseto>

128µ

g/dLat60minute