against Hepatic Encephalopathy.docx

against Hepatic Encephalopathy.docx

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againstHepaticEncephalopathy

hibitsGlutaminaseActivityandProtectsagainstHepaticEncephalopathy

∙JavierAmpuero,

∙IsidoraRanchal,

∙DavidNuñez,

∙María delMar Díaz-Herrero,

∙MartaMaraver,

∙JoséAntoniodelCampo,

∙ÁngelaRojas,

∙InésCamacho,

∙BlancaFigueruela,

∙JuanD.Bautista,

∙ManuelRomero-Gómez 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,NuñezD,Díaz-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）.ConsejeríadeEconomía.JuntadeAndalucía.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.

doi:

10.1371/journal.pone.0049279.t002

Theaimofthisstudywastodeterminewhetherthemetforminusewasassociatedwithdecreasedriskofhepaticencephalopathyindiabeticcirrhoticpatientsandtoanalyzetheabilityofmetformintoinhibitglutaminaseactivity invitro.

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Figure1. KaplanMeiercurveshowingtheimpactofmetforminuseonhepaticencephalopathy（n=82;logRank:

9.45;p=0.002）.

doi:

10.1371/journal.pone.0049279.g001

Methods

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.

doi:

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）.

doi:

10.1371/journal.pone.0049279.g002

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Figure3. Effectofmetforminonglutaminaseactivity invitro.

3A）Glutaminaseactivityinhibitionincellsassay（%）,accordingtometforminconcentration;3B）Ammoniaconcentrationincellsassay,accordingtometforminconcentration.Eachbarrepresentsthemean±SD（allexperimentswereconductedbytriplicate）.*p≤0.05vs.thecorrespondingcontrolsample.#p≤0.05vs.thesamegroupcollectedattheprevioustimepoint.

doi:

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>128µg/dLat60minute