Yale University EliScholar A Digital Platform for Scholarly Publishing at Yale Yale Medicine Thesis Digital Library School of Medicine January 2014 Impact Of Gain-Of-Function Mutations In The Low-Density Lipoprotein Receptor Related Protein 5 (lrp5 On Glucose And Lipid Homeostasis Dinah Foer Yale School of Medicine, dinah.foer@yale.edu Follow this and additional works at: http://elischolar.library.yale.edu/ymtdl Recommended Citation Foer, Dinah, "Impact Of Gain-Of-Function Mutations In The Low-Density Lipoprotein Receptor Related Protein 5 (lrp5 On Glucose And Lipid Homeostasis" (2014. Yale Medicine Thesis Digital Library. 1877. http://elischolar.library.yale.edu/ymtdl/1877 This Open Access Thesis is brought to you for free and open access by the School of Medicine at EliScholar A Digital Platform for Scholarly Publishing at Yale. It has been accepted for inclusion in Yale Medicine Thesis Digital Library by an authorized administrator of EliScholar A Digital Platform for Scholarly Publishing at Yale. For more information, please contact elischolar@yale.edu.
Impact of gain-of-function mutations in the low-density lipoprotein receptor related protein 5 (LRP5 on glucose and lipid homeostasis A Thesis Submitted to the Yale University School of Medicine in Partial Fulfillment of the Requirements for the Degree of Doctor of Medicine by Dinah Foer 2014
IMPACT OF GAIN-OF-FUNCTION MUTATIONS IN THE LOW DENSITY LIPOPROTEIN RECEPTOR RELATED PROTEIN 5 (LRP5 ON GLUCOSE AND LIPID HOMEOSTASIS. Dinah Foer, Meiling Zhu, Christine Simpson, Grace Lee, Rebecca Sullivan, and Karl L. Insogna. Section of Endocrinology, Department of Internal Medicine, Yale University, School of Medicine, New Haven, CT. Investigations in animal models suggest a physiologic link between LRP5 and glucose and lipid homeostasis. However, the data in human studies is far from consensus. While LRP5 has been shown to be critical to Wnt signaling, the molecular mechanism for the effect on metabolism is unknown. We hypothesize that a high bone mass (HBM mutation in LRP5 will lead to improved glucose and lipid homeostasis. We aim to measure the impact of these mutated receptors on human glucose and lipid metabolism and to investigate the pathway(s by which Wnt may affect glucose metabolism in vitro. Eleven subjects with HBM-causing LRP5 mutations were matched by gender, age, and body mass index (BMI to 22 unrelated control subjects. Hemoglobin A1c (HbA1c, estimated average glucose (eag, HOMA-B, HOMA-IR, and lipid panel were analyzed. An oral glucose tolerance test and 1 HMRS study of liver and skeletal muscle were performed on a subset of affected patients. Primary hepatocytes and HepG2 cells were treated in vitro with Wnt3a, and PEPCK-C mrna expression was measured by QPCR. INS-1 cells and human pancreatic islet beta-cells were assayed for glucose-stimulated insulin secretion using an ELISA technique. Statistical differences were analyzed using the unpaired Student t-test and one-way ANOVA. There were no statistically significant differences between affected and control populations for HbA1c, eag, insulin, HOMA-B and HOMA-IR. Differences in total cholesterol, triglycerides, and HDL, were not significant. However, LDL levels were significantly lower in affected subjects (p=0.04. Wnt did not effect PEPCK-C expression. Wnt did not significantly effect glucose-stimulated insulin secretion in INS-1 cells and human islets. In summary, this is the first investigation on the metabolic consequences of LRP5 mutations in human kindreds with HBM-causing mutations. Our data does not support the hypothesis that LRP5 improves glucose metabolism in these individuals. The data does suggest that there may be a specific, beneficial effect of LRP5 on LDL cholesterol, however additional studies need to be done to confirm the effect and elucidate the mechanism.
Acknowledgements.. ThisthesiswouldnothavebeenpossiblewithouttheinspiredparticipationoftheLRP5 kindredmembers.iamespeciallygratefulfortheenthusiasmofthelatedr.johnpackard, Sr.,Dr.JohnPackard,Jr.,andthecontinuedcommitmentofthenextgenerations. ThankyoutotheoutstandingYalescientistswhohelpedtostrengthenanddeepenthis work:dr.richardkibbeyandrebeccapongratzfortheirgenerositywithmaterials, knowledge,andcollaboration;anddr.kittfalkpetersenforherextraordinaryhelpwiththe OGTTandMRSstudies.ThankyoutoDr.ThomasCarpenterforhisinsightfulcomments duringthethesisreviewprocess. ThankyoutothewonderfulwomenoftheInsognaLab:MeilingZhu,BeckySullivan, ChristineSimpson,GraceLee,IreneSherman,CarrieThomas,JessicaBihuniak.Thankyouto BenhuaSunwhotaughtmehowtouseapipette Dr.KarlInsognahasbeenanexceptionalmentor,teacher,advocateandrolemodelforme overthepastfiveyears.iamdeeplygratefulforthetime,energyandconfidencehehas investedinmeandinthisproject. IamgratefulforthegenerousfundingprovidedbytheYaleOfficeforStudentResearch throughmultiplegrants.thisworkwassupportedbyanationalinstitutesofhealthtnhlbi MedicalStudentResearchFellowship,andfundingfromtheNationalCenterForAdvancing TranslationalSciencesoftheNationalInstitutesofHealthunderAwardNumber TL1TR000141.Thecontentissolelytheresponsibilityoftheauthoranddoesnot necessarilyrepresenttheofficialviewsofthenationalinstitutesofhealth. ThisthesisworkisdedicatedtoMeilingZhu,atrulyhumble,andgiftedteacher.
TABLE&OF&CONTENTS& & & Introduction.1 StatementofPurpose,Hypothesis,SpecificAims. 8 Methods... 9 HumanStudies..10 In#vitro#studies... 12 Results....18 HumanStudies.. 18 In#vitro#studies... 21 Discussion. 24 Conclusion....29 Figures&Tables...31 References....44
1 INTRODUCTION Thelowdensitylipoproteinrelatedprotein5(LRP5cametoclinicalattentionin 2001whenGongandcoworkersreportedthatlossoffunctionmutationsinthis receptorcausedprofoundosteoporosisandfractures(gongetal.,2001.in2002, YaleUniversityinvestigatorsreportedthatamutationinLRP5thatconferred resistancetoendogenousinhibitorssuchasdkk1,causedanotherdramaticskeletal phenotype.akindredwasidentifiedwithaninheritedsyndromeofhighbonemass thatwasphenotypicallycharacterizedbyareducedangleofthejaw,toruspalatinus, andextremelyhighbonedensitywhenmeasuredbydualenergyxray absorptiometry(boydenetal.,2002.thesyndromewasinheritedinanautosomal dominantpattern.interestingly,membersofthekindredreporteddifficultystaying afloatwhileswimming.affectedfamilymemberswereshowntohaveasinglebase pairmutationcausingaglycinetovalinechangeatposition171.glycine171is evolutionarilyconservedinlrp5andinitshomologues,indicatingacriticalrolefor thisresidue(heyetal.,1998.subsequentlyadditionalhighbonemass(hbm causingmutationsinlrp5havebeenidentifiedinotherkindreds(vanwesenbeeck etal.,2003;whyte,reinus,&mumm,2004.theexactmechanismbywhichthese highbonemasscausingmutationsleadtothephenotypedescribedisnot completelyunderstood.howeverasnoted,thelrp5g171vmutationisresistantto theactionsoftheendogenousinhibitordickopf1(dkk1.thus,whilethereceptor doesnotsignalintheabsenceofligandandappearstosignalnormallyinthe presenceofligand,whendkk1isaddedliganddependentsignalingislargely unaffected(boydenetal.,2002.
2 TheroleofLRP5skeletalhomeostasisisnowwellestablished(Williams&Insogna, 2009.Morerecently,animalmodelswithbothHBMandlowbonemass(LBM causingmutations,aswellashumanstudiesinpopulationswithlbmcausing mutations,implicatearoleforlrp5inglucosemetabolismandlipidhomeostasis (Fujinoetal.,2003;Saarinenetal.,2010.ThisintroductionwillreviewLRP5 s functionanditskeysignalingpathways,providingabackgroundforunderstanding thepotentialroleoflrp5inhumanmetabolism. LRP5.&TheLRP5geneislocatedonchromosome11q13.4.Theencodedproteinin vertebratesisasinglepasstransmembranemoleculethatisareceptorforlow densitylipoprotein(ldl(galoraetal.,2013.theproteinalsoservesasaco receptor,alongwithitshomologuelrp6andthefamilyoffrizzledtransmembrane receptors,forthewinglesstypemmtvintegrationsitefamilyofglycoproteins, commonlydenotedaswntproteins.thewntgenes,ofwhichtherearecurrently nineteen,encodesecretedglycoproteins,generatingsignaltransductioncascades thatarecriticaltonearlyeveryeventduringdevelopment(moon,2005.wnt signalingpersistspostnatallytoregulateexpressionofspecifictargetgenesinself renewingadulttissuessuchasgut,hairfollicle,bone,andthehematopoieticsystem. MutationsinWntsignalinghavesubsequentlybeenidentifiedinoncogenesis, notablyincoloncancerandliquidcancers(reya&clevers,2005,andisanactive areaofcancerresearch. & &
3 Wnt&signaling&pathways.&&ThreepathwaysareactivatedfollowingWntreceptor engagement:thecanonicalwntpathway,thenoncanonicalplanarcellpolarity pathway(pcp,andthewnt/ca 2+ pathway.thecanonicalpathwayisthebest studiedofthese(clevers,2006.inthispathway,wntligandsbindtoafrizzled/lrp complex,interactingwithdashandaxinproteins,andpreventingtheproteosomal degradationofbetacatenin,whichplaysamajorroleinthecanonicalpathway.as justnoted,intheabsenceofwntsignaling,betacateninistargetedfor phosphorylationdependentdestruction.stabilizedandunphosphorylated,beta5 cateninaccumulatesinthecytoplasmandtranslocatestothenucleuswhereitbinds transcriptionfactorsandtranscriptionallyactivatesawiderangeoftargetgenes (Tamaietal.,2004.SecretedDkkproteinsinhibitWntsignalingbydirectbindingto LRP5/6,promotinginternalizationandinactivationofthereceptors(Tamaietal., 2000.& & Bone.&&TheroleofLRP5inboneaccrualiswellestablished.InosteoblastsLRP5 transduceswntsignalingviathecanonicalpathway.lrp5deficientmice demonstratelowbonemassasaresultofimpairedboneformationattributableto defectsinosteoblastproliferationandmaturation(katoetal.,2002.ofnote,lrp5 deficientmicealsodemonstratepersistentembryoniceyevascularizationasaresult offailedmacrophagemediatedapoptosis.conversely,transgenicmiceexpressinga gainoffunctionlrp5alleleinosteoblastshaveincreasedbonedensityand increasedosteoblastactivity(babijetal.,2003.&
4 Asnoted,humanshomozygousforlossoffunctionmutationsinLRP5areconsistent withobservationsinanimalmodels.theseindividualshaveanautosomalrecessive disorder,osteoporosispseudoglioma(oppgsyndrome,characterizedbysevere osteoporosis,childhoodfracturesandblindnessduetoabnormalvitreous development(gongetal.,2001.commonpolymorphicvariantsinlrp5inavariety ofhumanethnicpopulationsandagegroupshavebeenshowntoinfluencebone densityinthegeneralpopulation,indicatingthatinadditiontoitsroleintherare inheritedsyndromesjustdescribed,ithasanimportantroleinregulatingnormal skeletalmetabolism(jiangetal.,2010;saarinenetal.,2010;zenibayashietal., 2008. ConsistentwithacriticalroleofWntsignalinginbonehomeostasisaretheskeletal phenotypesobservedwithlossoffunctionmutationsintheendogenousinhibitors ofwntsignalingdkk1(describedaboveandsclerostin.heterozygouslossof functionmutationsindkk1leadtohighbonemassinmice(morvanetal.,2006.in humans,lossoffunctionmutationsinsclerostinleadtoaconditioncalled sclerosteosis,characterizedbymassiveandprogressiveincreasesinbonemass resultingultimatelyinnerveentrapment(lietal.,2005;semenov,tamai,&he, 2005. Lipid&Homeostasis.&&AnimalmodelsofLRP5deficiencydemonstrateimpairedlipid homeostasisundercertainexperimentalconditions.lrp5deficientmicefedahigh fatdietdevelopedhighplasmacholesterollevels,markedbyimpairedhepatic
5 clearanceofchylomicronremnants.whenfedanormaldiet,thesemicehadnormal plasmalipidlevels(fujinoetal.,2003.however,micedeficientinbothapoeand LRP5fedanormaldietexhibitedapproximately60%higherplasmacholesterol levelscomparedwithapoeknockoutmice.analysisoflipidcomponentsshowed thatplasmaclearanceofdietderivedtriglycerideswasseverelyimpairedbylrp5 butnotapoedeficiency.thesedatasuggestthatinamurinemodel,lrp5mediates catabolismofplasmalipoproteinsthroughapoedependentandindependent mechanisms(magoorietal.,2003.& Inhumanstudies,animpactofLRP5onlipidmetabolismislesswellestablished. TwosinglenucleotidepolymorphismsinLRP5havebeenassociatedwithahigher riskforofabdominalaorticaneurysm.specifically,thesepolymorphismsresultin reducedexpressionoflrp5causingimpairedclearanceoflipoprotein(a,an atherothromboticriskfactor(giustietal.,2009.astudyofachinesehan populationfoundanassociationbetweentwosinglenucleotidepolymorphismsin LRP5andincreasedcirculatinglevelsoftotalcholesterol(Jiangetal.,2010.Ina prepubertalfinnishpopulation,variantsinlrp5wereassociatedwithhighertotal andldlcholesterollevels(lappalainenetal.,2009.however,incontrast,astudy of13finnishindividualsfromonefamilywiththreedifferentlbmcausinglrp5 mutationsdidnotdemonstratehypercholesterolemiaorelevatedtriglyceridelevels (Saarinenetal.,2010. ThefindingsofabnormallipidmetabolisminmurinemodelsofLRP5deficiencyand
6 insomehumanstudies,incombinationwiththeroleoflrp5inosteoporosis,have promptedinvestigationoflrp5asacandidategeneunderlyingthemetabolic syndrome.findingsfromonestudyinacaucasianpopulationsuggestedan associationofsnpsandhaplotypesinthelrp5gene,withobesity,althoughthe molecularmechanismwasnotdefined(guoetal.,2006.incontrast,astudyina Japanesepopulation,whichincludedthesameSNP,didnotfindanassociation betweenbodymassindex(bmiandthatsamepolymorphismorhaplotypes comprisingit.furthermore,otherstudieshavefoundnoevidenceofanassociation betweenlrp5polymorphismsandbmi,homeostaticmodelassessmentof estimatedbetacellfunction(homab,estimatedinsulinresistance(homair,or serumlipidsindiabeticversusnondiabeticcontrols(zenibayashietal.,2008. Consistentwiththis,Jingetal(mentionedabovefoundnoassociationofLRP5 polymorphismswithbmi(jiangetal.,2010. Glucose&metabolism.ApotentialroleforLRP5inglucosemetabolismwasfirst reportedinananimalmodel.lrp5deficientmice,fedanormaldiet,werefoundto haveimpairedglucosetoleranceandbluntedglucoseinducedinsulinsecretionby isolatedpancreaticbetacells.isletsisolatedfromthesemicehadreduced intracellularlevelsatpandca 2+, anddecreasedmrnalevelsofinsulinsignaling targetsincludingglucokinaseandhnf4understimulatoryconditions.wnt3afailed toinduceinsulinsecretioninlrp5deficientislets,whileinwildtypeisletsboth Wnt3aandWnt5astimulatedglucoseinducedinsulinsecretion(Fujinoetal., 2003.&
7 ThesuggestedlinkbetweenLRP5andglucosemetabolismpromptedarangeof humanstudies.however,findingstodatehavebeenlessdefinitivethanthosein animalmodels.nostatisticallysignificantassociationshavebeenfoundbetween geneticpolymorphismsinlrp5andglucosemetabolism,asassessedby HemoglobinA1c(HbA1c(Jiangetal.,2010,HOMAB,orHOMAIR,wasfoundin individualswithtype2diabetesorinnondiabeticsubjects(lappalainenetal., 2009;Zenibayashietal.,2008.However,thesinglenucleotidepolymorphismsthat giverisetothehbmphenotypeshaveneverbeenincludedinanyoftheseanalyses fortheobviousreasonthattheirfrequencyinthegeneralpopulationisexceedingly rare.astudyoffinnishindividualswhowerehomozygous,compound heterozygous,orheterozygousforthreedifferentdeletionallbmcausinglrp5 mutations,foundthatapproximately50%ofthesesubjectshaddiabetes,with abnormalitiesreportedineitherbloodhba1clevelsorinoralglucosetolerance tests(ogtt(saarinenetal.,2010.thosewithabnormalogttshadlowfirst phaseinsulinreleasebutwerenotinsulinresistant.theseinterestingobservations arelimitedbythelackofacontrolpopulation.forexample,thispopulationincluded overweightpatientsaswellaspatientswithsignificantcomorbidities,thatcould haveconfoundedtheresult.thestudyalsolackedstatisticalpowertodetect potentialdifferencesinmetabolicparameters. & LRP6.&WhilethisthesisfocusesonLRP5anditsroleinhumanmetabolism,recent studieshaveelucidatedaroleforlrp6infuelmetabolism.manietal.identifieda nonconservativelossoffunctionmutationinlrp6thatattenuatestranscription
8 factor7like2(tcf7l2dependenttranscriptionoftheinsulinreceptorgene(singh etal.,2013.affectedindividualshaveasyndromeofearlyonsettype2diabetes, coronaryarterydisease,metabolicsyndrome,andosteoporosisinheritedinan autosomaldominantpattern.carriersexhibithyperinsulinemiaandreducedinsulin sensitivityinperipheraltissues,particularlyskeletalmuscle,whichcanbedetected priortothedevelopmentofimpairedglucosetolerance(manietal.,2007. & STATEMENTOFPURPOSE,HYPOTHESIS,SPECIFICAIMSOFTHESIS AsreviewedintheIntroduction,studiesinanimalssuggestaphysiologiclink betweenlrp5andmetabolism.however,thereisnoconsensusopinioninhuman studies.inadditiontherehavebeennohumanstudiesonthemetabolic consequencesofhbmcausinglrp5mutations.thisthesisexplorestheroleoflrp5 inhumanglucoseandlipidmetabolismbystudyingvolunteerswithhbmcausing LRP5mutations.StudiesinthispopulationcouldprovideinsightsintoLRP5 srole,if any,incommondiseasessuchasdiabetesandhypercholesterolemia. WehypothesizethattheLRP5pathwayhasasimilarroleinhumans,asinmice,and thathbmcausinglrp5mutationsinhumanswillleadtoimprovedglucose homeostasisandlipidmetabolismcomparedtomatchedcontrols.thespecificaims ofthisthesisareto: 1.MeasuretheimpactofHBMcausingmutationsinLRP5onhumanglucoseand lipidmetabolism 2.Investigatethepathway(sbywhichWntmayaffectglucosemetabolismin&vitro
9 METHODS Study&Subjects.NinesubjectswiththeG171VHBMcausingLRP5mutationwere recruitedfromtwopreviouslyreportedkindreds(fig.1a,fig.1b(boydenetal., 2002;Leeetal.,2013.TwosubjectswithanN198S(asparaginetoserinemutation inlrp5associatedwithhbmwerealsostudied(leeetal.,2013.wealsorecruited 22gender,age,race,andbodymassindex(BMImatchedindividualsunrelatedto thekindredmemberstoserveascontrols. HbA1candinsulinweremeasuredinallparticipants.Twocontrolsubjectsmatched forgender,age,andbmiwererecruitedforeverystudysubject.fivesubjects,in total,fromtheaffectedandcontrolgroupsreportedtakinglipidlowering medicationsandwereexcludedfromthelipidanalysisportionofthestudy.recent datasuggestalinkbetweenhmgcoareductaseinhibitoruseandtype2diabetes (Carteretal.,2013.Asthisassociationhasnotestablishedcausality,wechosenot toexcludethesefivesubjectsfromtheglucoseanalysis.threesubjectsagreedto undergoanogtt,andtwounderwentmrsstudies.thesesubjectswerematched forage,race,bmi,andactivityleveltoalargecontrolgroup(n=54. ThestudycompliedwiththeWorldMedicalAssociationDeclarationofHelsinki EthicalPrinciplesforMedicalResearchInvolvingHumanSubjects.Thestudywas approvedbytheyalehumanresearchprotectionprogramirb.allsubjectsgave writtenconsenttoparticipateinthestudyafterthepurpose,natureandpotential risksofthestudieswereexplained.
10 IHumanStudies Theauthorofthisthesisrecruited,enrolledandstudiedallvolunteerswhosedata arereportedhereunlessotherwisespecified. Hemoglobin&A1c.Participantswereinstructedtofastfrommidnightonward.All bloodsamplingtookplacebetween7am10amontheyalenewhavenhospital ResearchUnit.HbA1cwasmeasuredinwholebloodbytheYaleNewHavenClinical ImmunologyLab,usingPremierHb9210boronateaffinityhighperformanceliquid chromatographywitheluatesmeasuredat413±2nmabsorbanceaccordingto manufacturersinstructions(trinitybiotech;jamestown,ny. Insulin.PlasmaconcentrationsofinsulinweremeasuredwithaHumanInsulin SpecificRIAKit(Millipore;St.Charles,MOintheYaleMineralMetabolism Laboratory.HOMABandHOMAIRweremathematicallyderivedfromthe measuredinsulinvalueandtheestimatedaverageglucose(eagfromthesame bloodsample(nathanetal.,2008. OGTT.WholebodyinsulinsensitivitywasassessedincollaborationwithDr.Kitt FalkPetersen(YaleUniversity,DepartmentofInternalMedicine,Sectionof Endocrinology;NewHaven,CTwitha3hrOGTT.Subjectswerefastedfrom8pm thenightpriortotesting.twentyminutesafterinsertionofanantecubitalivline, fastingbloodsampleswerecollectedfordeterminationofplasmaglucose.the75g dextrosedrink(glucola;curtinmathesonscientific,houston,txwasthen
11 administered,andbloodsamplescollectedat10,20,30,60,90,120,150,and180 minfordeterminationofplasmaglucoseandinsulinconcentrations.theinsulin SensitivityIndex(ISIwasusedtoestimateinsulinsensitivity(Petersenetal., 2006.PlasmaglucoseconcentrationsduringtheOGTTweremeasuredusingaYSI STAT2700Analyzer(YellowSpringsInstrumentCo.,YellowSprings,CA.Insulin concentrationsweremeasuredusingalincoriakit(emdmillipore;billerica, Massachusetts. Lipid&panel.Totalcholesterol,highdensitylipoprotein(HDL,andtriglycerides(TG concentrationsweremeasuredonanaceclinicalchemistrysystemautoanalyzer (AlfaWassermann;WestCaldwell,NJ.Lowdensitylipoprotein(LDL concentrationswerecalculatedfromtc,hdl,andtgwiththefriedewaldformula (Friedewald,Levy,&Fredrickson,1972. 1 H&MRS&Assessment&of&Intramyocellular,&Extramyocellular,&and&Hepatic&Lipid&Content.&& Intramyocellular(IMCLandextramyocellular(EMCLandhepaticlipidcontents weremeasuredusing 1 HMRSalsoincollaborationwithDr.KittFalkPetersenas previouslydescribed(petersenetal.,2006.musclelipidcontentwasmeasuredin thesoleusmuscleusingan8.5cmdiametercircular 13 Csurfacecoilwithtwin, orthogonalcircular13cm 1 Hquadraturecoils.Theprobewastunedandmatched andscoutimagesofthelowerlegwereobtainedtoensurecorrectpositioningofthe subjectandtodefineanadequatevolumeforlocalizedshimmingusingthe FASTMAPprocedure(Gruetter,1993.TheliverTGcontentwasmeasureby 1 H
12 respiratorygatedsteamspectroscopyina15x15x15mm 3 voxel.acquisitionwas synchronizedtotherespiratorycycleandtriggeredattheendofexpiration.a watersuppressedlipidspectrumandalipidsuppressedwaterspectrumwere acquiredinthreedifferentlocationsofthelivertoaccountforliverinhomogeneity. Aminimumofsixspectrawasacquiredforeachsubjectandthetotallipidcontent wasaveragedandcalculated.inaddition,hepaticlipidcontentwascorrectedfor transverserelaxation,usingthetransverserelaxationtimesof22msforwaterand 44msforlipid,aspreviouslydescribed(Rabol,Petersen,Dufour,Flannery,& Shulman,2011.& & IIIn#vitrostudies Theauthorofthisthesisdesignedandconductedallthein&vitroexperiments describedhere,unlessotherwisespecified. Materials.RecombinantmurineWnt3aandhumanWnt3awerepurchasedfrom R&DSystems(Minneapolis,MN.Referencesto Wnt intheseexperimentswillonly refertownt3ause.insulinhumalognd1048wasfromelilillyandcompany (Indianapolis,IN.GlucagonanddexamethasonewerepurchasedfromSigma Aldrich(St.Louis,MO.DMEM,heatinactivatedfetalbovineserum(FBS,andOpti MEMGlutaMAXwereallpurchasedfromGibcoLifeTechnologies(Carlsbad,CA.All reagentsusedforins1cellcultureswerepurchasedfromsigmaaldrich(st.louis, MOunlessotherwisespecified.
13 Cells.PrimaryhepatocytesisolatedfromSpragueDawley(SDratswerepurchased fromlifetechnologies(carlsbad,ca.hepg2cells(p76werepurchasedfrom AmericanTypeCultureCollection(ATCC;Manassas,VA.ExpressionlevelsofLRP5 transcriptsweremeasuredintissuesisolatedfromsdratpurchasedfromcharles River(Wilmington,MA.TheINS1832/13ratinsulinomacelllinewasgenerously providedbydr.richardkibbey(yaleuniversity;newhaven,ct,andinitialstocks wereoriginallyreceivedfromchristophernewgard(dukeuniversity;durham, NorthCarolina(Hohmeieretal.,2000. Primary&Rat&Islets.SpragueDawleyratpancreaticisletswerepreparedbyTamara DlugosintheYaleDiabetesEndocrinologyResearchCenter. Primary&Hepatocyte&Cell&Cultures.Pelletedcellswereresuspendedtwiceinmedia containinghighglucosedmem,10%fbs,1nmdexamethasone,1nminsulin,and P/S(1:100.Cellswereplatedataninitialdensityof5x10 5 incollagencoated6well platesandincubatedforfourhoursat37 C.Cellswerethenwashed1xwith phosphatebufferedsaline(pbsandincubatedat37 CovernightinOptiMEM GlutaMAX.Thenextmorningcellswerewashed1xwithPBSandincubatedfortwo hoursinglucosefreedmemcontainingsodiumbicarbonate.cellswerewashed againwithpbs,andthenreceivedoneofthefollowingtreatmentsinglucosefree, bicarbonatefreedmem: 1. 500nMdexamethasone 2. 100nMglucagonwith1mMsodiumpyruvate
14 3. 10nMinsulin,Wnt(100ng/mL 4. Wntanddexamethasone 5. Wntandglucagonwith1mMsodiumpyruvate 6. dexamethasoneandinsulin 7. glucagonandinsulin Mediawassampledat1,3,5,12,and24hrsaftertreatmentwasinitiated.The24 hourtimepointdatawereusedfortheanalysesreportedhere.cellswerewashed withpbsandrnaextractedusingtherneasykit(qiagen,valencia,ca. HepG2&Cell&culture.CellswerethawedandcentrifugedaccordingtotheATCC recommendedprotocol.theywerethentreatedwiththesamereagentsunder identicalconditionsasdescribedaboveinthesectiononprimaryhepatocytecell Cultures. INS51&Cell&Culture.ThisexperimentwasperformedincollaborationwithDr.Richard Kibbey(YaleUniversity;NewHaven,CTandRebeccaPongratzfromhislaboratory. INS1&cellsweremaintainedasmonolayersinRPMI1640completemediumwith 11.1mMDglucosesupplementedwith10%(v/vfetalbovineserum,antibiotics (10,000units/mlpenicillinand10mg/mlstreptomycin,10mMHEPES,4mML glutamine,1mmsodiumpyruvate,and50mmbetamercaptoethanol.for experiments,cellswereplatedinto6wellplatesatadensityof2.0x10 5 cellsper wellandculturedat37 Cuntil80%confluent.Cellswerethenrandomlyassignedto
15 threeconditions:wntpretreatment,nowntpretreatment,ornownttreatment.for pretreatmentwnt(100ng/mlwasaddedthenightpriortoglucosestimulation. EquivalentamountsofPBSwereaddedtothenonpretreatmentcellstoensure equivalentvolumesofmedia.onthedayoftreatmentcellswerewashedwithpbs andthenpreincubatedinbasalmediafor90minutes.basalmediawasglucosefree DMEMcontaining3.7g/LNaHCO3,4mMglutamine,10mMHEPES,2.5mMdextrose and0.2%96%fattyacidfreebovineserumalbumin.wntwasaddedtothemediaof cellsselectedforwnttreatment.mediawasthenchangedtobasalmediacontaining either2.5mmdextrose±wnt,orstimulationmediacontaining9mmdextrose±wnt for45minutes.followingtheincubation,mediafractionswerecollectedandplaced oniceuntilanalyzedforinsulincontent.theremainingmediawasaspirated,cells werewashedwithpbsx2andthen1mloficecold0.1%tritonx100wasadded. Celllysateswerecollectedandassayedforprotein. INS51&insulin&analysis.Insulinconcentrationintheconditionedmediawasmeasured usingthehighrangeratinsulinelisakitin96wellplates(alpcodiagnostics, Salem,NH.Allmediaextractswerenormalizedtoacellproteinconcentrationusing amicrobcaproteinassaykit(thermoscientific;waltham,maandthe manufacturer srecommendedprotocol. Human&islet&perfusion.ThisexperimentwasalsoperformedincollaborationwithDr. KibbeyandMs.Pongratz.HumanisletswereobtainedfromtheUniversityofAlberta IsletCore,AlbertaDiabetesInstitute,Edmonton(Pigeauetal.,2009.Isletswere
16 culturedincompletecmrlmediaat37 Cwith5%CO2,95%airuntilthetimeof theexperiment.priortoperfusion,approximately160islets(40islets/per replicatewerepretreatedwithwnt(75ng/mlfor19hours.another160islets weredesignatedasthecontrol(nowntpretreatment.isletswereperfusedwith DMEMbasalmediacontaining2.5mMglucose(D5030,Sigma.Fortyisletsper chamberwereperifusedwithbasalmediafor45minutestoequilibratetheisletsto thecolumnandinstrument.afterthepreincubation,isletswereperifusedwiththe differenttreatmentsand100ulofperifusatecollectedperminute.theperifusion protocolconsistedofabasalperiodof2.5mmglucosefor20mininwhichwnt (75ng/mLwasaddedbacktothetreatmentgroupisletsafter10minand maintainedforthedurationoftheperifusion.thebasalperiodwasfollowedbya 9mMglucosestimulationfor45min.Theglucoseconcentrationwasthenreducedto 2.5mMglucosefor15minfollowedbyasecondstimulationwith30mMKClat 2.5mMglucose(5min.toevaluateinsulinsecretionindependentofglucose metabolism.theisletswerethenallowedtorecoverinbasalmediafor5min. InsulinlevelsintheperfustateweremeasuredwithanELISAforhumaninsulin(80 INSHUE01,ALPCODiagnostics,Salem,NHandnormalizedtotheDNAcontentof thecelllayer(p7589,molecularprobes. Quantitative&Real&Time&PCR.TotalRNAwasisolatedusinganRNeasykit(Qiagen, Valencia,CAperthemanufacturersinstructions.cDNAsynthesiswascarriedoutat 43 Cfor45minfollowedbydenaturationat95 CforfiveminutesusingacDNA synthesiskit(stratagene,lajolla,ca.quantitativerealtimepcr(qpcrwas
17 performedwithtaqmanpcrreagentkits(stratagene,lajolla,causing40ngof RNAwithareactionvolumeof20uLusingtheOpticon2DNAEngineContinuous FluorescenceDetectionSystem(MJR,Waltham,MA.Specificprimer/probesetsfor targetgeneswerepurchasedfromappliedbiosystemslifetechnologies(carlsbad, CA(seeTable1.AllqPCRreactionswereperformedinduplicate,andcycling conditionswere95 Cfor20sand60 Cfor1minfor39cycles.Theamplification signalfromthetargetgenewasnormalizedtoabeta5glucuronidasesignalinthe samereaction.therelativeexpressionlevelsweredeterminedusingthe comparativectmethod(alsoknownasthe2ctmethod.dataarepresentedasthe relativemrnalevels. Statistical&Analyses.Weestimatedwecouldrecruit11subjectsfromtheaffected kindredssuitableforanalysistobepairedwith22matchedcontrolsina2:1ratio. Basedonpreliminarycontroldata,weestimatedthatthestandarddeviation(SD forhba1cwouldbe0.22(%.sincewedidnotknow,a&priori,whatthesdwouldbe forhba1cintheaffectedindividualsweconservativelyestimateditat0.44(%. UsingtheseestimatesforSDs,wewouldhave99%powertoseeadifferencein HbA1cvaluesof0.5%orgreater,and80%powertoseeadifferenceof0.3%or greater(graphpadstatmate2.0:graphpadsoftwareinc.,lajolla,ca. StatisticaldifferenceswereanalyzedusingtheunpairedStudentttest,andoneway ANOVAwhereappropriate(GraphPadPrismversion6.00forWindows,GraphPad Software;LaJollaCaliforniaUSA,www.graphpad.com.Datapresentedaremean
18 ±standarderrorofthemean(sem.differenceswereconsideredstatistically significantwhenp<0.05. RESULTS IHumanStudies Baselinecharacteristicsoftheaffectedsubjectsandtheage,gender,andBMI matchedcontrolgroupareshownintable2.meanbmiofthesegroupswere 27.4±1.8and27.6±1.1,respectively. HbA1c&is¬&significantly&different&in&individuals&with&HBM&mutations&in&LRP5&& Theparametersofglucosemetabolismmeasuredinthestudysubjectsare presentedintable3.twoaffectedsubjectswereknowndiabeticstakinginsulin. Thesesubjectsandtheirfourmatchedcontrolswereexcludedfromanalysis.With9 affectedindividualsand18controls,wehad99%powertoseea1.0%difference and80%powertoseea0.6%differenceinhba1cvalues.changesofthismagnitude wouldhavebeenclinicallymeaningful.however,wedidnotobserveanydifference inthisparameter.&therewerenostatisticallysignificantdifferencesbetween affectedandcontrolpopulationsforhba1c(p=0.06,eag(p=0.06,insulin(p=0.82, HOMAB(p=0.34andHOMAIR(p=0.66.MeanHbA1cforaffectedandcontrol groupswere5.8±0.3%and5.4±0.5%respectively.oneaffectedsubjectandone controlsubjectwerefoundtohaveelevatedhba1clevelsinthediabeticrange ( 6.5whentestedforthepurposesofthisstudy.HbA1cvaluesaredepictedin Figure2.&
19 OGTT&results&were¬&significantly&different&in&individuals&with&HBM&LRP5&mutations&& ThreesubjectswiththeHBMLRP5mutationunderwentanOGTT.Bloodglucose (Fig.3A,insulin(Fig.3Bandcpeptide(Fig.3Clevelswerenotconsistently differentfromthecontrolgroup,suggestingthatthereisnodifferenceininsulin productionaswellasininsulinsensitivityinthesethreeindividualswhen comparedtoalargecontrolgroup. IMCL&and&liver&TG&is¬&significantly&different&in&individuals&with&HBM&mutations&in& LRP5& AspartoftheplannedinvestigationintotheroleofLRP5inglucosemetabolism,we alsomeasuredintramyocytelipidasanindexofinsulinsensitivity.inparticular, thereisasubstantialliteratureonassessingintramyocellularlipid(imclcontent asabiomarkerofinsulinsensitivity:magneticresonancespectroscopy(mrs studiesindiabeticandnondiabeticpatientshaveshownthatinsulinresistancecan beattributedtopostreceptordefectsininsulinsignaling,forexample,reduced Glut4transporteractivitycausedbymetabolitesofintracellularlipids(Morino, Petersen,&Shulman,2006;Petersenetal.,2007.Studiesinahealthy,leanelderly individuals,leanandobeseadolescents,andlean,insulinresistantoffspringof patientswithtype2diabetes(petersenetal.,2003;petersen,dufour,befroy,garcia, &Shulman,2004;Sinhaetal.,2002,allsupporttheconclusionthatinsulin resistanceresultsfromtheintracellularaccumulationoffattyacylcoas,and diacylglycerolinskeletalmyocytesandhepatocytes.theseadverseeffectscanbe attenuatedwithweightloss(petersenetal.,2005;petersenetal.,2012;raboletal.,
20 2011. TwooftheLRP5affectedsubjectsunderwentMRSoftheirskeletalmuscleandliver (Table4.TheInsulinSensitivityIndex(ISIwaspredictablyhigherinSubjectBwho hadalowerbmi,lowerpercentbodyfat,andhigheractivitylevel(notshownthan SubjectA.TheISIofbothsubjectscomparedtothecontrolgroupsvariespredictably withbmiandbodyfatpercentage,withsubjectahavingalowerpercentbodyfat andhigherisicomparedwithcontrols,andsubjectbhavingahigherbmiandlower ISIcomparedwithcontrols.TheIMCLinbothsubjectswasnotsignificantlydifferent comparedtothecontrolgroup.therewasalsonoconsistenttrendinlivertg towardshigherorlowerpercentagelipidscomparedwithcontrols. LDL&cholesterol&levels&are&lower&in&affected&study&subjects& LipidprofilesfortheaffectedindividualsarepresentedinTable5.Threeaffected andtwocontrolsubjectswereknowntohavehypercholesterolemiaandwere takingcholesterolcontrollingmedicationsatthetimeofstudy.thesesubjects,and theirmatchedcontrols,wereexcludedfromtheanalysis. Therewerenosignificantdifferencesintotalcholesterol,TGs,andHDL,between controlandaffectedsubjects(figs.4ac.howeverasshowninfigure4d,ldl levelsweresignificantlylowerinaffectedsubjects(p=0.04.
21 IIIn#vitrostudies Tissue&survey& AsaninitialsteptoexploretheroleofWntsignalinginglucosemetabolism,we surveyedtheexpressionoflrp5inseveraltissuesinvolvedinfuelmetabolism. LRP5expressionwashighestinliver,andlowestinbone(Fig.5.&Thesedatalargely agreewithpreviouslypublishedtissueexpressionsurveysoflrp5(heyetal., 1998.& Testing&the&hypothesis&that&Wnt&suppresses&gluconeogenesis&by&inhibiting&PEPCK5C& HBMmutationsinLRP5arepositedtoexerttheireffectontheskeletonbycausinga functionalincreaseinwntsignaling.thisisnotduetoligandindependentreceptor signaling,butratherduetoaresistanceofthesemutantreceptorstoendogenous inhibitorssuchasdkk1.thus,invivo,thesemutantreceptorstransmitagreater signalinthepresenceofendogenousinhibitorsthanthewildtypereceptor.froma functionalstandpoint,therefore,thesemutationsconstitutegainoffunctioninwnt signaling.wethereforesoughttodeterminetheeffectofwntsignalingonglucose metabolismbytestingtwohypotheses. ThefirstwasthatWntsignalingwouldsuppressgluconeogenesisbyinhibitingone ofthekeyenzymesinthegluconeogenicpathway,thecytosolicformof phosphoenolpyruvatecarboxykinase(pepckc.pepckccatalyzesthefirst committedstepofgluconeogenesis,specificallytheconversionofoxaloacetate+gtp >phosphoenolypyruvate+gdp+c02.itisnotregulatedposttranslationallyor
22 allostericallybutentirelytranscriptionallysuchthatenzymeactivityisdirectly proportionaltotheleveloftranscriptexpressedintheliver.(chakravarty,cassuto, Reshef,&Hanson,2005.Previousworkinprimaryhepatocytes,hepatomacell lines,andanimalmodelshasexploredtheeffectofcanonicalwntsignalingon PEPCKCandmodulationofthegluconeogenicresponse(Hall,Wang,George,Koch, &Granner,2007;Liuetal.,2011.WhilethePEPCKpromoterhasnoknown canonicalwntbindingmotifs,pepckcisinhibitedbyglucagonsynthasekinase3 (GSK3adownstreamtargetforcanonicalWntsignaling.ActivationofWntsignaling suppressesgsk3activity(frame&cohen,2001.thereforewehypothesizedthat Wntsignalingwouldsuppressgluconeogenesis,byinhibitingPEPCKCexpression. Wnt&does¬&effect&PEPCK5C&expression&in&primary&hepatocytes& AsdepictedinFigures6AC,andasexpected,PEPCKexpressioninprimary hepatocyteswassuppressedbyinsulin,andinducedbydexamethasoneand glucagon.pepckinductionbyglucagonwasbluntedbyinsulin.theseareallknown effectsofinsulin,dexamethasoneandglucagonandconfirmthatourin&vitroassayis sensitiveenoughtodetectchangesinducedbyhormonesandcytokines.however, theadditionofwntdidnothaveasignificantsuppressiveeffectonpepckc expressionascomparedtothecontrol(fig.7anordiditblunttheeffectof glucagon(fig.7c.unexpectedly,wntseemedtoenhancetheeffectof dexamethasonethoughthechangewasnotstatisticallysignificant(fig.7b.these resultswereconfirmedinexperimentsunderthesameconditionsusingcellsfrom thehepg2cellline.
23 Testing&the&hypothesis&that&Wnt&augments&glucose5stimulated&insulin&secretion&in& pancreatic&islets& TheeffectofWntoninsulinreleasefrompancreaticisletbeta5cellswasalso exploredasanalternativemechanismbywhichwntsignalingcouldaffectglucose metabolism.asnotedearlier,wntfailstostimulateinsulinsecretionfromislets isolatedfromlpr5knockoutmice;conversely,exposureofwildtypeisletstownt stimulatedglucoseinducedinsulinsecretion(fujinoetal.,2003. WeinitiallyundertookstudiesusingtheINS1832/23celllinesincethesecellshave importantsimilaritiestopancreaticbetacellsincludingthoserelatedtoinsulin secretion(cline,lepine,papas,kibbey,&shulman,2004;hohmeieretal.,2000;lu etal.,2002;pongratz,kibbey,shulman,&cline,2007.thereforeweexpectedthat theresultsinthesecellswouldlikelymirrorthoseinprimaryhumanislets. Wnt&does¬&augment&glucose5stimulated&insulin&secretion&in&INS51&cells& 9.0mMglucosestimulatedinsulinsecretioninINS1celltwofoldcomparedtothe secretoryrateunderbasalconditions(2.5mmglucose.addingwnttoeitherbasal ortostimulatoryglucoseconcentrationshadnostatisticallysignificanteffecton insulinsecretion(fig.8. GivenpriorreportsthatWntisactiveinpancreaticbetacellswenextsoughtto confirmlrp5expressionintheins1832/23cells.forthesestudiesthelevelof LRP5transcriptexpressionintheINS1832/23cellswascomparedtothatin
24 primaryratislets.lrp5expressionissignificantlylowerintheins1832/23cells whencomparedtotheprimarycells(fig.9.thissuggeststhatwntdoesnotaffect insulinsecretioninins1832/23cellsperhapsinpartbecausethelevelofreceptor expressionistoolow.& Wnt&does¬&augment&glucose5stimulated&insulin&secretion&in&primary&human&islets& InviewofthefindingthatLRP5wasonlyexpressedatalowlevelinINS1832/23 cells,wenextexaminedtheeffectofwntoninsulinsectioninprimaryhumanislets. HumanisletsareknowntohaverobustexpressionofLRP5(Heyetal.,1998.That notwithstanding,glucoseinducedinsulinsecretioninprimaryhumanisletswasnot affectedbywnt(fig.10.whilethesedataareconsistentwithourfindinginthe INS1cells,theyareatvariancewithpriorstudiesusingmurineisletsinwhichWnt treatmentledtoadoublingofglucoseinducedinsulinsecretion(fujinoetal.,2003. DISCUSSION Theprevalenceandreachofdiabetesacrossgenerationsandpopulationsiswell known.overeightpercentoftheunitedstatespopulationhasdiabetes.in particular,thereisanincreasedincidenceamongadolescentandnonwhite populations(http://www.cdc.gov/diabetes/pubs/pdf/ndfs_2011.pdf.hospitaland healthsystemsfacesoaringdirectmedicalcosts,whichtopped$176billionin2012. Theaverageannualcostofcaringforadiabeticis2.3timeshigherthananon diabetic,multiplyingthecostofthediseaseacrosshospitalizationsandoutpatient visits("economiccostsofdiabetesintheu.s.in2012,"2013.notably,these
25 estimatesdonotaccountfortheadditional,significantcostofreducedproductivity andcaregiverburden. WntsignalingthroughLRP5representsanewlyappreciatedmetabolicpathwaythat mightbeatargetfordrugdiscoveryintype2diabetes.inpreadipocyteswntand insulinsignalinghavebeenshowntointeractwithlrp5exertingapositiveeffecton insulinsignaling(palsgaardetal.,2012.studiesinrodentssupporttheconclusion thatlrp5mediatesglucoseandlipidmetabolism(fujinoetal.,2003;magoorietal., 2003.However,datafromhumanstudiesinLBMcausingmutationspopulations hasnotbeenasconsistent,withsomestudiessuggestingthattheseindividualshave impairedglucosemetabolismandothersfailingtofindasimilartrend(saarinenet al.,2010;zenibayashietal.,2008. Inviewoftheseconflictingdataandbecauseavarietyofmethodologieshavebeen usedtomeasureglucoseandlipidmetabolismwerevisitedtherelationshipsina humanpopulationthathasnotyetbeenstudied:thosewithhbmcausingmutant LRP5receptors. Ourdatashowthatcomparedtomatchedcontrols,individualswithHBMcausing LRP5mutationsdidnotdemonstrateimprovedglucosehomeostasis.HbA1c,a commonclinicaltestfordiagnosisandclinicalmonitoringofdiabetes,wasnot significantlydifferentbetweencontrolandaffectedpopulations.similarly,therewas nodifferenceineag,insulinlevels,homab,andhomairscores.asnoted,our
26 studyincludedmembersofmultiplekindreds,encompassingtwodifferenthbm causingmutationsinlrp5.whilethestudywassufficientlypoweredtodetecta differenceinhba1cbetweenthetwostudygroups,itwasnotpossibletodoa meaningfulsubanalysisofthesmallnumberofindividualswiththen198s mutationtodetermineiftheirresponsedifferedfromthatintheindividualswith theg171vmutations.however,asubanalysisofg171vmutationkindredmembers alonealsodemonstratednosignificantdifferencefromthecontrolgroup(p=0.07 (datanotshown.inotherstudiesinthesekindredsithasbeensuggestedthatthein vivoactionsofthesetwomutationdiffered(leeetal.,2013. AsmallersampleofaffectedLRP5subjectsunderwentanOGTTandMRSofliver andskeletalmuscle.inagreementwiththedatainthelargerstudygroup,blood glucose,insulinlevels,andcpeptidelevelswerenodifferentinthesethree individualscomparedtocontrols.theisivariedappropriatelywithbmi,percent bodyfatandactivitylevel,andtherewasnotrendtoindicateimprovedinsulin sensitivityinthetwoaffectedindividualswhoparticipatedintheseanalyses. PreviousstudieshavefoundastrongrelationshipbetweenIMCLcontentand muscleinsulinresistance,suggestingthatintracellularlipidmetabolitesmightbea causalfactorinitspathogenesis.mrsmeasuresoftginmuscleandliverareuseful proxiesforassessingspecificfattyacidcontent(i.e.diacylglycerolsthatcould otherwiseonlybeevaluatedwithinvasivetechniquessuchasmusclebiopsy.we hypothesizedthatonemechanismbywhichlrp5affectedindividualscouldhave improvedglucosemetabolismwasthroughimprovedinsulinsensitivityresulting
27 fromreducedimclcontent.givenoursmallsamplesize,wewouldhaveneededto seeaveryrobustdifferencebetweenthefindingsinthetwostudysubjectsandthe entirecontrolgroup.however,insulinsensitivityinthesetwostudysubjectswasno differentfromthatofthecontrolsubjects.itisworthrecallingthattwoaffected subjectsweretakinginsulinandwereexcludedfromtheseanalyses.athirdaffected subjectwasfoundtohaveaserumhba1cinthediabeticrange.theseobservations supportthefindingsthathbmcausingmutationsdonotimpactglucose homeostasis.consistentwiththat,ourdataalsoindicatethathbmcausinglrp5 mutationsdonotimproveinsulinsensitivityorinsulinproduction.iflrp5playsa roleinglucosehomeostasis,thenthisstudysuggeststhatitsimpactisminimalin comparisontomoredominantfactorsthuskeepingusfromobservingitinasmall population. Inadditiontoeffectsonglucosehomeostasis,animalstudiesalsopointtoarolefor LRP5inlipidhomeostasis.LRP5 / micefedhighfatdietshadelevatedcholesterol, andimpairedplasmaclearanceofdietderivedtriglyceridesimplyingapossible ApoEindependentroleforLRP5intriglyceridehydrolysis(Magoorietal.,2003. However,studiesinhumanLBMcausingmutationpopulations,havenotfound differencesinlipidlevels.ofnote,thesestudieswerenotcontrolmatched,andone studyincludedmultiplesubjectsonlipidloweringdrugs.(saarinenetal.,2010; Zenibayashietal.,2008. Ouranalysisexcludedindividualsonlipidloweringdrugsandcontrolledforgender, age,andbmi.totalcholesterol,triglyceridesandhdlwerenotdifferentbetween
28 affectedandcontrolspopulations.however,ldlwassignificantlylowerinthe affectedsubjects.thisfindingisparticularlyinterestingasthelrp5receptoris knowntobindapoecontaininglipoproteinsin&vitro&(kimetal.,1998. LRP5isknowntobehighlyexpressedintheliverandinhepatocytes(Heyetal., 1998;Kimetal.,1998.Weconfirmedthisinthecurrentstudy.Wehypothesized thattheadditionofwntwouldinhibitpepckc,aratelimitingstepinthe gluconeogenicpathway.sinceacceleratedgluconeogenesisplaysanimportantrole intype2diabetes,thishypothesishadconsiderableclinicalrelevance.howeverour in&vitro&studiesconsistentlyshowednoeffectwntonpepckcinprimary hepatocytes.neithertheinductionofpepckbydexamethasoneorglucagonorthe suppressionbyinsulinwasaffectedbywnt.itremainspossiblethatwntsignaling throughlrp5hastargetsgenesotherthanpepcktomodulategluconeogenesis, thatwerenotconsideredinthecurrentstudy. WealsoexaminedtheeffectofWnttreatmentinINS1cellsandhumanisletsto assesstheimpactthishasoninsulinsecretion.ratins1cells,consideredavalid systemtostudyinsulinsecretiondynamics(hohmeieretal.,2000,responded appropriatelytoglucosestimulationwitharobustincreaseininsulinsecretion. However,theydidnotfurtherincreaseinsulinproductioninresponsetoWnt treatment.sincethesecellshadlowlevelsoflrp5comparedwithprimaryratislets, thesestudieswereextendedtohumanislets.wnttreatmentfor16hoursdidnot changehumaninsulinproductionunderbasalorglucosestimulatedculture
29 conditions.thesefindingsareatoddswiththefindingthatwntincreasedglucose inducedinsulinsecretioninmurineislets(fujinoetal.,2003.thereasonforthese divergentfindingsisnotclear.oneobviouspossibilityisthatmurineandhuman isletsresponddifferentlytownt.whythendoesmurineboneapparentlyfaithfully recapitulatetheeffectsofwntonthehumanskeleton?thismaybebecausethe macroarchitectureofboneaswellastheinteractionsofbonecellsareverysimilar inmouseandman.incontrast,thearchitectureofthemurineisletaswellasits cellularcompositionisdifferentfromthehumanislet(steineretal.,2010,andso maybethebiologicalmechanisminvolvedinthedevelopmentandmaintenanceof thesetissues. Conclusion& StudiesofLBMcausingmutationsinLRP5inbothexperimentalmodelsandin humanswithoppgsuggestthatlossoffunctioninthisreceptorisassociatedwith impairedglucosetolerance.howeverthesestudiesarelimitedbythelackofcontrol groupsandsmallnumbersofsubjectsstudied.ourstudy,thefirstinhumanswith HBMcausingmutationsinLRP5,includedacarefullymatchedcontrolgroupanda largernumberofaffectedindividuals.thesefindingsprovidestrongevidencefora limitedroleoflrp5inglucosemetabolismaswecouldnotdemonstratean interactionbetweenthetwo. TheHBMcausingmutationsstudiedinourkindredsarenottissuespecificintheir actions(asdemonstratedbyourexpressionsurvey,andshouldchangewnt
30 signalinganywherethatwntacts.ourstudypopulationincludedtwohbmcausing mutations,eachwithextremeskeletalphenotypes.giventherobustphenotypein bone,weanticipatedaconsiderablephenotypeinglucosehomeostasis.although thesamplesizefortheogttandmrssubanalysesweretoosmalltodemonstrate statisticalsignificance,wecouldidentifynotrendtowardincreasedinsulin sensitivityoralteredinsulinproduction.infact,threeofthekindredmembers screenedforinclusioninthisstudyhaddiabetesand/orinsulinresistance.these resultsareindirectcontradictiontothefindingsinpreviouslyreportedanimal models,underscoringthepotentiallimitationsinextendingsomefindingsinmiceto humans. Whileourfindingsdonotdirectlyevaluatetheroleoflossoffunctionmutationsin LRP5onglucosemetabolism,theydomakeLRP5alessattractivetargetfordrug discoveryindiabetes.however,itdoesappearthatthereisaneffectonldl metabolism,amajorriskfactorforcoronaryarterydisease.todetectastatistically significanteffectonldlevenwithoursmallsample,certainlysuggeststhatthe molecularmechanismunderpinningthiseffectwarrantsfurtherstudy. Insummary,ourdatadoesnotsupportthehypothesisthatLRP5improvesglucose metabolisminindividualswithhbmcausinglrp5mutations.thedatasuggestthat theremaybeaspecific,beneficialeffectoflrp5onldlcholesterol.additional studiesshouldfocusonclarifyingthecellularandmolecularmechanismsbywhich theseoccur.
31 FIGURES&TABLES Figure1A.G171VKindredPedigreeofFamilyA Affectedkindredmembers,indicatedbydarksymbols,haveasinglebasepair mutationcausingaglycinetovalinechangeatposition171inthelowdensity lipoproteinrelatedreceptorprotein5(lrp5.thisresultsinahighbonemass (HBMphenotype. Figure1B.G171VKindredPedigreeofFamilyB Affectedkindredmembers,indicatedbydarksymbols,haveasinglebasepair mutationcausingaglycinetovalinechangeatposition171inthelowdensity lipoproteinrelatedreceptorprotein5(lrp5.thisresultsinahighbonemass (HBMphenotype.
32 Table1.TaqManassaysusedforqPCRreactions. GeneSymbol NCBIgenereference TaqManassayID Pck1 NM_198780.3 Rn01529014 Alp1 NM_007431.2 Rn01516028 LRP5 NM_001106321.2 Rn01451428 Actb NM_031144.2 Rn00667869 QPCRwasusedtoanalyzePEPCKCmRNAexpressioninexperimentsusingRNA fromprimaryhepatocytesandhepg2cells.lrp5mrnaexpressionwasanalyzedin avarietyofrattissues,ins1cells,andprimaryratislets. Table2.Baselinecharacteristicsofallstudyparticipants Variable Affected MatchedControls pvalue (n=11 (n=22 Age(years 63±7 63±4 0.96 Male(n,% 9(82% 18(82% n/a Female(n,% 2(18% 4(18% n/a BMI(kg/m 2 27.4±1.8 27.6±1.1 0.92 Theterm affected referstothosestudysubjectswiththehbmcausinglrp5 mutations.therewerenostatisticaldifferencesinageandbmibetweenthetwo groups.therewereequivalentnumbersofmalesandfemalesenrolledinboth groups.thistabledemonstratesthatthecontrolgroupwasappropriatelymatched forthesecharacteristicstotheaffectedindividuals.dataisshownasmean±sem.
33 Table3.Measurementsofglucosemetabolisminaffectedstudyparticipants Pt# LRP5 BMI HbA1c eag A Insulin HOMA B [ HOMA[B genotype (kg/m 2 (% (uu/ml IR 1* g171v 22.3 6.4 137 5.5 1.9 26.8 2 g171v 20.8 5.6 114 8.0 2.3 56.5 3 g171v 35.0 8.2 188.6 14.9 6.9 42.7 4 g171v 34.0 5.2 102.5 17.8 4.5 162.2 5 g171v 29.4 5.6 114 10.6 3.0 74.8 6 g171v 24.8 5.4 108.3 9.9 2.7 78.7 7 g171v 32.7 5.9 122.6 24.4 7.4 147.4 8 g171v 26.7 5.5 111.2 5.8 1.6 43.3 9* g171v 34.7 6.8 148.5 12.1 4.4 51.0 10 n198s 21.2 5.7 116.9 12.4 3.6 82.8 11 n198s 20.1 5.4 108.3 7.0 1.9 55.6 Mean±SEM& 27.4±1.8& 6.0±0.3& 124.7±7.6& 11.7±1.7& 3.6±0.6& 74.7±13.0& AEstimatedaverageglucose=28.7xHbA1c46.7(Nathanetal.,2008 BHomeostaticModelAssessment *Subjecttakesinsulin Thistablesummarizestheresultsofthestudiesofglucosemetabolisminthe affectedindividuals.twosubjectsusinginsulinwereexcludedfromsubsequent analysis.
34 Figure2.HbA1cresultsinstudyparticipants HbA1cvaluesarenotsignificantlydifferentbetweenLRP5affectedindividualsand age,gender,andbmimatchedcontrols(p=0.06.errorbarsrepresentthestandard errorofthemean. Figure3A.ChangesinbloodglucoseduringtheOGTTinthreestudy participants ThreeaffectedsubjectsunderwentanOGTT.Therewasnoconsistentdifferencein glucoselevelsinthesethreeindividualscomparedtoagroupofpreviouslystudied age,race,bmi,andactivitymatchedcontrols(n=54.thetestingandcontroldata weregenerouslyprovidedbydr.kittfalkpetersen.
35 Figure3B.ChangesininsulinlevelsduringtheOGTTinthreestudy participants ThreeaffectedsubjectsunderwentanOGTT.Therewasnoconsistentdifferencein insulinlevelsinthesethreeindividualscomparedtoagroupofpreviouslystudied age,race,bmi,andactivitymatchedcontrols(n=54.thetestingandcontroldata werekindlyprovidedbydr.kittfalkpetersen.subjectbhasmoreintramyocyte lipidandislikelysomewhatinsulinresistant(seetable4. Figure3C.ChangesinC[peptidelevelsduringtheOGTTinthreestudy participants ThreeaffectedsubjectsunderwentanOGTT.Therewasnoconsistentdifferencein cpeptidelevelsinthesethreeindividualscomparedtoagroupofpreviously studiedage,race,bmi,andactivitymatchedcontrols(n=54.thetestingand controldatawaskindlyprovidedbydr.kittfalkpetersen.
36 Table4.Intramyocellularlipidandhepaticlipidtriglyceridecontentby 1 HMRS Subject BMI (kg/m 2 BodyFat(% ISI IMCL(% LiverTG(% SubjectA 27.8 25.3 3.1 1.4 4.3 SubjectB 25.7 19.4 6.6 1.7 0.6 Control(n=54 23.8±0.4 27.6±1.4 4.7±0.2 1.3±0.1 1.5±0.3 & Twoaffectedsubjectsunderwent 1 HMRSofskeletalmuscleandliverforlipid content.subjectswerecomparedtoacontrolgroup(n=54matchedforage,race, BMI,andactivitylevel.Whilethesampleistoosmallforstatisticalanalysis,there wasnotrendobservedinthedatatowardshigherorlowerpercentlipidcontent, whichhasbeenshowntobeanimportantmarkerofinsulinresistance(petersenet al.,2007. 1 HMRStestingwasgenerouslyconductedbyDr.KittFalkPetersen.Data isshownasmean±sem. Table5.Lipidlevelsinaffectedindividuals Pt# A,B BMI (kg/m 2 Total Cholesterol (mg/dl TG (mg/dl HDL (mg/dl LDL (mg/dl 2 20.8 115 49 40 65.2 4 34.0 180 112 58 99.6 6 24.8 185 66 71 100.8 7 32.7 163 104 45 97.2 8 26.7 149 44 68 72.2 9 34.7 143 140 49 66.0 10 21.2 209 132 101 81.6 11 20.1 212 96 80 112.8 Mean±SEM& 26.9±2.2& 169.5±11.8& 92.9±12.9& 64.0±7.2& 86.9±6.4& AGenotypesaslistedinTable3. BStudysubjects#1,#3,#5wereexcludedforreasonsdescribedintext. CLDL=TCHDL(TG/5(Friedewaldetal.,1972.
37 Figure4A.Comparisonoftotalbloodcholesterolinaffectedandcontrol individuals Individualstakinglipidloweringdrugswereexcludedfromtheanalysis.Total cholesterollevelswerenotsignificantlydifferent(p=0.43.dataisshownas mean±sem. Figure4B.Comparisonofbloodtriglyceridelevelsinaffectedandcontrol individuals Individualstakinglipidloweringdrugswereexcludedfromtheanalysis. Triglyceridelevelswerenotsignificantlydifferent(p=0.56.Dataisshownas mean±sem.
38 Figure4C.Comparisonofhighdensitylipoprotein(HDLlevelinaffectedand controlindividuals Individualstakinglipidloweringdrugswereexcludedfromtheanalysis..HDLlevels werenotsignificantlydifferent(p=0.32.dataisshownasmean±sem. Figure4D.Comparisonoflowdensitylipoprotein(LDLlevelsinaffectedand controlindividuals Individualstakinglipidloweringdrugswereexcludedfromtheanalysis.LDLlevels weresignificantlylowerinaffectedindividuals(p=0.04.dataisshownas mean±sem.
39 Figure5.RelativeexpressionofLRP5inrattissues LRP5expressionwashighestintheliver,andlowestinbone.Transcriptexpression isdefinedasfoldchangeincomparisontoliver. Figure6A.RegulationofPEPCK[Cexpressioninprimaryrathepatocytesby insulin Phosphoenolpyruvatecarboxykinase(cytosolicform(PEPCKCexpressionin primaryrathepatocyteswasmeasuredinthepresenceofknowninducersand inhibitorstoconfirmthesensitivityofourin&vitroassay.insulinpredictably suppressedpepckcexpressioncomparedtothecontrol(p=0.02.dataisshownas mean±sem.
40 Figure6B.RegulationofPEPCK[Cexpressioninprimaryrathepatocytesby dexamethasone PEPCKCexpressioninprimaryrathepatocyteswasmeasuredinthepresenceof knowninducersandinhibitorstoconfirmthesensitivityofourin&vitroassay. DexamethasonepredictablyinducedPEPCKCexpressioncomparedtothecontrol (p=0.05.dataisshownasmean±sem. Figure6C.RegulationofPEPCK[Cexpressioninprimaryrathepatocytesby glucagon,insulinanddexamethasone PEPCKCexpressioninprimaryrathepatocyteswasmeasuredinthepresenceof knowninducersandinhibitorstoconfirmthesensitivityofourin&vitroassay. GlucagonpredictablyinducedPEPCKCexpressioncomparedtothecontrol.This experimentconfirmedtheinductionofpepckcbydexamethasone,andits
41 suppressionbyinsulinrelativetocontrol.theeffectofglucagonwasbluntedby insulin(p=0.01.dataanalyzedusingonewayanova.dataisshownasmean±sem. Figure7A.EffectofWntonPEPCK[Cexpressioninprimaryrathepatocytes PEPCKClevelsweremeasuredaftertreatmentfor24hourswith100ng/mlofWnt. WnthadnoeffectonPEPCKCtranscriptexpression(p=0.42.Dataisshownas mean±sem. Figure7B.EffectofWntandDexamethasone[inducedexpressionofPEPCK[C inprimaryrathepatocytes PEPCKCtranscriptlevelsweremeasuredaftertreatmentwithWntorWntin combinationdexamethasone.dexamethasonealoneinducedpepckcexpression. TheadditionofWntdidnotsignificantlychangetheeffectofdexamethasone (p=0.42.dataisshownasmean±sem.
42 Figure7C.EffectofWntandglucagononPEPCK[Cexpressioninprimaryrat hepatocytes PEPCKClevelsweremeasuredinaftertreatmentwithWntorincombinationwith glucagon.glucagonaloneinducedpepckcexpression.theadditionofwntdidnot significantlyaltertheresponsetoglucagon(p=0.90.dataisshownasmean±sem. Figure8.TheeffectofWntonglucose[inducedinsulinsecretioninINS[1cells INS1cellsweretreatedwithWntinthepresenceofbasal(2.5mMandstimulatory (9.0mMlevelsofglucoseHighextracellularglucosesignificantlyincreasedinsulin secretion.however,theadditionofwntdidnotchangebasalorglucosestimulated insulinsecretion.dataisshownasmean±sem.
43 Figure9.LRP5transcriptexpressioninINS[1cellsandprimaryratislets. LRP5transcriptexpressionintheINS1cellswascomparedtothatinprimaryrat islets.lrp5expressioninins1cellswaslessthanhalfofthatintheprimaryrat islets. Figure10.EffectofWntonglucose[inducedinsulinreleasefromprimary humanislets HumanisletswereperifusedwithWntandexposedtobasal(2.5mMand stimulatory(9.0mmconcentrationsofglucose.glucoseinducedinsulinsecretion wasnotaffectedbywnttreatment.