ENERGY BALANCE-OBESITY Inducible Brown Adipose Tissue, or Beige Fat, Is Anabolic for the Skeleton Sima Rahman, Yalin Lu, Piotr J. Czernik, Clifford J. Rosen, Sven Enerback, and Beata Lecka-Czernik DepartmentsofOrthopaedicSurgery(S.R.,Y.L.,P.J.C.,B.L.-C.)andDepartmentofPhysiologyand D Pharmacology(B.L.-C.)andCenterforDiabetesandEndocrineResearch(S.R.,B.L.-C.),Universityof o w ToledoHealthSciencesCampus,Toledo,Ohio43614;MaineMedicalCenterResearchInstitute(C.J.R.), n lo Scarborough,Maine04074;andDepartmentofMedicalandClinicalGenetics(S.E.),Instituteof a d Biomedicine,TheSahlgrenskaAcademy,UniversityofGothenburg,Gothenburg,SwedenSE40530 e d fro m h It is known that insulin resistance and type 2 diabetes mellitus are associated with increased ttp fcriaatcetudrwesitahntdypteha2tdbiarobwetnesa.dBiyptohseeutissesuoefF(BoAxCT2)co(cid:1)u/nTgtemraiccets,amwaenlyl-iefsntaobtliaslhleodfmthoedseylmfoprtoinmdsucatsisoon- s://ac AD a ofBAT,orbeigefat,wepresentdataextendingthebeneficialactionofbeigefattoalsoinclude de m apositiveeffectonbone.FoxC2AD(cid:1)/Tgmiceareleanandinsulin-sensitiveandhavehighbonemass ic.o duetoincreasedboneformationassociatedwithhighboneturnover.InducibleBATislinkedto u p activation of endosteal osteoblasts whereas osteocytes have decreased expression of the Sost .c o m transcriptencodingsclerostinandelevatedexpressionofRankl.Conditionedmedia(CM)collected /e fromforkheadboxc2(FOXC2)-inducedbeigeadipocytesactivatedtheosteoblastphenotypeand nd o increasedlevelsofphospho-AKTand(cid:1)-catenininrecipientcells.Inosteocytes,thesamemedia /a decreased Sost expression. Immunodepletion of CM with antibodies against wingless related rtic le MMTVintegrationsite10b(WNT10b)andinsulin-likegrowthfactorbindingprotein2(IGFBP2) -a b raensdul(cid:1)te-cdaitnenthine.loCsosnovfeprsreol-yo,sCteMobdlearsitviecdacftriovimty,caenllsdothveerleoxspsorefsisnicnrgeaIGseFBinPt2hoerleWveNlsTo10fbphroesstpohroe-dAoKsT- strac t/1 teoblasticactivityinrecipientcells.Inconclusion,beigefatsecretesendocrine/paracrineactivity 5 4 thatisbeneficialfortheskeleton.(Endocrinology154:2687–2701,2013) /8 /2 6 8 7 /2 4 Recentadvancesinthecharacterizationofbrownadi- lismseenwithaging,diabetes,andobesity(6).Thesecon- 2 3 7 pose tissue (BAT) function in postnatal life suggest ditions are associated with a decrease in bone mass, an 8 6 that, besides its role in nonshivering thermogenesis and increase of fat volume in bone marrow cavity, and an by g energydissipation,BATisessentialforthedetermination increase in fractures (reviewed in Ref. 7). In contrast, u e s ofinsulinsensitivityandregulationofenergymetabolism high BAT activity in healthy young women correlates t o n (1,2).GeneticablationofBATinrodentsresultsindiet- positivelywithhighbonemineraldensity(8),andthere 0 3 inducedobesity,diabetes,andhyperlipidemia(3).Uncou- is a positive association between BAT volume, the A p plingprotein1(UCP1)deficiencyrendersanimalssensi- amountofbone,andcross-sectionalsizeofthefemurin ril 2 0 tive to cold and increases their susceptibility to diet- childrenandadolescents(9).Inaddition,thebonemass 19 inducedobesitywithaging(4,5).Inhumans,BATactivity inwomenrecoveringfromwastingdiseasessuchasan- correlatesnegativelywithimpairmentinenergymetabo- orexia nervosa is higher in those who possess cold- ISSNPrint0013-7227 ISSNOnline1945-7170 Abbreviations:ALK,alkalinephosphatase;BALP,bone-specificalkalinephosphatase;BAT, PrintedinU.S.A. brownadiposetissue;BFR,boneformationrate;BMAT,bonemarrowadiposetissue;BMP, Copyright©2013byTheEndocrineSociety bonemorphogenicprotein;CM,conditionedmedia;FABP4/aP2,fattyacidbindingprotein ReceivedNovember25,2012.AcceptedMay10,2013. 4;FoxC2,forkheadboxc2;IGFBP2,insulin-likegrowthfactorbindingprotein2;IGTT,ip FirstPublishedOnlineMay21,2013 glucosetolerancetest;IITT,ipinsulintolerancetest;mCT,microcomputedtomography; MSC,mesenchymalstemcell;Opg,osteoprotegerin;pAKT,phosphorylatedAKT;RANKL, Foreditorialseepage2579 receptoractivatorofnuclearfactorkappa-Bligand;TRAP5b,tartrate-resistantacidphos- phataseform5b;UCP1,uncouplingprotein1;WAT,whiteadiposetissue;Wnt10b,wing- lessrelatedMMTVintegrationsite10b. doi:10.1210/en.2012-2162 Endocrinology,August2013,154(8):2687–2701 endo.endojournals.org 2687 2688 Rahmanetal BeigeFatIsAnabolicforBone Endocrinology,August2013,154(8):2687–2701 inducedBATfociascomparedwiththosewholostBAT lean, insulin-sensitive, and resistant to diet-induced obe- function (10). sity(21,26).Consistentwiththemetabolicfunction,the These observations suggest that energy metabolism levels of FOXC2, and mitochondria gene expression, regulates bone turnover. Indeed, bone homeostasis and which are decreased in adipocytes of type 2 diabetic pa- remodelingarecloselylinkedtotheosteoblasticresponse tients, can be normalized in response to the antidiabetic toinsulin(11,12).Insulininducesosteoblastogenesisand therapywiththeinsulinsensitizerrosiglitazone(27). receptor activator of nuclear factor kappa-B ligand Inthepresentstudy,wehaveanalyzedtheendocrine/ (RANKL)productionleadingtohighbonemass,whichis paracrineactivityofbeigefattowardregulationofbone associatedwithincreasedboneturnover(11,12).Incon- mass using the FoxC2 (cid:1)/Tg murine model of induced AD trast, conditions of insufficient insulin signaling due to BATactivitytargetedbyexpressionofFOXC2infatcells. eitherdeficiencyininsulinproductionintype1diabetesor We have demonstrated, for the first time, that beige fat D o w resistancetoinsulinintype2diabetesareassociatedwith producesfactorsthatmaybesecretedtocirculationoract n lo low bone turnover and correlate with high levels of cir- directlyinthebonemarrowenvironmentandinduceos- a d e culatingsclerostin,anegativeregulatorofboneremodel- teoblast differentiation and osteocyte support for bone d ing, and low numbers of circulating osteoprogenitors formationandboneturnover.Twoofthesefactors,insu- from (13–16). lin-like growth factor binding protein 2 (IGFBP2) and h ttp IthasbeenrecognizedthatBATmaycomefrom2dif- winglessrelatedMMTVintegrationsite10b(WNT10b), s ferent origins. The classical preformed BAT originates are of special interest because they function in the regu- ://ac a fromMyf5-positivedermomyotomalprogenitors,which lation of both bone remodeling and energy metabolism. de m alsogiverisetoskinandmuscleandfunctioninnonshiv- Theirpotentialcontributiontotheanaboliceffectofbeige ic .o eringthermogenesis(17).Incontrast,theMyf5-negative fatonboneispresentedinthismanuscript. up progenitors can differentiate to white adipocytes with .co m function in energy storage or to beige adipocytes, which /e n havecharacteristicsofbothbrownandwhitefatcells(18). Materials and Methods do /a TheBAT-likephenotypecanbeinducedinwhiteadipose rtic tissue (WAT)-type adipocytes by several mechanisms Plasmids le -a comprising either endocrine action of fibroblast growth TheexpressionconstructforhumanFOXC2waspreparedat bs factor 21 (19) or irisin (20) or action of transcriptional theUniversityofGothenburg,andtheIgfbp2constructwaspur- trac regulatorsincludingforkheadboxc2(FOXC2)(21)and cwhhaesreedasfrtohmeWOrnitG1e0nbeeTxepcrhesnsoiolongcieosn,sItnrcuc(Rtwocaksvbilales,edMoanryclDanNdA), t/154 PRdomain-containingprotein16(22)and,asshownre- obtainedfromAddgene(Cambridge,Massachusetts).Allcon- /8/2 cently,SirT1-mediateddeacetylationofperoxisomepro- structswerepreparedinpcDNA3.1vector(Invitrogen,Grand 68 7 liferator-activatedreceptorgamma(23). Island,NewYork). /2 4 Bone marrow adipose tissue (BMAT) represents an- 23 othertypeoffatthatplaysanimportantroleinmodula- Protein analysis 786 The following antibodies were used for immunochemistry b tionofmarrowenvironmentsupportingboneremodeling. y and immunodepletion studies: anti-(cid:1)-catenin (BD Biosciences, g BMATvolumeincreaseswithaging,estrogendeficiency, San Jose, California), anti-(cid:1)-actin (Sigma-Aldrich, St. Louis, ues diabetes, and anorexia nervosa, and this expansion cor- Missouri), anti-AKT and anti-phospho-AKT (Cell Signaling t o n relateswithadecreaseinbonemassandincreaseinfrac- Technology, Beverly, Massachusetts), anti-WNT10b and 0 3 tures(reviewedinRef.7).Interestingly,themetabolicpro- IGFBP2 (Santa Cruz Biotechnology, Santa Cruz, California). A p fileofBMAThasbothWATandBATcharacteristicsin WesternblotrelativebanddensitywasmeasuredwithImageJ ril 2 NationalInstitutesofHealth(NIH)software.UCP1immuno- 0 respecttotheexpressionofgenemarkersanditsfunction 1 histochemistrywasperformedusinganti-UCP1(Sigma)andthe 9 (24).BAT-likefeaturesofBMATarecompromisedwith immunoperoxidase system provided by Vector Laboratories aging and in diabetes, suggesting a positive correlation (Burlingame,California). between the BAT-like metabolic profile of BMAT and bonemass(24). Gene expression analysis using quantitative real- The transcription factor FOXC2 promotes brown fat time RT-PCR analysis development by sensitizing cells to the (cid:1)-adrenergic The reaction was prepared as described (24) using Power cAMP-proteinkinaseApathwayandbyregulationofmi- SYBRGreenandprocessedwithStepOnePlusSystem(Applied Biosystems,Carlsbad,California).Relativegeneexpressionwas tochondrialmetabolism(21,25).Targetedexpressionof determinedbythe(cid:2)(cid:2)(cid:3)Ctmethodusing18SRNAlevelsfornor- FoxC2inadipocytesunderthecontroloffattyacidbind- malization.AllprimersusedinthisstudyarelistedinSupple- ingprotein4(FABP4/aP2)promoterconvertsepididymal mentalTable1(publishedonTheEndocrineSociety’sJournals WATintoBAT-likeorbeigefatandresultsinmicethatare Onlinewebsiteathttp://endo.endojournals.org). doi:10.1210/en.2012-2162 endo.endojournals.org 2689 Animals osteocyteswasakindgiftofDr.Bonewald(UniversityofMis- FoxC2 (cid:1)/Tgmiceweredescribedpreviously(21).Thecol- souri, Kansas City, Missouri). Primary bone marrow cultures AD onyofFoxC2 (cid:1)/Tg andwild-type(WT)(FoxC2 (cid:1)/(cid:1))mice were established from femur marrow aspirates and differenti- AD AD were maintained at the University of Toledo Health Science atedasdescribed(34).Theosteoclastogenesisassaywascarried Campus.Theanimaltreatmentandcareprotocolsconformedto using primary bone marrow nonadherant cells as a source of NIHGuidelinesandwereperformedusingaUniversityofTo- osteoclastprogenitorsandeitherU-33/(cid:3)2cellsoradherentbone ledoHealthScienceCampusInstitutionalAnimalCareandUti- marrowcellsassupportingosteoclastrecruitmentanddifferen- lizationCommitteeprotocol.Fatandleanmasswereevaluated tiation(28).After8daysofgrowthinthepresenceof10(cid:3)8M usingaminispecmq10NMRanalyzer(Bruker,Billerica,Mas- 1,25-hydroxyvitaminD ,osteoclastswerestainedwiththeleu- 3 sachusetts).Foodconsumptionwasdeterminedbymonitoring kocyteacidphosphatase(TRAP(cid:1))kit(Sigma). foodintakeof4-month-oldanimalsduringa1weekperiod.For glucose(ipglucosetolerancetest[IGTT])andinsulin(ipinsulin ALP activity in cell culture D tolerancetest[IITT])tolerancetestseither2g/kgglucoseor0.75 ALP enzyme activity was measured as described (28) after o w U/kginsulinwereinjectedipafter4hoursfasting.Bloodglucose normalization to cell number assessed by the Cell Titer 96 nlo w(AabsbmotetaLsaubreodrautosirniegs,tNheormthurCinheic-sapgeoc,ifIilclinAolips)h.aRTaRnAdKomsylesvteemls AMQadueiosousn,nWonirsacdoinosainct)i.ve cell proliferation assay kit (Promega, aded ofseruminsulinweremeasuredattheUniversityofMichigan fro m DiabetesResearchCenterCoreFacility(AnnArbor,Michigan), Collection and testing activity of conditioned h whereasserumlevelsforIGF-1andIGFBP2weremeasuredby ttp media s bRootchk,ArAkraknasnassasC)hialdnrdenbs’yHMosEpCitOalRREeseLaarbcohraIntostriytut(eBa(nLgitotlre, Conditioned media (CM) were collected either from AD2 ://ac a Maine),andadiponectinbyMaineResearchInstitute(Scarbor- cellstransientlytransfectedwithFoxC2,Igfbp2,orWnt10bex- de ough,Maine).Serumlevelsofbone-specificalkalinephospha- pressionconstructsorfromtheorgancultureofepididymalfat mic tase(BALP)weremeasuredusingthealkalinephosphatase(ALP) after 3 days incubation with the media. For depletion studies, .o diagnostic kit (Sigma-Aldrich) as described (28), and tartrate- CMwasincubatedwith1(cid:2)g/mlanti-WNT10boranti-IGFBP2 up.c resistantacidphosphataseform5b(TRAP5b)usinganELISA ornonspecificgoatIgGfor2hoursat4°Cfollowedby2hours om provided by Immunodiagnostic Systems Inc (Scottsdale, incubationwithProteinA/GPLUS-Agarosebeads(SantaCruz /e n Arizona). Biotechnology). do /a Bone analysis Statistical analysis rticle Microcomputedtomography(mCT)ofthetibiaeandL4ver- Statisticalanalysisofcellcultureexperimentswasconducted -ab s tebraewasperformedusingthe(cid:2)CT-35system(ScancoMedical with2-tailedStudent’sttest,whereasanimalexperimentswere tra AG,Bassersdorf,Switzerland)aspreviouslydescribed(29).The analyzedwithSPSSStatisticsversion17.0softwareusing1-way ct/1 analysis of bone microstructure conformed to recommended ANOVA and post hoc Tukey’s test. All data shown represent 54 guidelines(30). meansandSD.APvalue(cid:5).05wasconsideredsignificant. /8/2 To permit static and dynamic bone histomorphometry, 68 7 4-month-oldanimalswereinjectedwith30mg/kgtetracycline7 /2 4 and2daysbeforetheywerekilled,andundecalcifiedtibiaewere 2 Results 3 embeddedinmethylmethacrylate,sectioned,andstainedwith 78 6 either Goldner’s trichrome or Von Kossa/McNeal by the His- FoxC2 (cid:1)/Tg animals are lean and insulin-sensitive b tology Core at the Department of Anatomy and Cell Biology, AD y g ThemurinemodelofFOXC2transcriptionalregulator u Indiana University (Indianapolis, Indiana). The histomorpho- e s metricexaminationwasconfinedtothesecondaryspongiosaof ectopically expressed in adipocytes under the control of t o n proximal tibia and was performed using the Nikon NIS-Ele- Fabp4/aP2promoter/enhancerhasbeendescribedprevi- 0 3 ments BR3.1 system. The measurements were collected under ously(21).Forthestudiespresentedhere,thephenotype A (cid:4)Th4e0tmeramginniofilcoagtyioannfdroumnit6sruesperdesweenrteattihvoesfeierledcsopmemrbeonndeedsabmypthlee. otofFthoexCm2eAtaDb(cid:1)o/lTigcmpaicreamhaestebrese,ngaronwaltyhzepdaattgearinn,wanitdhgreegnadredr pril 201 HistomorphometryNomenclatureCommitteeoftheAmerican 9 differences (Figure 1). At 1 month of age, mice with SocietyforBoneandMineralResearch(31). FoxC2 (cid:1)/Tggenotype,bothmales(Figure1)andfemales AD Extraction of osteoblast- and osteocyte-enriched (notshown),havelowerbodyweightthanWTcontrols, fractions andthisdifferenceincreaseswithageprogression(Figure Cellfractionsenrichedineitherosteoblastsorosteocyteswere 1A). Although smaller, the FoxC2 (cid:1)/Tg mice consume AD isolatedbysequentialcollagenasedigestionoffemorabone,ac- morefood(Figure1B).Alongwithhighercaloricintake, cordingtopreviouslydescribedprotocol(32). these mice accrue up to 15% more lean mass than WT controlovera4-monthgrowingperiod(Figure1C).The Cell culture experiments miceareleanwithnearlyabsentfattissueasmeasuredby Murine marrow-derived cell lines representing adipocytic (AD2cells)andosteoblastic(U-33cells)phenotypeshavebeen nuclearmagneticresonance(Figure1D).InWTcontrols, previously described (33). The MLO-A5 cell line representing bodycompositionchangedwithincreasingageduetorel- 2690 Rahmanetal BeigeFatIsAnabolicforBone Endocrinology,August2013,154(8):2687–2701 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /e n d o /a rtic le -a b s tra c t/1 5 4 /8 Figure1. BodycompositionandmetabolicparametersofmaleFoxC2 (cid:1)/Tg(Tg)ascomparedwithwildtype(WT)control(n(cid:7)6miceper /2 AD 6 group).A,Changesinbodyweightovera5-monthgrowthperiod.B,Dailyfoodintakemonitoredover1weekin4-month-oldmice.C–F, 8 7 Changesinthelean(C)andfatmass(D)andpercentcontributionoflean(E)andfat(F)masstobodyweightmeasuredoveraperiodof4months /2 4 bynuclearmagneticresonance.G–H,WeightsofepididymalWAT(G)andinterscapularBAT(H)in5-month-oldmice.I–J,Randomglucose(I) 2 3 andinsulin(J)levelsin4-month-oldmice.K,GlucosedisposalmeasuredinIGTTinresponsetoinjectionof2g/kgglucose.L,Glucosedisposal 78 6 measuredinIITTinresponsetoinjectionof0.75U/kginsulin.OpencirclesandgraybarsrepresentWT,andblackcirclesandblackbarsrepresent b FoxC2AD(cid:1)/Tgmice.*,P(cid:5).05vsWT. y g u e s ativedecreaseinlean(Figure1E)andrelativeincreasein highcopynumberofatransgenicconstructthattendsto t o n fat(Figure1F)mass.Incontrast,thebalancebetweenlean bearrangedasunstablemultimersinaheadtotailfashion 0 3 and fat mass remains similar over the 4-month growth and leads to the loss of transgene copies with increasing A p period of FoxC2AD(cid:1)/Tg mice (Figure 1, E and F). This numberofgenerations. ril 2 suggeststhatFoxC2 (cid:1)/Tgmiceareprotectedfromage- Despite increased food consumption (Figure 1B), 01 AD 9 relatedchangesinbodycomposition. FoxC2 (cid:1)/Tg mice maintained a level of serum glucose AD Thereducedfatmassin4-month-oldFoxC2 (cid:1)/Tgis similartoWTmice(Figure1I)paralleledwithsignificantly AD primarilyduetosignificantreductioninthemassofepi- reducedlevelsofinsulin(Figure1J);however,serumglu- didymal WAT (Figure 1G); however, the mass of inter- cose levels after fasting were significantly reduced in scapular BAT is also reduced (Figure 1H), which differs FoxC2 (cid:1)/Tg mice as compared with WT (87.0 (cid:6) 13.1 AD fromtheoriginalphenotypecharacterizedbyreductionin mg/dl vs 137.3 (cid:6) 15.9 mg/dl) (Figure 1L). Glucose dis- WATbutincreaseinBATmass(21).Thismightbedueto posal, measured in either IGTT or IITT, is significantly thefactthatmiceusedinthepresentstudywerebredfor higher in FoxC2 (cid:1)/Tg than in WT mice, indicating in- AD more than 30 generations from original mice, which re- creased insulin sensitivity (Figure 1, K and L). On that sulted in a decrease of initially high copy number of the note, the rapid glucose clearance in FoxC2 (cid:1)/Tg upon AD FoxC2 transgene. This is frequently seen in mice with a insulinchallengeinIITTresultedinseverehypoglycemia doi:10.1210/en.2012-2162 endo.endojournals.org 2691 within30minutesfrominsulininjection(Figure1L).No 16 (Prdm16) and (cid:1)3-adrenergic receptor (ADRB3) re- gender differences were noted in the body composition vealed modest, but consistent upregulation as compared andmetabolicparametersofFoxC2 (cid:1)/Tgmice. withcontrolmice.Interestingly,levelsofleptinexpression AD weresimilarinWATofFoxC2 (cid:1)/TgandWTmice. AD Ectopic expression of FoxC2 induces acquisition of ToassesswhetherBATphenotypemaybeinducedin beige phenotype in vivo in the epididymal WAT marrow adipocytes, FoxC2 was ectopically expressed in and in vitro in cells representing marrow AD2 cells representing immortalized marrow cells com- adipocytes mittedtotheadipocytelineage(33).Similartoitseffectin It was previously shown that ectopic expression of WAT,FOXC2inducedtheexpressionofBATmarkersin FoxC2 transcription factor in adipocytes induces BAT- marrow adipocytes. However, in contrast to WAT, likegeneexpressioninWATandenhancesmitochondrial D biogenesis (21, 25). Epididymal WAT of FoxC2 (cid:1)/Tg FOXC2 increased the expression of leptin in AD2 cells ow AD n (Figure 2B). Changes in BAT gene markers expression lo miceshowedsignificantlyincreasedexpressionofseveral a d markersofbrownadipocytes(Figure2A).Amongtested were proportional to the levels of FOXC2 expression in ed markers, UCP1 and type II iodothyronine deiodinase WATandAD2cells(Figure2,AandB).BecauseFOXC2 fro m (Dio2)showedthelargestincreaseat52-and16-foldover increasesinsulinsensitivityinadipocytesasreportedpre- h age-matchedWTcontrols,respectively,whereastheother viously(21),wehavetestedwhetherithasthesameeffect ttps markersofBATincludingPRdomain-containingprotein on marrow adipocytes. As shown in Figure 2C, ectopic ://ac a d e m ic .o u p .c o m /e n d o /a rtic le -a b s tra c t/1 5 4 /8 /2 6 8 7 /2 4 2 3 7 8 6 b y g u e s t o n 0 3 A p ril 2 0 1 9 Figure2. MetabolicprofileofFOXC2-expressingadipocytesinvivoandinvitro.A,Quantitativereal-timePCRanalysisofBATgeneexpressionin epididymalWATisolatedfrom6-month-oldwildtype(WT)andFoxC2 (cid:1)/Tg(Tg)mice(n(cid:7)4micepergroup).B,BATgeneexpressioninAD2cells AD transfectedwitheitheremptyvector(EV)orFoxC2expressionvector(FC2).C,ExpressionofinsulinsignalinggenesinAD2cellstransfectedwith eitheremptyvectororFoxC2expressionvector.D,ProteinlevelsofpAKTinemptyvectorandFoxC2expressionvectortransfectedAD2cells treatedwitheithervehicle(V)or100nMinsulin(Ins)for30minutesaftera2-hourperiodofserumdepletion.Valuesforrelativebanddensity measuredwithImageJNIHsoftwareareindicatedbelowimages.GraybarsrepresentWTmiceoremptyvector;blackbarsrepresentFoxC2 (cid:1)/Tg AD miceorFoxC2expressionvector.*,P(cid:5).05vsWTmiceoremptyvector-transfectedcells. 2692 Rahmanetal BeigeFatIsAnabolicforBone Endocrinology,August2013,154(8):2687–2701 expressionofFoxC2inAD2cellsresultedinanincreased servedwithrespecttothehigherbonemassandstructural expressionofinsulinreceptorsubstrate1(IRS1),aposi- differencesinvertebraofFoxC2 (cid:1)/Tgmice.Thediffer- AD tiveregulator,andadecreasedexpressionofsuppressorof encebetweenboneacquisitioninvertebraandtibiacanbe cytokinesignaling3(Socs3),anegativeregulatorofinsu- attributedtobothadifferenceinthesympatheticnervous linsignaling.Insulinsensitivitywasmeasuredasprotein systemcontrolofboneswithdifferentembryologicalor- levelsofphosphorylatedAKT(pAKT)inresponsetoin- igin(35)andtheincreasedperiostealactivityinvertebraof sulinchallenge.InAD2cells,FOXC2increasedbasallev- FoxC2 (cid:1)/Tgmice,whichmayresultfromtheincreased AD elsofpAKTupto4-foldandupto8-folduponstimulation sensitivity to the sympathetic nervous system-controlled withinsulin,ascomparedwiththeemptyvectorcontrol (cid:1)-adrenergic/cAMP/proteinkinaseAsignaling(21). (Figure2D).ThesedataindicatethatFOXC2inducesBAT Histomorphometricanalysisoftrabecularboneinthe phenotypeandincreasesinsulinsensitivityinbothWAT proximal tibia confirmed mCT measurements of high D o w andbonemarrowadipocytes. bonemass(Figure4A).The3-foldincreaseintrabecular n area (trabecular area/tissue area) in FoxC2 (cid:1)/Tg mice loa AD d FoxC2 (cid:1)/Tg mice have high bone mass that is was associated with increased bone remodeling due to ed associaAtDed with increased bone formation rate and increased activity of its cellular components, osteoblasts fro m high bone turnover andosteoclasts.Theosteoblastnumber(perbonesurface) h FoxC2 (cid:1)/Tg mice, males and females, have signifi- intibiaofFoxC2 (cid:1)/Tgmicewas3.6-foldhigher,andthe ttps cantlyhighAeDrtrabecularbonemassascomparedwithage- trabecularsurfacAeDoccupiedbyactiveosteoblasts(osteoid ://ac a matched WT animals (Figure 3 and Supplemental Table surface/bone surface) was almost 3 times larger than in de m 2).Thehigherbonemass(bonevolume/tissuevolume)in WT animals (22.6% vs 61.9%). At the same time, oste- ic .o proximaltibiawasnoticeablein2-month-oldanimalsand oclast number (per bone surface) was also elevated by u p showedasustainedandprogressiveincreasewithanimals’ 3-fold, indicating increased bone turnover in .co m age.Itwasaccompaniedbyanoverallincreaseinthetissue FoxC2 (cid:1)/Tgmice.Consistentwithahighnumberofos- /e AD n volume of proximal tibia and increased trabecular bone teoblasts, the mineral apposition rate was increased by do /a vboolnuemmea(sSsuipnpbleomthenmtaalleTsaabnldef2e)m.Aalte2swmaosnathsssoocfiaatgeed,whiigthh 23--ffoollddainndFtohxeCb2one(cid:1)fo/Trgmmatiicoenarsaatess(eBsFsRed)wbyasdiynncraemasicedhbisy- rticle AD -a thehighnumberoftrabeculae,highconnectivity,andde- tomorphometryoftetracycline-labeledbone(Figure4,A bs creasedseparationbetweentrabeculae.Thephenotypeof andB).Inaddition,tetracyclinelabelingofnewlydepos- trac highbonemassnotonlypersistedbutwasevenmorepro- itedbonewasrelativelystrong(Figure4B)andextended t/15 nouncedwithaging,althoughitwasaccompaniedbydif- to64%ofthetrabecularsurfaceinFoxC2 (cid:1)/Tgmiceas 4/8 AD /2 ferentstructuralchangesinmalesandfemales.Although comparedwith30%inWTanimals.IncreasedBFRcor- 6 8 7 highbonemassin5-month-oldmaleswasstillduetomore responded to a 3-fold increase in the thickness of new /2 4 numerous trabeculae, a high bone mass in 5-month-old osteoid(Figure4C)andtothepresenceofactivatedcuboi- 23 7 femaleswasduetomuchthickertrabeculae(Figure3,C dalosteoblastsalongthesurfaceoftrabeculae(Figure4D). 86 b andD,andSupplementalTable2).Female,butnotmale, The number of adipocytes in proximal tibia of y g bonehadlargerendostealarea,whichcorrelatedwithin- FoxC2AD(cid:1)/Tg mice was elevated, whereas their average ues creasedstrengthtoresisttorsionandbendingasmeasured sizewassignificantlysmallerascomparedwithWTadi- t o n bypolarmomentofinertia(SupplementalTable3).High pocytes(SupplementalFigure1).Mostimportantlyandin 0 3 bone mass in long bones of FoxC2 (cid:1)/Tg males and fe- contrasttoWTanimals,UCP1proteinwaspresentinthe A AD p males was restricted to the trabecular bone, because no marrowofFoxC2AD(cid:1)/Tgmice(Figure4E).Inconclusion, ril 2 differenceswereobservedineithercorticalthickness(Sup- the bone phenotype of FoxC2 (cid:1)/Tg mice is associated 01 AD 9 plementalTable2)orbonelength(notshown)oftibiaand withbothhighboneturnover,increasedboneapposition, femurbetweenWTandFoxC2 (cid:1)/Tgmice. andexpressionofBATmarkerUCP1protein.Insupport AD Intheaxialskeleton,thevertebralbodywaslargerin of increased bone turnover, levels of both BALP and size in FoxC2 (cid:1)/Tg mice than in WT animals and pos- TRAP5b were significantly elevated in sera of AD sessedincreasedtrabecularbonemassduetoahighnum- FoxC2 (cid:1)/Tgmice(Table1).Nogenderdifferenceswere AD beroftrabeculae(Figure3BandSupplementalTable2). noticedinmeasuredparameters. This feature was observed in both 2- and 5-month-old To confirm that osteoblasts in the bone of malesandfemales.Interestingly,theincreaseinbonemass FoxC2 (cid:1)/Tgmicearehighlyactivated,weanalyzedtheir AD withagewasratherduetoanincreaseinthevolumetric phenotypeinvivointhefractionofcellsassociatedwith sizeofvertebrabutnotduetochangeinthedensityand endosteal bone surface. As shown in Figure 4F, the thickness of trabeculae. No gender differences were ob- FoxC2 (cid:1)/Tg-derived osteoblasts had higher expression AD doi:10.1210/en.2012-2162 endo.endojournals.org 2693 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /e n d o /a rtic le -a b s tra c t/1 5 4 /8 /2 6 8 7 /2 4 2 3 7 8 6 b y g u e s t o n 0 3 A p ril 2 0 1 9 Figure3. mCTanalysisoftrabecularboneinproximaltibiaandL4lumbarvertebra.AandB,mCT-generatedcoronalsectionimagesoftrabecular boneinproximaltibia(A)andL4vertebralbody(B)of5-month-oldmalewildtype(WT)andFoxC2 (cid:1)/Tg(Tg)mice.C,Boneparametersof AD proximaltibiaandL4lumbarvertebrain2-and5-month-oldmales.D,BoneparametersofproximaltibiaandL4lumbarvertebrain2-and5- month-oldfemales.Abbreviations:BV/TV,bonevolume/tissuevolume;Tb.N,trabecularnumber;Tb.Th,trabecularthickness;Tb.Sp,trabecular spacing.WhiteboxesrepresentWT,andgrayboxesrepresentFoxC2 (cid:1)/Tgmice;n(cid:7)4–10animalspergroup.*,P(cid:5).05vsage-matchedWT AD control. 2694 Rahmanetal BeigeFatIsAnabolicforBone Endocrinology,August2013,154(8):2687–2701 D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /e n d o /a rtic le -a b s tra c t/1 5 4 /8 /2 6 8 7 /2 4 2 3 7 8 6 b y g u e s t o n 0 3 A p ril 2 0 1 9 Figure4. Measurementofcellularbonecompartments.A,Staticanddynamichistomorphometryoftrabecularboneinproximaltibiaof5-month-old malemice.Abbreviations:MAR,mineralappositionrate;N.Ob/BS,osteoblastnumber/bonesurface;N.Oc/BS,osteoclastnumber/bonesurface;OS/BS, osteoidsurface/bonesurface;OS.Th,osteoidthickness;Tb.A/TA,trabeculararea/tissuearea.B,Doubletetracyclinelabelingoftrabecularsurface (magnification,(cid:4)10).C,Goldner’strichromestainingofosteoid(magnification,(cid:4)20).D,Microphotographofactivatedosteoblastsonthesurfaceof trabeculae.StainingwithVonKossa/McNealtovisualizemineralizedbone(black)andosteoidandmarrowcells(blue),respectively(magnification,(cid:4)40). E,UCP1proteinexpressioninproximaltibia,counterstainedwithmethylgreen(magnification,(cid:4)40).BlackarrowsindicateUCP1-positivestaining.F, Osteoblast-specificgeneexpressioninosteoblast-enrichedfractionisolatedfromfemoraof4-month-oldmales.G,Exvivoanalysisofthenumberof MSCsabletoformfibroblast-likecolonies(CFU-F),osteoblastcolonies(CFU-OB),andadipocytecolonies(CFU-AD).H,Osteoblast-specificgeneexpression inexvivoculturedprimarybonemarrowcellsderivedfromWTandFoxC2 (cid:1)/Tgmiceharvestedfromfemoraof4-month-oldmales.I,Analysisof AD osteoclastprogenitornumberinthenonadherentfractionofbonemarrowderivedfromWTandFoxC2 (cid:1)/TgmiceandcoculturedwithU-33/(cid:3)2cells.J, AD AnalysisofosteoclastprogenitordifferentiationpotentialinthepresenceofadherentprimarybonemarrowcellsfromWTandFoxC2 (cid:1)/Tgmice. AD Multinucleatedcells(morethan2nuclei)thatstainedpositivelyforTRAPwereconsideredasdifferentiatedosteoclastsandwereenumerated.Graybars representwildtype(WT),andblackbarsrepresentFoxC2 (cid:1)/Tg(Tg)mice;n(cid:7)3animalspergroup.*,P(cid:5).05vsWT. AD doi:10.1210/en.2012-2162 endo.endojournals.org 2695 conditions. The mRNA expression levels of cyclin D1, Table 1. BoneTurnoverMarkersinSerumofWTand FoxC2 (cid:1)/TGa distal-lesshomeobox5(Dlx5),osteocalcin,andcollagen AD werenotdifferentinFoxC2 (cid:1)/TgmarrowMSCsascom- Marker WT FoxC2 (cid:1)/TG AD AD paredwithWTcells(Figure4H).Thesedataexcludedthe BALP,U/L 5.12(cid:6)1.31 8.61(cid:6)0.12b possibilitythatthehighnumberandincreasedactivityof TRAP5b,U/L 1.92(cid:6)0.38 2.62(cid:6)0.15c osteoblastinmiceectopicallyexpressingFoxC2infatare aResultsarefromn(cid:7)4animalspergroup. duetointrinsiccellularchangesatthelevelofMSClineage bP(cid:8).01vsWT. commitment. cP(cid:8).05vsWT. Similarly, the number of osteoclast progenitors and their differentiation did not differ between WT and ofcyclinD1,amarkerofcellproliferation,andincreased FoxC2 (cid:1)/Tganimalsasassessedinacocultureexperiment Do expression of distal-less homeobox 5, osteocalcin, and AD w ofnonadherentbonemarrowcellswitheitherU-33/(cid:3)2cells n collagen,markersofosteoblastdifferentiationandmatu- lo a (Figure4I)oradherentbonemarrowcellsderivedfromeither d ration. This profile is consistent with the observed high e number and activity of differentiated osteoblasts in WTandFoxC2AD(cid:1)/Tgmice(Figure4J). d fro FoxC2AD(cid:1)/Tgmice(Figure4A). The rate of bone turnover is regulated by osteocytes, m h To determine whether the increase in the number of which produce 2 essential proteins, sclerostin and ttp s osteoblastswasduetointrinsicchangesthatwouldaffect RANKL,forregulationofosteoblastandosteoclastfunc- ://a c tions,respectively.Becauseboneturnoverisincreasedin a lineage commitment of the marrow mesenchymal stem d cceolllosn(MiesSaCnsd),dthifefenruemntbiaetreotfoMwaSrCdsewitihtheraopsoteteonbtliaaslttoorfoardmi- FFooxxCC22AADD(cid:1)(cid:1)//TTggwmiitche,WwTeocsotmeopcayrteeds.tThheeinenvriivchomaecntitvoitfyoosf- emic.ou p pocytelineagewasanalyzedexvivousingacolony-form- teocytesisolateswasassessedbythelevelofexpressionof .c o ingunitassay(33).AsshowninFigure4G,thenumberof Dmp1andSostgenemarkers(Figure5A).Asshown,the m /e MSCswiththeabilitytoformfibroblast-likecoloniesand expressionofDmp1andSostintheosteocytefractionwas nd o thenumberofcolonieswithpotentialtodifferentiateto- 2to3ordersofmagnitudehigherthantheexpressionof /a ward either osteoblasts or adipocytes were not different these markers in osteoblast fraction, indicating relative rticle between FoxC2AD(cid:1)/Tg and WT animals. Next, we ana- homogeneity of analyzed cells (Figure 5A). When com- -abs lyzedthephenotypeofFoxC2AD(cid:1)/Tgosteoblastinexvivo paredwithosteocytesderivedfromWTanimals,theiso- trac t/1 5 4 /8 /2 6 8 7 /2 4 2 3 7 8 6 b y g u e s t o n 0 3 A p ril 2 0 1 9 Figure5. AnalysisofprimaryosteoblastsandosteocytesisolatedfromfemoraofFoxC2 (cid:1)/TgandWTmice.A,Determinationofhomogeneityof AD osteocyte(Ot)-enrichedfractionascomparedwithosteoblast(Ob)-enrichedfractionisolatedfrommurinefemora.Valuesbelowthegraph representthethresholdcycle(Ct)atwhichthetargettranscriptwasdetectedinthereal-timePCR.B,Sost,Rankl,andDmp1geneexpressionin osteocyte-enrichedfraction.C,RelativeexpressionofRANKLinosteoblast-andosteocyte-enrichedfractions.D,RelativeexpressionofOPGin osteoblast-andosteocyte-enrichedfractions.E,RANKLtoOPGratioinprimaryosteoblastsandosteocytes.Graybarsrepresentwildtype(WT), andblackbarsrepresentFoxC2 (cid:1)/Tg(Tg)mice;n(cid:7)3animalspergroup.*,P(cid:5).05vsWTmice. AD 2696 Rahmanetal BeigeFatIsAnabolicforBone Endocrinology,August2013,154(8):2687–2701 lates from bones of FoxC2 (cid:1)/Tg mice showed reduced pressing FoxC2 (donor cells) and transferred to the cul- AD expressionofSOSTby2-foldandincreasedexpressionof tures of the preosteoblastic U-33 cells (recipient cells) RANKL by 3-fold (Figure 5B). Although SOST and induced ALP activity (Figure 6A) and the expression of RANKLexpressionweredifferent,theexpressionofthe osteoblast-specificgenemarkersintherecipientcells(Fig- Dmp1markerofosteocytematurationdidnotdifferbe- ure6B).Thisprofileofexpressionwasremarkablysimilar tweenFoxC2 (cid:1)/TgandWTmice,indicatingthatosteo- totheexpressionprofileofosteoblastsisolatedfromthe AD cyte function, but not differentiation, is altered in bone of FoxC2 (cid:1)/Tg mice (Figure 4F). Moreover, CM AD FoxC2 (cid:1)/Tg mice (Figure 5B). Based on recent reports fromFOXC2-expressingdonorcellsdecreasedtheexpres- AD that osteoclast differentiation and function in bone re- sion of suppressor of cytokine signaling 3 and increased modeling is controlled by osteocytes rather than osteo- theexpressionofinsulinreceptorsubstrate1,indicating blasts(36),wecomparedthelevelsofRANKLexpression increasedinsulinsensitivityofrecipientcells(Figure6B). D o intheosteocyteandosteoblastfractions.AsshowninFig- Consistently,thebasallevelsofboth(cid:1)-cateninandpAKT, wn lo ure5C,RANKLexpressionwasalmost40-foldhigherin twomajorcellularmediatorsofpro-osteoblasticandin- a d e the osteocyte fraction as compared with the osteoblast sulin signaling activity was increased in recipient osteo- d fraction in WT animals and over 120-fold higher in os- blasticcells(Figure6C). fro m teocytesofFoxC2 (cid:1)/Tgmice(Figure5C).Onthatnote, ThesameCMfromFOXC2-expressingadipocytesde- h AD ttp a similar difference in the RANKL expression as shown creased expression of SOST in the osteocytic MLO-A5 s between WT osteocyte and osteoblast fraction has been cells;however,theexpressionofRANKLandDMP1were ://ac a d recently reported (37). Next, we analyzed the levels of not affected in the recipient cells (Figure 6D). A similar e m expressionofosteoprotegerin(OPG),theRANKLdecoy effectwasobservedwhenCMwascollectedfromanorgan ic receptor and a negative regulator of osteoclastogenesis. cultureofepididymalfatisolatedfromFoxC2 (cid:1)/Tgan- .ou AD p.c Interestingly, OPG was expressed at similar levels as imals.Indeed,CMfrombeigefatdecreasedexpressionof o m RANKL in osteoblasts derived from either WT or SOSTintherecipientosteocyticcellswithouteffectingthe /e n FoxC2 (cid:1)/Tg(Figure5D),resultinginaRANKLtoOPG expressionofRANKLandDMP1(Figure6D).Thissug- do AD /a ratiocloseto1(Figure5E).Incontrast,primaryosteocytes geststhat,incontrasttosclerostin,elevatedexpressionof rtic expressednegligiblelevelsofOPG,leadingtoaveryhigh RANKLinprimaryosteocytesofFoxC2 (cid:1)/Tgmiceisnot le AD -a RANKLtoOPGratio(Figure5,DandE).Theseresults aresultofadirecteffectoffactorssecretedfrombeigefat bs argueforamajorroleofosteocytesinregulationofbone butinsteadanindirectsystemiceffect.Anotherpossibility trac remodelingandindicatethatincreasedboneturnoverin is that the beige fat-induced mechanism leading to in- t/15 4 FoxC2 (cid:1)/Tgmiceresultsfromosteocytesupportforos- creasedRANKLexpressionisnotactiveinMLO-A5cells. /8 AD /2 teoblastandosteoclastfunction. Takentogether,cellsthatacquirethebeigefatpheno- 68 7 Thedecreasedexpressionofsclerostin,anegativereg- type due to ectopic expression of FoxC2 secrete factors /2 4 2 ulatorofosteoblastdifferentiation,andtheincreasedex- thatincreasebone-formingactivityeitherbydirectlyac- 3 7 pressionofRANKL,apositiveregulatorofosteoclastdif- tivating osteoblast bone-forming capabilities or through 86 b ferentiation, were consistent with increased osteoblast increasedsupportofosteocytesforosteoblastsasaresult y g numberandincreasedosteoclastnumbershowninFigure ofdecreasedproductionofsclerostin. ue s 4A and indicate that FoxC2 (cid:1)/Tg mice have increased t o AD n boneremodelingduetoalteredactivityofosteocytes.In Beige fat endocrine/paracrine activity comprises 0 3 conclusion,osteoblastandosteocyteactivityisalteredin bone-anabolic factors including IGFBP2 and A p FoxC2AD(cid:1)/Tgmice,butthisalterationresultsratherfrom WNT10b ril 20 systemiccuesasopposedtointrinsicchangesintheMSC To characterize factors secreted by beige adipocytes, 1 9 or hematopoietic stem cells potential to differentiate to- which may regulate osteoblast and osteocyte activity ei- wardeitherosteoblastsorosteoclasts,respectively. therinanendocrineorinaparacrinemanner,weprofiled fat tissue of FoxC2 (cid:1)/Tg mice and marrow adipocytes AD Beige fat secretes factors that activate osteoblasts ectopicallyexpressingFoxC2fortheexpressionoffactors and modulate Sost expression in osteocytes recognizedfortheirbone-anabolicactivity.Ifapplicable, Because ectopic expression of FoxC2 in fat of the we determined the protein levels of these factors in the FoxC2 (cid:1)/Tg mice positively correlates with increased serum.Adiponectin,IGF-1,andIGFBP2wereselectedas AD bonemassandincreasedinvivobone-anabolicactivityof candidates for endocrine activity of fat to regulate bone osteoblasts and osteocytes, we tested the possibility that mass, whereas activators of pro-osteoblastic WNT and beigeadipocytesreleasefactorsthatregulatetheseactiv- bone morphogenic protein (BMP) signaling, and angio- ities. CM collected from AD2 adipocytes ectopically ex- genicfactorangiopoietin2,wereselectedaspotentialcan-