185

European Journal of Endocrinology -21-0267 Rienk Nieuwland Jurrien Prins Barend W Florijn extracellular vesiclemicroRNA-224/452 associates withreducedcirculating Estradiol-driven metabolismintranswomen mechanistic understandingofmetabolismfollowinggender-affirmin g estradioltherapy. Conclusion: uptake (brownadipose). (miR target)genesareassociatedwithlipogenesis(white adipose)andmitochondrialrespirationglucose and mitochondrialrespiration(brownadipose).Inwhitebrownadiposetissue,differentiallyexpressed systemic silencinginmiceandculturedadipocytesincreasedlipogenesis(whiteadipose)butreducedglucoseuptake Results: mechanistic studiesinculturedadipocytes. was followedbyRNA-seq-basedgeneexpressionanalysisofbrownandwhiteadiposetissueinconjunctionwith we identifiedmiR-224andmiR-452.SubsequentsystemicsilencingofthesemiRsinmaleC57Bl/6Jmice( Methods: involvedinmetabolismmice. understood, thisstudyassessedcirculatingestradiol-drivenmicroRNAs(miRs)intranswomenandtheirregulation of compounds) couldaffectmetabolismaswell.Giventhattheunderlyingpathophysiologicalmechanismsarenotfully metabolism. Intranswomen,gender-affirminghormonetherapylikeestradiol(incombinationwithantiandrogenic Objective: Abstract *(R BijkerkandAJanvanZonneveldarejointseniorauthors) Erasmus MedicalCenterRotterdam,TheNetherlands University MedicalCenter,Amsterdam,TheNetherlands,and Experimental ClinicalChemistry,DepartmentofChemistryandVesicleObservationCenter,Amsterdam Biology (SectionElectronMicroscopy),LeidenUniversityMedicalCenter,Leiden,TheNetherlands, Amsterdam, TheNetherlands, Medical Center,Leiden,TheNetherlands, 1 and Department ofInternalMedicine(Nephrology), https://doi.org/ https://eje.bioscientifica.com Clinical Study Anton Jan van Zonneveld EstradiolintranswomenloweredplasmamiR-224and-452carriedextracellularvesicles(EVs)whiletheir FollowingplasmamiR-sequencing(seq)inatranswomendiscovery( Sexsteroidhormoneslikeestrogenshaveakeyroleintheregulationofenergyhomeostasisand Thisstudyidentifiedanestradiol-drivepost-transcriptional networkthatcouldpotentiallyoffer a 10.1530/EJE 1 , 2 , HuayuZhang 6 -21-0267 , Bas Bvan Rijn 1 , 2 , Jacques M G JDuijs 4 Department ofInternalMedicine(Endocrinology), 4 1 1 , , 2 3 2 , Hetty C MSips B WFlorijnandothers Department ofInternalMedicine,Division ofEndocrinology,VUUniversityMedical Center, 7 , Ton JRabelink Published by Bioscientifica Ltd. 2 Einthoven LaboratoryforVascularandRegenerativeMedicine,LeidenUniversity Printed inGreat Britain 1 , 2 © , MaartjeKlaver 2021Theauthors 7 Department ofObstetrics andFetalMedicine, 2 , 4 , WendyStam 1 , 2 , Patrick C NRensen Estradiol-derived miRs 3 5 , Eline NKuipers Department ofCellandChemical 1 , 2 , MaaikeHanegraaf 6 2 Laboratory of , n 4 , Martinden Heijer

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5 via freeaccess , European Journal of Endocrinology https://eje.bioscientifica.com can befound. materials on (seesection methods Supplementary canbefound methods inthe ofthe A fulldescription Methods inmice. metabolism transwomen ofgenes involved regulation andtheir in estradiol-drivenstudy assessed circulating miRsin related genesof functionally this inmetabolism, (estradiol) regulation post-transcriptional associate with sites atdistal ( metabolism tissue miRs ( adipose tissue-derived to whole-bodyadipose tissue ( metabolism increasing evidence islinkingestrogenthat in regulation andprocessing ( ofmiRtranscription regulation X- and (ii) estrogensilencing ofthe X-chromosome located (X-linked) miRsdueto incomplete result viaatleast two (i)doubledosage of mechanisms: ( at 3 complementarity ofbasepairing expression viathe miR-mediated of coordination addressed this demand ( response ofcellsto inmetabolic achange functional the related togethersets shape genes, offunctionally that binding expression coordinate ofmultiple the of non-codingRNAs asmicroRNAs such (miRs)andRNA networks interplay anintricate through transcriptional are notfully understood. However, underlying the pathophysiological mechanisms future cardiovascular disease(CVD)intranswomen ( for risk type2diabetes increases and the health metabolic sensitivity ( (HOMA-IR) ( ( metabolism affect energy by increasing bodyfat total compounds) antiandrogenic with intranswomen may Also, regular estradiol administration (incombination known to affect adiposity andinsulinsensitivity( α ( homeostasis and metabolism of energy regulation likeSex estrogens hormones have akey role inthe Introduction 8 3 1 (ESR1)orER Clinical Study ). Interestingly, sex-specific expression ofmiRsmay ), fasting insulin,andHOMAofinsulinresistance), fasting estrogen). Particularly uponbindingthe receptor Therefore, to how understand trans-hormones post- It has become increasingly that clear ′ -untranslated regions (3 given at the end of this article). Below a summary Below asummary article). given endofthis atthe 7 ). Also in human adipose tissue, studies have). Also in human adipose tissue, 5 ). This sex hormone-associated decline in). This sexdecline hormone-associated 4 ), thereby insulin reducing peripheral β (ESR2) in adipose tissue, estrogen (ESR2)inadiposetissue, is 11 ′ -UTR) oftarget mRNAs 12 ) altercould potentially B WFlorijnandothers ). 10 supplementary supplementary ), sex-specific, 9 ). Given 6 2 ). ). ).

blood pressure. HDL, highdensitylipoprotein;SBP,systolic bloodpressure;DBP,diatolic HDL-cholesterol (mmol/L) Triglyceriden (mmol/L) Cholesterol (mmol/L) Creatinine (µmol/L) Insulin (pmol/L) Glucose (mmol/L) Hematocrit (L/L) Hemoglobin (mmol/L) DBP (mmHg) SBP (mmHg) Testosterone (nmol/L) Estradiol (pmol/L) BMI (kg/m Age (years) transwomen cohort. Table 1 Library preparationandnext-generation Kit (Qiagen). from 200 Plasma RNA sample patient was isolated from each Plasma RNAisolation and transwomenthe 1 and 2 are displayed cohort in daily estradiol of administration. Patient characteristics oftreatment andafter 1year start were atthe of obtained week, aspreviously ( described estradiolh transdermal (Systen and 4mgestradiol valerate (Progynova®, Bayer) or100 µg/24 50mgcyproterone acetateboth (CPA) (Androcur Transwomen received oral adaily treatment with doseof Patients described ( described exclusion column,aspreviously chromatography (SEC) size- human plasmato mLSepharose a3.64 CL-2B Plasma EVs (70 nm)were isolated by applying 125 Plasma EVisolation Denmark. at ExiqonServices, Illumina NextSeq system. 500 were Experiments conducted according to protocol. Samples were sequenced onthe Plasma miR sequencing was by performed Exiqon sequencing ofplasmamiRs Estradiol-derived miRs 2 , respectively. Clinical characteristicsofthepilot 2 ) 14 μ L EDTA-plasma RNeasy by Micro usingthe ). Downloaded fromBioscientifica.com at10/09/202104:45:22AM 1.38 1.06 4.59 0.47 10.2 19.9 25.2 133 5.6 Baseline 79 72 86 96 35 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 13 0.37 0.42 0.86 9 44 0.5 0.02 0.5 11 10 8.7 21 4.1 13 ® , Janssen–Cilag) twicea ). Paired plasmasamples 185 1.16 0.97 4.05 0.43 24.5 105 128 310 5.5 9.0 0.8 Estradiol 79 82 36 n :4

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European Journal of Endocrinology (TG)-rich emulsion particles (80nm) were prepared as emulsionparticles (TG)-rich Glycerol tri[ lipoprotein-like particles Clearance ofradiolabeled glucoseand (Mercodia, 10-1247). werePlasma insulinconcentrations measured by ELISA Plasma ELISA Systems).TSE in fully automated cages metabolic (LabMaster System, Five days before injection, micewere individually housed orscrambled miRsequence(antimiR-452), (scramblemiR). (LNA)-modified miR-224 antisense (antimiR-224), miR-452 received two s.c.injections acid of25mg/kglocked nucleic River Nederland) were and groups randomized inthree Male C57Bl/6J mice( Animal experiments Tablesequences are listed inSupplementary 2. manufacturer’s protocols.the Target gene mRNA primer 4427975, Fisher Thermo Scientific) were usedaccording to were usedfor plasmamiRsequencing. Taqman (Cat. primers pooledsamplesindividual samplescomprised the that that Selected miRswere validated using quantitative PCR, with mRNA expression qPCR validationofplasmamiR,EVmiRs,and blood pressure. HDL, highdensitylipoprotein;SBP,systolicbloodpressure;DBP,diatolic HDL-cholesterol (mmol/L) Triglyceriden (mmol/L) Cholesterol (mmol/L) Creatinine (µmol/L) Insulin (pmol/L) Glucose (mmol/L) Hematocrit (L/L) Hemoglobin (mmol/L) DBP (mmHg) SBP (mmHg) Testosterone (nmol/L) Estradiol (pmol/L) BMI (kg/m Age (years) transwomen cohort. Table 2 Clinical Study Clinical characteristicsofthevalidation 2 ) 3 H]oleate-labeled lipoprotein-like triglyceride n

=

10 age pergroup, 78.2 50.1 0.45 20.4 23.5 127 1.4 1.1 4.7 5.4 9.8 Baseline 80 86 34 ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.3 0.5 1.1 8.8 30.9 0.7 0.03 0.5 9 10 6.3 22 6.1 12 B WFlorijnandothers 73.1 71.8 0.42 24.9 122 275 1.1 0.9 5.2 8.8 0.8 Estradiol 75 35 = 4

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the alignmentofsamples.the The reads were mappedto the Mus musculusreference version GRCm38.p4 was usedfor Mapping andanalysisofRNA-seqdata standard protocols. were hematoxylin stained with andeosin(H&E) using embedded inparaffin, andcutinto 5- gonadal WAT (gWAT) were dehydrated in70% EtOH, Formalin-fixed BAT interscapular (iBAT), s.c.WAT, and Tissue histologyandimmunohistochemistry (Perkin Elmer). were harvested anddissolved overnight at56 vein. tail mg TG permouse)viathe After 15 min,organs mice were injected 200 with DG) was addedina previously ( described adipocytes were 100 incubated with nM17- Differentiated 3T3-L1 adipocytes and differentiated brown Cell treatment (IBMX) andPPAR penicillin andstreptomycin, 3-isobutyl-1-methylxanthine (Life Technologies Europe), human insulin,dexamethasone, HEPESpH7.4,supplemented with 10% FBS heat-inactivated medium(DMEM/Ham’sgrowth F-12 medium(1:1, v/v) 3T3-L1 preadipocytes (Zenbio) were differentiated in wereExperiments ondays performed 12–14 ofdifferentiation. ( described depots of5-week-old maleC57BL/6J miceaspreviously Brown preadipocytes were isolated from BAT interscapular 3T3-L1 whiteadipocytes Cell cultureofmurinebrownadipocytesand Analysis (IPA) software. Pathway analysis outusingIngenuity Pathway was carried Pathway analysis values were calculated. perkilobaseofexonfragments permillion reads mapped) v2.15.3. R platform the Additionally, RPKM/FPKM (reads/ v1.10.1, DESeqpackage into the within package astatistical Burrows–Wheeler Transform. The read countswere loaded reference alignerbasedonthe sequences usingashort-read Estradiol-derived miRs 16 ), anduponconfluence,cells were differentiated. γ agonist rosiglitazone. 3 H: 14 15 Downloaded fromBioscientifica.com at10/09/202104:45:22AM C ) and[ =

4:1 ratio. After 6hoffasting, 4:1 ratio. μ L of emulsion particles (1 L ofemulsionparticles https://eje.bioscientifica.com 14 C]deoxyglucose ([ μ 185 m sections. Sections Sections m sections. :4 ° C inSolvable β estradiol 541 14 via freeaccess C] European Journal of Endocrinology https://eje.bioscientifica.com All patients gaveAll patients informed written consent.Allanimal ofHelsinki. Declaration ofthe principles ethical the with Medical Center (Leiden,The Netherlands) andcomplied (Amsterdam, Leiden University The Netherlands) andthe VUUniversity the Boards ofboth Medical Center These studies were approved Review Institutional by the Study approval ANOVA Bonferroni’s with using atwo-tailed paired orunpaired Student’s analysisStatistical GraphPad was with performed software wasregression used to adjust for possible confounders. linear multivariable Inaddition, for distribution. normal was tested Kolmogorov–Smirnovdistribution usingthe test are expressed data asmean All other in expression levels between samples (TMM normalization). was usedbasedonlog-fold andabsolute gene-wise changes M-values method meanofthe trimmed the normalization, software (Bioconductor). For package EdgeR statistical the next generation sequencing was doneusing experiment Differential expression analysisthe ofplasmamiRsin Statistical analysis manufacturer’s according to protocol. the Aldrich/Merck) assay kitusing aglucoseuptake colorimetric (Sigma– transfected LNA-antimiR-224 with and-452was performed brown murine adipocytesGlucose uptake by immortalized Glucose uptakeexperiments DMEM/F12 FBS. (Sigma) without and MitoTracker RedCMXRos Fisher) (250nM;Thermo in MitoTrackermin with Green FM(125 Fisher) nM;Thermo LNA-antimiR-224with and-452incubated for 30 brown murine adipocytes wereImmortalized transfected Mitotracker experiment XF96 analyzer (AgilentTechnologies). rate (ECAR) acidification the Seahorse were measured using Oxygen consumption rate andextracellular (OCR) acidification ofmurinebrownadipocytes Oxygen consumptionandextracellular Trizol PBScombinedwith washed with to isolate RNA. (E2758, Sigma–Aldrich) for 48h.After 48h,cellswere Clinical Study post hoc B WFlorijnandothers test. ±

s.e.m. Variable t -test or miRs intranswomen Identification ofcirculatingestradiol-responsive Results were generated study. current oranalyzed the during for access:ahmfqeqglzwphcf). No applicableresources andare accessibleunderGSE147966(GEO) (reviewer token study arecurrent available Omnibus in the The generated datasets and/oranalyzed the during during Data andresourceavailability Netherlands). LeidenUniversityof the MedicalCenter (Leiden,The animal welfare authorities veterinary committee ofthe andprotocolsexperiments were approved by the the pilotstudy,the before ( samples sametranswomen was usedin ofthe that cohort Fig. 1)by(Supplementary RT-qPCR individual in the step process. validation First, we reassessed plasmamiRs To miRs, we validate circulating conducted athree- these independent transgendercohorts Validation ofestrogen-responsivemiRsin (based on to +/ ( groups based on(i)asufficient level across sample of regulation -874, -30b,-483,-539, and-652.These miRswere selected miR-224,validation: -122, -23a,-452, -139, -133b, -215, -9, ( 5p, -3198, -3940-3p, -625-3p, and miR-6786-3p increased 3p, -let-7d-3p, -432-5p,-139-3p, -6747-3p, -433b-3p,-584- -483-5p, -let-7b-5p, -6787-3p, -184, -3679-5p, -370-5p, -615- decrease, while levels circulating ofmiR-3138, -215-5p, 3p, 30b-5p,andmiR-490-3p levels displayed asignificant -452-5p, 23b-3p,-3913-5p, -144-5p, -331-5p, -766-3p, -874- Specifically, miR-224, -122-5p, -539-5p, 23a-3p,-133b, 1). 33 were Data differentially expressed (Supplementary estradiol 667 treatment, identified miRs( five transwomen, before ( (pooled basedonage andestradiol from concentration) of plasmamiRsinfour pooledEDTA-plasma samples to surgicaltransition ( prior in transwomen who received 1year ofestradiol suppletion We a pilot study performed assessing plasma miR profiles Estradiol-derived miRs Fig. 1B − 1.0 log-FoldChange) and(ii)differential expression ). The following miRswere selected for RT-qPCR > P 2-fold up-ordown-regulation, corresponding -value) asaresult of1-year estradiol treatment. n Downloaded fromBioscientifica.com at10/09/202104:45:22AM n

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European Journal of Endocrinology https://eje.bioscientifica.com marked anasterisk), andsomekey with metabolism- BAT andWAT Fig. genes 6,metabolic (Supplementary plotted inheatmaps,many were ofthem upregulated in canonical pathways. When miR-224/452 target genes were asupstream couldserve regulators ofdifferentialthat top IPA miR-224/452 allpotential to identify target genes and insulinreceptor signaling.We subsequentlyused the HIPPO-pathwayaffected (adipocyte differentiation) hypertrophy) were affected, treatment while antimiR-452 3(STAT3)transcription (controls lipogenesis andadipocyte andsignaltransducermetabolism) of andactivator kinase(AMPK) signaling (regulates adipocyte WAT from antimir-224 tissue treated mice,AMP-activated pathways Fig. affected 5).Incontrast, (Supplementary in receptor signaling in BAT as significant top canonical dysfunction andinsulin tool, wemitochondrial identified applying pathwaysmetabolic bymiRs ( these of a multiple coordinated regulation indicating potentially forstrongly metabolism, associate with genes enriched that (IPA), we miR-224/452 found both that target genes are Interestingly,changes. usingIngenuity Pathway Analysis BAT metabolism-associated gene expression to identify next RNA performed sequencing (RNA-seq) ofWAT and uptake in WAT and decreased glucose uptake in BAT, we miRsstimulated fattyacid Given silencingofthese that signaling, glucosemetabolism,andlipogenesis mitochondrial energymetabolism,insulin miR-224 andmiR-452targetgenesareinvolvedin was seeninantimiR-224 and-452treated cells( depolarization adecrease inmitochondrial depolarization, intensity (MTR/MTG), which measures mitochondrial respectively. of relative fluorescence After quantification independent of- anddependenton membrane potential, red (MTR;active) ( using Mitotracker (MTG; andMitotracker total) green brownand -452 treated murine adipocyte cell cultures 2M proton leakinantimiR-224 and-452treated cells( inantimiR-224 cellsandadecreasebasal respiration in affected,was notsignificantly adecrease weobserved in treated brown maximalrespiration adipocytes. Although the culture mediaofantimiR-224 andantimiR-452 flux in we by respiration alsomeasured mitochondrial proton glycolytic ( respiration mitochondrial fluxmaintain ( respirometry Clinical Study ). Next, inantimiR-224 we mitochondria stained the in silico Fig. 2L analysis IPA RNA-seq the ofthe with data Fig. 2N ). Becauseglucoseconsumption and ), which stain mitochondria ), which stain mitochondria B WFlorijnandothers Fig. 3A ). By further ). By further Fig. 2O ). 19 Fig. ), with more FAwith uptake ( decreased ( decreases inkey insulinsignaling genes like genes namely we alsovalidated lossofBAT-specific the glucose uptake Given the and whose losspredisposes to insulinresistance ( ( a reduced expression confirmed of validation qPCR With regard in BAT, metabolism energy to mitochondrial have amajor by impact metabolism onenergy RT-qPCR. expression of several genes are known whose to functions is displayed in differentially expressed genes involvedthese pathways in 2,while Data aselected subset of in Supplementary ( lipid metabolism ( insulin signalingandglucosemetabolism and miR-452silencing genes involvedinmetabolismfollowingmiR-224 Adipose tissue-specificdifferentialexpressionof gain andadiposity ( insulin resistance like as higher expression oflipogenesis-associated genes, such ( or exacerbate insulinsensitivity like as such lipid metabolisms treatment coordinately upregulated regulate genes that silencing. Interestingly, inWAT ( BAT ( thermogenesis and obesity ( in BAT ( miR-224of the targets and related target genes couldbevalidated by ( qPCR metabolism, the expression of the metabolism, ( decreased expression of of inBAT,in lipidmetabolism we validated alower expression were inantimiR-224 observed treated WAT ( ( metabolism energy genes inmitochondrial volcano differential expression plots of confirming pathways. To end,we first that generated gene-distribution top altered inthe canonicalmetabolic function that Next, differential expression we of genes aimedto identify Estradiol-derived miRs Fig. 3E 37 27 Pnrc2 Cidea ), andincreased ). Similarly, treated antimiR-452 WAT displayed tissue C ). 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metabolism Mitochondrial energy Ucp2 Ppargc1a Ppargc1a Slc25a15 ). It is also important to note). Itisalsoimportant Lhpp Slc25a21 Ndufa8 Ndufa4 Ndufa11 Cox7b Cox6c ↑ , Cox7a2l ↑ ↑ , ↑ ↑ ↓ ↓ , Slc25a15 , 185 ↑ , ↑ , , Cox7a2 Cyc1 , ↑ Slc25a21 , Ndufa6 ↓ Atp6voa2 Cox5b Ppa2 , , Ndufa2 :4 Ucp3 ↑ ↑ , , ↑ Gadd45b Ndufa1 ↑ ↑ , ↓ ↓ , , Sdhb , , Cox6b1 ↑ ↑ Cox7a2 ↑ Ucp2 ↓

, , , Ndufa7 Ndufa3 Cox6c ↑ ↑ , ↑ , ↑ 548 ↑ l ↑ , ↑ , 47 ↑ , ↑ via freeaccess , , ) European Journal of Endocrinology brown adiposeandresultsinmoretriglyceride-derivedfattyaciduptakewhiteskeletalmuscle. lower miR-224andmiR-452inextracellularvesicles.ThelossofbothmiRslowersmitochondrialrespirationglucoseuptake in brown adiposeandlipiduptakeinwhiteleanmetabolismisdisrupteduponsupraphysiologicallevelsofestrogen that Proposed mechanismbywhichmiR-224andmiR-452affectmetabolismintranswomen.Tightlybalancedglucoseuptake in Figure 4 female testosterone transgenders. Itcouldbearguedthat miRs in male- because we both mice, particularly identified to bodycompositionin relation over changes time. and affectedsignificantly in transwomen this study ( in alter miR plasma levels as well ( composition may may andassuch affect health metabolic levels ofestradiol Lastly, changes. andmetabolic body between relationship high the supporting shown) further list ofdifferentiallythe expressed pathways not (data ( (GDM) compared from control to subjects adipose tissue from women diabetes gestational tissue mellitus with analyses differential proteome adipose ofomental ofthe Interestingly, metabolism. energy when we IPA performed mediated effects onglucoseuptake and mitochondrial 48 Clinical Study ) we dysfunction topped found mitochondrial that 2 We conducted miR-224 and-452silencing inmale ). Still, future). Still, studies shouldinvestigate miR-224/452 49 B WFlorijnandothers ), although BMI was not ), although Tables 1 repeated elimination and compositionrepeated oftriglycerides). elimination expenditure (energy dueto a nutrients cycling ofthose glucose toward lipid consumption) andpossibly futile from (change partitioning resulted inashiftnutrient onsetexpenditure, ofinsulinresistance the we that suspect we donothave for elevated adirectexplanation the energy indicate beneficialeffects to prevent lipotoxicity. Although expenditure andincreased fattyaciduptake could also inmiceregarding increased energy Our observations of sex-specific miR effectspotential in the tissue ( BAT following dysfunction inBAT, mitochondrial suggesting obese andshowed impaired systemic insulinsensitivity female ratsparticularly were previously found to become our hypothesis. However, it hasbeendemonstrated that wouldintact, be morefor appropriate direct testing of whiledata, female mice,inwhich estrogen signalingis micemay havesignaling inthese confounded Estradiol-derived miRs Downloaded fromBioscientifica.com at10/09/202104:45:22AM https://eje.bioscientifica.com 185 :4 the invivo the 549 50 via freeaccess ). ).

European Journal of Endocrinology https://eje.bioscientifica.com the useofandtechnicalsupportat electronmicroscopefacilities. Biology, Section Electron Microscopy, Leiden University Medical Center) for The authorsthankProfAbrahamJKoster (DepartmentofCellandChemical Acknowledgement M Kcontributedequally.RBandAJvZsharedseniorauthorship. and D J G M J analyses. data the of accuracy the and data the of integrity such, had full access to all the data in the study and responsibility for the manuscript. BWF,AJVZ,andRareguarantorsofthisworkand, as and RBcontributedtothediscussionreviewededited Z, V J A R, N C P R, J T N, R data. the researched S W and Y, Y B, A T, D G L, J manuscript. M K, E N K, R W A L L, and S K acquired and analyzed data. J D, B W F conducted experiments, acquired and analyzed data, and wrote the Author contributionsstatement project 459001002toAJVZandBWF). Foundation R B)andtheDutchDiabetesResearch (ZonMw, Doorbraak to 16OKG16 grant (KOLLF Foundation Kidney Dutch the B), R and Z V J A R), the EuropeanFund for theStudy of Diabetesand Boehringer Ingelheim (to N C (P CVON-GENIUS-2 and Z), V J (A CVON-RECONNECT 2013/T084), Foundation in the contextofconsortia:Queen of Hearts(AJVZ,BWF, This study was supported by funding provided by the Netherlands Heart Funding be could that interest of conflict perceived asprejudicingtheimpartialityofthisstudy. no is there that declare authors The Declaration ofinterest EJE-21-0267. This islinkedtotheonline version ofthepaperat Supplementary materials (adipocyte) inmice. metabolism miRsare neededto investigateof both of regulation their ( factor involved in redox and tumor suppression regulation thioredoxin protein interacting (TXNIP), akey transcription targeted miRs in whichmelanomas, both in malignant simultaneously controls was cellularmetabolism found ( relatedregulate genes functionally insimilarprocesses ( density lipoprotein (viaits (LDL)metabolism target PCSK9) ( apoptosis uponinflammation of3T3-L1adipocytesmetabolism ( of reduced observed expressioninsulin resistance ordueto the assessment ofwhether glucoseuptake isindeeddueto for example, by doingglucosetolerance testing and this miR-mediated phenotype, are neededto confirm negatively. health metabolic experiments additional Still, and reduced BAT glucoseuptake were shown to impact genes in BAT of thermogenic reduction the Furthermore, 55 54 53 Clinical Study Glut4 ). A striking example). Astriking miR-224/452 inwhich the cluster ) is consistent with the notion that miRs coordinately that notion ) isconsistent the with ). However, more studies (synergistic) silencing with . The fact that miR-224. The that isknown fact to control FA B WFlorijnandothers 52 ) andcontrols low- 51 ), prevents 3T3-L1 https://doi.org/10.1530/

References

Estradiol-derived miRs 12 10 14 13 11 5 4 3 2 1 9 8 7 6 Thomou T, Dreyfuss JM, Konishi M, Mori MA, Sakaguchi M, Auer MK, Ebert T, Pietzner M, Defreyne J, Fuss J, Stalla GK &T’Sjoen G. Shadid S, Abosi-Appeadu K, Defreyne J, DeMaertelaere AS, Klaver M, deBlok CJM,Wiepjes CM, Dekker MJHJ, Nota NM, de Zhou Z, Moore TM, Drew BG, Ribas V, Wanagat J, Civelek M, Nelson MD, Santos RS, Criollo A, Morselli E, Palmer BF &Clegg DJ. Florijn BW, Duijs JMGJ, Levels JH, Dallinga-Thie GM, Wang Y, van Velzen DM, Paldino A, Klaver M, Defreyne J, Nota NM, Hovingh GK, Kim JH, Cho HT&Kim YJ. The role ofestrogen inadiposetissue Florijn BW, Bijkerk R, van derVeer EP &van Zonneveld AJ. Genderand &Kulyte A.Arner P MicroRNA networks regulatory inhumanadipose &Cohen SM.MicroRNAsHerranz H networks: andgene regulatory Wierckx K, Heylens G, Declercq E, DeCuypere G, Elaut E, Taes Y, Shi Z, Zhao C,Guo X,Ding H,Cui Y, Shen R&Liu J. Differential Clinical Endocrinology andMetabolism Clinical Endocrinology transgender individuals:focus cytokines. onmetabolic the metabolic syndromeEffects in treatment on ofsex hormone (https://doi.org/10.2337/dc19-1061) responses intransgender people. therapygender-affirming oninsulinsensitivity andincretin hormone Veldeman L, Holst JJ, Lapauw B,Vilsboll T &T’Sjoen G. Effects of (https://doi.org/10.1530/EJE-17-0496) prospective study. therapy:using cross-sex results from hormonal amulticenter in regional bodyfat,leanmassandshapeintrans persons Schreiner T,Mutsert R, Fisher AD, T’Sjoen G &denHeijer M. Changes 2020 remodeling. polg1 andmitochondrial α Segawa M, Wolf DM, Norheim F, Seldin MM org/10.1038/nrendo.2017.12) health. The effects ofoestrogenstheir oncardiometabolicreceptors and microRNAs Among extracellular vesicles, HDL,andAgo-2. angiogenic ofcirculating distribution nephropathy the alters Yuana Y,Boing AN, Stam W, Limpens RWAL, Au YW 1937–1947. transwomen. of testosterone intransmen andestrogen pluscyproterone acetate in Thijs A, Simsek S,T’Sjoen G &den Heijer M. Cardiometabolic effects nature21365) tissues. in other et al. Wolfrum C, Rao TN, Winnay JN, Grinspoon SK Garcia-Martin R, insightsmetabolism: into glucosehomeostasis regulation. cvr/cvx223) Cardiovascular Research inwomen? fraction preservedforce ejection failure with behindheart cardiovascular disease:are sex-biased microRNA networks adriving (https://doi.org/10.1038/nrendo.2015.25) andobesity.tissue Development impact ofnoiseinbiologicalsystems.managing the 169 acase-control study.persons: oftrans therapy cross-sex inalarge hormone cohort cancer during &T’Sjoen G.Kaufman JM Prevalence ofcardiovascular diseaseand org/10.1210/jc.2017-01559) 2019 2019 155 expression inestrogen-induced insulinresistance. diabetes mellitus subjects reveals miR-222 asaregulator ofERalpha expression ofmicroRNAs from gestational adiposetissue inomental Journal controls inwhite metabolism andbrown adipocytes by regulating 1982–1990. 471–478. Adipose-derived miRNAs circulating regulate gene expression 68 12 2014 2014 Nature Reviews: Endocrinology 2287–2300. eaax8096. (https://doi.org/10.1210/jc.2018-02138) 2010 2010 61 Journal of Clinical Endocrinology andMetabolism Journal ofClinicalEndocrinology (https://doi.org/10.1530/EJE-13-0493) 1055–1067. Nature (https://doi.org/10.1210/en.2013-2046) 24 European Journal ofEndocrinology Nature Reviews: Endocrinology (https://doi.org/10.1126/scitranslmed.aax8096) (https://doi.org/10.2337/db18-1360) 1339–1344. 2018 2018 2017 2017 (https://doi.org/10.1507/endocrj.ej14-0262) Downloaded fromBioscientifica.com at10/09/202104:45:22AM 542 114 European Journal ofEndocrinology 450–455. 210–225. (https://doi.org/10.1101/gad.1937010) Diabetes Care 2017 2017 2018 2018 Science TranslationalScience Medicine 13 (https://doi.org/10.1093/ 103 (https://doi.org/10.1038/ 185 et al. 352–364. 352–364. 2020 790–802. 2015 2015 :4 Estrogen receptor . Endocrinology 2018 2018 43 Genes and Genes et al. 11 411–417. (https://doi. Journal of 276–288. 178 (https://doi. Diabetic Diabetic 2019 2019 Endocrine Endocrine Diabetes 163–171. 2013 2013 550 2014 2014 104

via freeaccess

European Journal of Endocrinology

20 29 28 25 23 22 24 26 27 21 18 19 15 17 16 Clinical Study Bazuine M, Stenkula KG, Cam M, Arroyo M &Cushman SW. Guardian Zhou D, Shen R,Ye JJ, Li Y, Tsark W, Isbell D, Tso P &Chen S.Nuclear De Luca M,Klimentidis YC, Cho R, Casazza K,Chambers MM, Wilfling F,Mejhert N, Esteve D, Galitzky J, Pellegrinelli V, Kolditz CI, Moest H, Frei AP, Geiger M, Wollscheid B Bhattacharya I, & Davies JC, Bain SC&Kanamarlapudi V. ADP-ribosylation factor 6 Liu Y, Zhou D, Abumrad NA &Su X.ADP-ribosylation factor 6 Kanzleiter T, Schneider T, Walter I, Bolze F, Heldmaier G, Eickhorst C, Henegar C, Tordjman J, Achard V, Lacasa D, Cremer I, Guerre-Millo M, Winther S, Isidor MS,Basse AL,Skjoldborg N, Cheung A,Quistorff B Ludwig N, Leidinger P, Becker K, Backes C, Fehlmann T, Pallasch C, Dekker MJ, Wierckx K, Van Klaver M, Caenegem E, Kreukels BP, Elaut E, Liu J, Sips HCM,Dorleijn JC, Kuipers EN, van Dam AD, Rensen PC, van Dijk MC, Havenaar EC, Bijsterbosch MK, Kruijt JK & Cutchins A, Harmon DB, Kirby JL, Harmon DB, Cutchins A, Doran AC, Skaflen M, Oldham SN, Lipidology fatcell. ahypothesis onp53 signaling inthe of corpulence: doi.org/10.1074/jbc.M703234200) adiposity. receptor PNRC2 expenditure regulates energy and org/10.1371/journal.pone.0011286) whole-body metabolism. energy roleconserved of for regulation syndecan family inthe members Harbison ST, Jumbo-Lucioni P, Zhang S,Leips J&Fernandez JR. A 2013 novel adipokinelinked to extracellular composition. matrix Viguerie N, Tordjman J, Trayhurn P Naslund E, 1831 changes. quantitative surfaceome Wolfrum C. ofadipocytes inobesity islinked Malfunctioning to bcp.2014.06.012) Pharmacology lipolysisregulates endothelin-1-induced inadipocytes. org/10.1152/ajpcell.00541.2009) Journal ofPhysiology: Cell Physiology modulates adrenergic stimulated lipolysis inadipocytes. 37–44. effector ofbrownthermogenesis. fat Klaus S &Klingenspor M. Evidencefor Nr4a1asacold-induced (https://doi.org/10.1161/ATVBAHA.111.234856) Arteriosclerosis, Thrombosis, andVascular Biology mediates highdifferentiation-3 fatdiet-induced visceral fat expansion. Klibanov AL, Keller SR, Meller N, Garmey J (https://doi.org/10.1186/gb-2008-9-1-r14) extracellular inhumanobesity. matrix relevance signaturetranscriptomic highlights pathological of the Poitou C, Basdevant A, Stich V, Viguerie N ajpendo.00218.2017) and Metabolism and oxygen consumption. glycolysis& Hansen JB.Restricting impairs brown adipocyte glucose 3865–3877. miRNA expression across humantissues. Rheinheimer S, Meder B,Stahler C, Meese E doi.org/10.1016/j.jsxm.2016.03.371) part. endocrine European network for investigation the ofgender incongruence: Fisher AD, van Trotsenburg MA, Schreiner T, denHeijer M org/10.1016/j.bbalip.2019.08.007) Molecular andCell ofLipids Biology proliferative anddifferentiated states. brown preadipocytesConditionally immortalized canswitch between Christodoulides C, Karpe F, Zhou G,Boon MR,Rensen PCN Medicine B. chylomicrons: to hepatitis approach anew therapeutic van Berkel TJ. Selective liver by ofantivirals recombinant targeting 56 1208–1216. (https://doi.org/10.1152/physiolgenomics.00204.2005) 1792–1801. 1995 2009 2009 Journal ofBiologicalChemistry (https://doi.org/10.1093/nar/gkw116) 2014 2014 1 2018 2018 221–225. 4 Journal of Sexual Medicine Journal ofSexual 231–243. (https://doi.org/10.1016/j.bbalip.2013.04.001) 90 (https://doi.org/10.1007/s00125-013-2931-z) 314 406–413. E214–E223. (https://doi.org/10.1038/nm0395-221) American Journal of Physiology: Endocrinology Journal ofPhysiology:American Endocrinology (https://doi.org/10.2217/clp.09.2) (https://doi.org/10.1016/j. PLoS ONE 2019 2019 Biochimica etBiophysica Acta 2010 2010 Physiological Genomics (https://doi.org/10.1152/ Biochimica etBiophysica Acta: B WFlorijnandothers Genome Biology Genome 1864 2008 2008 298 2016 2016 Nucleic Acids Research et al. 2010 2010 et al. et al. 158511. C921–C928. 283 et al. 13 Adipose tissue Adipose tissue 2012 2012 Inhibitor of 5 Distribution of Distribution 994–999. 541–553. e11286. Semaphorin 3C is a 3Cisa Semaphorin 32 (https://doi. 2008 2008 Biochemical 317–324. American American Nature et al. (https://doi. (https://doi. 2005 2005 Diabetologia (https:// (https:// Clinical et al. 9 R14. 2016 2016 2013 2013 A

24

44

Estradiol-derived miRs 44 36 35 33 32 30 43 39 38 42 34 40 41 31 37 Pramfalk C, Eriksson M &Parini P.Pramfalk C, Eriksson M Role ofTG-interacting factor (Tgif) Puri V, Ranjit S, Konda S, Nicoloro SM, Straubhaar J, Chawla A, Fasshauer M, Benito M, Klein J, White MF Ueki K,Kriauciunas KM, Olsen JM, Sato M, Dallner OS,Sandstrom AL, Pisani DF, Chambard JC, Kleiner S, Mepani RJ, Laznik D, Ye L, Jurczak MJ, Jornayvaz FR, Estall JL, Dib L, Bugge A &Collins S.LXRalpha fuelsfattyacid-stimulated White UA, Stewart WC, Mynatt RL&Stephens JM. Neuropoietin Schmeier S, MacPherson CR, Essack M, Kaur M, Schaefer U, Kaur M, Essack M, Suzuki H, MacPherson CR, Schmeier S, Florijn BW, Valstar GB, Duijs J, Menken R, Cramer MJ, Teske AJ, Zhou X, Wang X, Huang Z,Xu L, Zhu W&Liu P. AnER-associated Harada N, Oda Z, Hara Y, Fujinami K, Okawa M, Yonemoto M, Ohbuchi K, Osuga J, Ishibashi S,Oka T, Yagyu H, Tozawa R, Fujimoto A, Shionoiri F, Eissing L, Scherer T, Todter K, Knippschild U, Greve JW, Buurman WA, Ellis JM, Li LO, Wu PC, Koves TR, Ilkayeva O, Stevens RD, Watkins SM, Kristensen H, Haldrup C, Strand S, Mundbjerg K,Kristensen H, Haldrup C, Mortensen MM, Chouinard M, Lin C, Burkart A, Corvera S Chouinard M, Lin C,Burkart A, (https://doi.org/10.1074/jbc.M004046200) adipocytes. andglucoseuptake inbrown ofglut4translocation stimulation role ofinsulinreceptor substrate-2 Essential & Kahn CR. ininsulin org/10.1083/jcb.201403080) translocation. fat cellsisdependentonmTOR complex 2-promoted GLUT1 &Bengtsson T. Hutchinson DS Amri EZ, Glucoseuptake inbrown PNAS PGC-1alphainadiposetissues. of insulinresistance inmicelacking Chatterjee Bhowmick D, Shulman GI&Spiegelman BM. Development 2014 oxygen consumption inwhite adipocytes. (https://doi.org/10.1016/j.bbalip.2014.07.019) in lipidmetabolism. org/10.1074/jbc.M710462200) Journal ofBiologicalChemistry attenuates adipogenesis andinducesinsulinresistance inadipocytes. with lipiddropletswith andinsulinsensitivity inhumans. microvascular injury. left ventricular diastolic dysfunction orHFpEF associate with et al. Ghossein-Doha C, Rutten FH, denRuijter HM Spaanderman MEA, BMC Genomics microRNA differentiation. genes humanmonocytic expressed during of Hayashizaki Y circuitry &Bajic VB. transcriptional the Deciphering doi.org/10.1186/s13046-014-0094-5) Research andClinicalCancer Journal ofExperimental miRNA inER-positive signature predictsprognosis breast cancer. 27 in liver-specific mice. ACC1-deficient Ikeda Y, Ohwaki K, Aragane K org/10.1073/pnas.97.2.787) hypertrophy, butnotinobesity. hormone-sensitive lipaseresults inmalesterility andadipocyte Yahagi N, Kraemer FB, Tsutsumi O org/10.1038/ncomms2537) health. metabolic lipogenesis inhumanfatandliver islinked to ChREBP-beta and Pinnschmidt HO, Rensen SS, Wolf AM, Bartelt A Metabolism acids toward beta-oxidation andisrequired for coldthermogenesis. Muoio DM &Coleman RA.Adipose acyl-CoA directs fatty synthetase-1 7833–7838. doi.org/10.1158/1078-0432.CCR-13-2642) prostatectomy. afterin prostate radical recurrence cancerpredicts biochemical GABRE~miR-452~miR-224 ofthe Hypermethylation promoter Thorsen K, Ostenfeld MS, Wild PJ, Arsov C, Goering W org/10.1038/s41598-020-70848-8) 1881–1888. Sex-specific microRNAs in diabeteswomen with and 55 2012 2012 247–257. 2010 2010 109 Journal ofBiologicalChemistry (https://doi.org/10.1073/pnas.0802063105) 2009 2009 (https://doi.org/10.1128/MCB.01122-06) Journal ofCell Biology 9635–9640. Clinical Cancer ResearchClinical Cancer 12 (https://doi.org/10.1194/jlr.M043422) Nature Communications 53–64. 10 Biochimica etBiophysica Acta Scientific Reports Scientific 595. (https://doi.org/10.1016/j.cmet.2010.05.012) Downloaded fromBioscientifica.com at10/09/202104:45:22AM (https://doi.org/10.1073/pnas.1207287109) (https://doi.org/10.1186/1471-2164-10-595) et al. 2008 2008 PNAS Hepatic denovo Hepatic lipogenesis ispresent et al. 283 2014 2014 Molecular andCellular Biology https://eje.bioscientifica.com 2020 2000 2014 2014 Targeted of disruption 22505–22512. 2000 207 2013 2013 et al. Journal ofLipidResearch 10 97 185 20 365–374. 13945. 787–792. Cideaisassociated 2169–2181. 4 275 :4 1528. et al. 2015 2015 2014 2014 25494–25501. Denovo PNAS (https://doi. (https://doi. 1 et al. (https://doi. (https://doi. 33 851 (https://doi. 94. (https:// 2008 2008

9–12. (https:// 2007 2007 551 105

Cell Cell via freeaccess

European Journal of Endocrinology https://eje.bioscientifica.com

50 49 45 48 46 47 Clinical Study Nadal-Casellas A, Bauza-Thorbrugge M, Proenza AM, Gianotti M Circulating &Martínez JA. Riezu-Boj JI,Milagro FI Assmann TS, Ma Y, Gao J, Yin J, Lu H,Yang Y, Gu L,Liu X,Chen S,Huang Q, Zhou H Gava G, Mancini I,Alvisi S, Acomparison &Meriggiola MC. Seracchioli R Zarotsky V, Huang MY, Carman W, Morgentaler A, Singhal PK, Mauvais-Jarvis F, Clegg DJ &Hevener AL. The role ofestrogens in mitochondrial biogenesis andinsulinsignaling parametersmitochondrial in & Llado I.Sex-dependent differences in rat browntissue adipose 2020 diet obesity. insubjects with adiposity-related microRNAs response to alow-fat ofthe aspredictors 628–637. analysis adiposetissue. ofomental associated protein diabetes (APMAP)ingestational mellitus by proteomic et al. 2013 balanceandglucosehomeostasis. control ofenergy Endocrinology estradiol with intranswomen.in combination of 5-year administration ofcyproterone acetate orleuprolide acetate 2014 comorbidities, andconsequences ofhypogonadism inmen. Coffin D & Jones TH. Systematic literature riskfactors, the review of Identification ofanovel ofadipocyte plasmamembrane- Identification function 2 34 24 819–834. 309–338. 2956–2967. (https://doi.org/10.1021/acs.jproteome.5b01030) 2020 (https://doi.org/10.1111/andr.274) (https://doi.org/10.1210/er.2012-1055) 183 (https://doi.org/10.1111/jcmm.14920) 561–569. Journal ofCellular andMolecularMedicine (https://doi.org/10.1530/EJE-20-0370) Journal of Proteome ResearchJournal ofProteome B WFlorijnandothers European Journal of European Journalof Endocrine Reviews Endocrine 2016 2016 Andrology 15

Received 20March2021 Accepted 3August2021 Revised versionreceived10June2021

Estradiol-derived miRs 52 55 54 51 53 Knoll S, Furst K, Kowtharapu B, Schmitz U, Marquardt S, Peng Y, Xiang H,Chen C,Zheng R,Chai J, Peng J &Jiang S.MiR-224 Tsang JS, &van Oudenaarden A. Genome-widedissection Ebert MS Naeli P, MirzadehAzad F, Seidah NG&Mowla SJ. Malakootian M, Qi R, Huang J, Wang Q, Liu H,Wang R, Wang J &Yang F. MicroRNA- response to anobesogenic diet. 2014 2014 expression to TXNIP drive EMTthrough downregulation. Wolkenhauer O, &Putzer BM.E2F1inducesmiR-224/452 Martin H molcel.2010.03.007) signatures. networksof microRNA andcotargeting usinggene functions set fgene.2017.00189) miR-224. ofPCSK9byPost-transcriptional regulation miR-191, miR-222, and 233 activation. controlling NF-kappaB 224-5p regulates adipocyte apoptosis inducedby TNFalpha via biocel.2013.04.029) Cell Biology acid metabolism. impairs adipocyte early andregulates fatty differentiation 2013 1236–1246. 15 373 1315–1329. Frontiers inGenetics 125–135. Molecular Cell 2013 2013 (https://doi.org/10.1002/jcp.25992) 45 International Journal of Biochemistry and JournalofBiochemistry International (https://doi.org/10.1007/s11010-012-1481-x) (https://doi.org/10.15252/embr.201439392) 1585–1593. 2010 2010 2017 2017 Downloaded fromBioscientifica.com at10/09/202104:45:22AM 38 Molecular andCellular Biochemistry 8 (https://doi.org/10.1016/j. 140–153. 189. Journal ofCellular Physiology (https://doi.org/10.3389/ (https://doi.org/10.1016/j. 185 :4 EMBO Reports

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