3,5-T2 Alters Murine Genes Relevant for Xenobiotic, Steroid, and Thyroid Hormone Metabolism

56:4

j lietzow and others 3,5-T alters murine genes 56:4 311–323 Research 2

3,5-T2 alters murine genes relevant for xenobiotic, steroid, and thyroid hormone metabolism

Julika Lietzow1, Janine Golchert2, Georg Homuth2, Uwe Völker2, Wenke Jonas3,4 and Josef Köhrle1

1 Institut für Experimentelle Endokrinologie, Charité – Universitätsmedizin Berlin, Berlin, Germany Correspondence 2 Interfaculty Institute for Genetics and Functional Genomics, Department of Functional Genomics, should be addressed University Medicine Greifswald, Greifswald, Germany to J Köhrle 3 Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, Email Nuthetal, Germany [email protected] 4German Center for Diabetes Research (DZD), Helmholtz Center Munich, Neuherberg, Germany

Abstract Key Words The endogenous thyroid hormone (TH) metabolite 3,5-diiodo-l-thyronine (3,5-T2) acts as ff thyroid hormone a metabolically active substance affecting whole-body energy metabolism and hepatic ff 3,5-diiodo-l-thyronine lipid handling in a desirable manner. Considering possible adverse effects regarding (3,5-T2) thyromimetic action of 3,5-T treatment in rodents, the current literature remains largely 2 ff transcriptome analysis controversial. To obtain further insights into molecular mechanisms and to identify novel ff drug metabolism target genes of 3,5-T in liver, we performed a microarray-based liver tissue transcriptome 2 ff constitutive androstane analysis of male lean and diet-induced obese euthyroid mice treated for 4 weeks with receptor (CAR)

a dose of 2.5 µg/g bw 3,5-T2. Our results revealed that 3,5-T2 modulates the expression ff steroid metabolism of genes encoding Phase I and Phase II enzymes as well as Phase III transporters, which ff cholesterol metabolism Journal of Molecular Endocrinology play central roles in metabolism and detoxification of xenobiotics. Additionally, 3,5-T2 changes the expression of TH responsive genes, suggesting a thyromimetic action of

3,5-T2 in mouse liver. Interestingly, 3,5-T2 in obese but not in lean mice influences the expression of genes relevant for cholesterol and steroid biosynthesis, suggesting a novel

role of 3,5-T2 in steroid metabolism of obese mice. We concluded that treatment with

3,5-T2 in lean and diet-induced obese male mice alters the expression of genes encoding hepatic xenobiotic-metabolizing enzymes that play a substantial role in catabolism and inactivation of xenobiotics and TH and are also involved in hepatic steroid and lipid

metabolism. The administration of this high dose of 3,5-T2 might exert adverse hepatic

effects. Accordingly, the conceivable use of 3,5-T2 as pharmacological hypolipidemic Journal of Molecular agent should be considered with caution. Endocrinology (2016) 56, 311–323

Introduction

Thyroid hormones (TH) regulate multiple metabolic heart, bone, or muscle restrict their use as therapeutically processes and play an essential role in normal growth, active compounds for obesity-related diseases (Angelin & development, and energy homeostasis. Moreover, they Rudling 2010). are powerful agents in counteracting hyperlipidemia and In the last few decades, several TH derivatives have been reducing body weight. However, unwanted effects on designed as treatment options to counteract obesity and

http://jme.endocrinology-journals.org © 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-15-0159 Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 03:47:03PM via free access

10.1530/JME-15-0159 Journal of Molecular Endocrinology is theendogenousTHmetabolite3,5-diiodo- chronic treatmentwith3,5-T side effectsatothertargets.Inarecentlypublishedstudy, effects knownfortheclassicalTH3,5,3′ accumulation and steatosis without inducing thyromimetic rats fedwithahigh-fatdiet(HFD)preventedhepaticlipid HFD inamousemodel(Goldberg etal.2012). metabolism ineuthyroidmammals onstandardor (T (3,5-T results on miceand rats treated with 3,5-T andtheheart,areinagreementwithpublished pituitary related thyromimetic actions, among others also on the status (Jonas improving hepatic lipid metabolism and systemic lipid at the dose effectively heart weight was observed (HPT)axisaccompaniedbyincreased pituitary–thyroid negative feedbackinhibitionofthehypothalamus– But in contrast to these beneficial effects, an undesired of bodyfatmassandimprovementhepaticlipidstatus. in diet-inducedobeseeuthyroidmiceresultedreduction central effectsof3,5-T 2009, (2014) stronglysuggestthat 3,5-T 1995, Goldbergetal.2012Padron2014). ( effects eveninadose-escalatingpilotstudytwovolunteers related diseases,withoutinducingadversethyromimetic et al.1997). et al.2013, (TR) asshownforteleosts(Garcia possibly throughbindingandactivatingtheTHreceptor influences theexpressionofTHresponsivegenes 2012, et al. rats (deLangeetal.2011, obesity orchronichypothyroidisminrodents,mainly inexperimental modelsof acid oxidationwereobserved mitochondrial function,lipidmobilization,andfatty In vivoandinvitro studiesrevealedthat3,5-T attention becauseofitsmarkedeffectsonmetabolism. energy metabolism,3,5-T brown adiposetissue(Lombardi and vascularization of consequences such as innervation signaling havebeendemonstratedwithperipheral mitochondrial respiration.3,5-T hepatic fattyacidoxidationandincreasescellular only scarce dataexistregardingtheeffectsof3,5-T Antonelli DOI: 10.1530/JME-15-0159 http://jme.endocrinology-journals.org Research 3 ) and The resultsindiet-inducedobesemicebyJonas Most ofthebeneficialeffects3,5-T However, apartfromtheseunambiguouseffectson 2 de Langeetal.2011, ) (Lehmphul l -thyroxine (T et al.2011, Cavallo Navarrete-Ramirez ). Such observations ondose- et al.2014).Suchobservations ), which has attracted scientific et al.2014),whichhasattractedscientific et al.2013, Coppola 4 2 ) (Lanni on adrenergic and sympathetic onadrenergicandsympathetic 2 mightalsoexertthyromimetic Grasselli Moreno 2 (2.5 Silvestri et al. 2015). et al.2014)andrats(Ball ). One of these et al.2014).Oneofthese 2 et al.2005, administered mainly to administeredmainlyto j

et al.2010),while and others Printed inGreatBritain 2 2 reportedon Mollica (Horst 2 l l stimulates stimulates -thyronine -thyronine -thyronine Lombardi Mendoza et al. et al. et al. 2 on

in leanaswellobesemice,aneffectof3,5-T of genes encoding xenobiotic-metabolizing enzymes cholesterol andbileacidsynthesis. proteins involvedinthemodulationofendogenous additionally alterstheexpressionofgenesencoding Interestingly, onlyindiet-inducedobesemice3,5-T hepatocytes. (CAR) targetgeneSult1a1inmurineprimary demonstrated fortheconstitutiveandrostanereceptor 3,5-T euthyroid leanandobesemice. .5 or2.5 0.25 daily i.p.eitherwith male micewereinjectedfor 14 days Germany). Health andConsumerProtection(StateofBrandenburg, ethics committeeoftheStateAgencyEnvironment, animalsandwere approvedbyhandling oflaboratory followed established guidelines for the care and 3.8 HFD: 50.2 ± ( housed beforestartingtheexperiment.Ineachgroup HFD were randomlydivided into groups andsingle- 90% lard,D12492,Research Diets)adlibitum. USA) orahigh-fatdiet(HFD;60 44% lard,D12450B,Research Diets,NewBrunswick,NJ, fed eitheranormaldiet(ND:10 Soest, Germany)wasassured.Afterweaning,micewere Free access to water and a standard diet for mice (ssniff, standard conditions(22°C,12 Rehbrücke, Nuthetal,Germany. Micewerekeptunder of the German Institute of Human Nutrition Potsdam- Laboratories andwerefurtherbredattheanimalcareunit C57BL/6J breedingpairswereobtainedfromJackson Animals andtreatment Materials andmethods obvious that3,5-T treated with either 0.25 or 2.5 transcriptomes ofdiet-inducedobesemicethatwere unknown tobeTHresponsive,wecomparedlivertissue 2.5 liver tissue from lean mice treated with the higher dose of we performedacomparativetranscriptomeanalysisof (Sigma-Aldrich) orwiththe solventcontrol(Fig.1A). physiological salinesolution containing0.15%NaOH 3,5-T n Published byBioscientifica Ltd. 2.3 g, 6–8), bw was normally distributed (ND: 29.9 ± = µg/g bw3,5-T To identifynoveltargetgenesof3,5-T The results obtained suggest a specific signatureof We carriedoutan initialexperimentinwhichHFD Twenty weeksafterweaning,malemiceonNDor 2 2 altersmurinegenes -dependent geneexpression.Furthermore,itbecame µg/g bw3,5-T g). Allexperimentalproceduresdescribed 2 tocomparetheeffectsof3,5-T 2 treatmentinducestheexpression Downloaded fromBioscientifica.com at09/24/202103:47:03PM 2 (purity>99.9%)dissolved in µg/g bw 3,5-T h light:12 kJ% fat,55%soybeanoil, kJ% fat;9%soybeanoil, 56 h darknesscycle). : 4 2 . Furthermore, . Furthermore, 2 previously previously 2 between between 2 312 also via freeaccess 2

Journal of Molecular Endocrinology 2 or4 weeks.(B)Groupsofmicefednormaldiet(ND)for Experimental design.(A)Groupsofmicefedhigh-fatdiet(HFD)foreither Figure 1 was performedusing the 2 article. Calculationoftherelative mRNAconcentration section on for RT-qPCR Table areshowninSupplementary 1 , see Information. The characteristics of gene-specific primers Primer-Blast toolofNationalCenterforBiotechnology Jonas and RT-qPCR weremonitoredasdescribed inastudyby concentration aswellperformanceofcDNAsynthesis following themanufacturer’s instructions.RNApurityand hepatocytes wasisolatedusingtheAurumKit(Bio-Rad) and storedat−80°Cuntiluse.Total RNA fromprimary (Invitrogen) followingthemanufacturer’s protocol Total RNAfromliverwasisolatedbyTRIzolreagent quantitative real-time PCR Total RNApreparation, cDNAsynthesis,and snap-frozen inliquidnitrogenandstoredat−80°Cuntiluse. were immediatelyexcised,weighed,andsubsequently before beingkilledunderanesthesiawithisoflurane.Livers lipid metabolism(Jonasetal.2014). publication, withrespecttotheirHPTfeedbackaxisand identical tothosealreadydescribedinaprevious (Fig. 1A and B). These treatment groups were partially phosphoribosyltransferase. encodinghypoxanthine–guanine normalized toHprt Schmittgen 2001).Foldchangesforeach gene were The lowdoseof3,5-T of 3,5-T to studylong-termtreatmentandcomparetheeffects 3,5-T HFD aswellNDmiceweretreatedwithahigherdoseof application. Therefore,inasecondandthirdexperiment, change hepaticgeneexpressioncomparedwithsaline that usedinpreviousratexperiments,didnotsignificantly http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0159 Research After treatment,micewererestrictedfromfoodfor6 2 (2.5 et al.(2014).Theprimersweredesignedwiththe 2 in mice receiving different dietary fatsupply inmicereceivingdifferentdietary µg/g bw)orwiththesolventcontrolfor28 days supplementary data given at the end of this supplementary 2 (0.25 µg/g bw),correspondingto -ΔΔCT j

and 100 n 4 ×10 and et al.2015). collagenase typeII,asdescribedpreviously(Rathmann were isolatedbydigestionoftheliverwithperfusion 20–30 Three-month-old C57BL/6Nmalemiceweighing Primary culture ofmousehepatocytes at 37°C and 5% CO data analysiswasperformedusingRosettaResolver Expression Consolesoftware(Affymetrix).Microarray Quality assessmentofhybridizationswasmonitoredusing (GeneAtlas WT Expression Kit User Manual, Affymetrix). Expression KitandGeneChipWTTerminal LabelingKit according tothemanufacturer’s instructionsusingtheWT Target preparationandarrayhybridizationwereperformed using GeneChip Mouse Gene 1.0 ST Arrays (Affymetrix). 3) weresubjectedtotranscriptomeanalysis samples (n = 2100 Bioanalyzer(AgilentTechnologies), individualRNA total RNApurificationandqualitycontrolusinganAgilent with homogenizationinabeadmilldismembrator. After modified phenol extraction with TRIzol reagent combined Total RNAwasextractedfromfrozenlivertissuesusinga Microarray analysis DMSO, 100 DMEM containing4.5 in FCS-freemediumfor24 For CA, USA). Pathway Analysis (Ingenuity Systems, Inc. Redwood City, commercial systemsbiologyorientedpackage,Ingenuity differentially expressedgenes wascarried out usingthe 0.05), andfold-change≥1.5-fold. (Ratio Builder, P ≤ 0.05), signalcorrectionstatistics rate(P ≤ false discovery criteria: one-wayANOVA withBenjaminiandHochberg the mRNAlevelwereidentifiedusingfollowingcut-off Document1. Supplementary For adetaileddescriptionofmicroarrayanalysisrefer to software system(RosettaBiosoftware,Seattle,WA, USA). M 3,5-T 10 µM penicillin G, 100 1% Berlin, Germany) supplemented with4.5 6-well platesandculturedinDMEM(BiochromGmbH, 3,5-T Published byBioscientifica Ltd. stimulation, primary hepatocytes were starved hepatocyteswerestarved stimulation,primary ex vivo Cell viability was assessed by trypan blue staining blue staining Cell viabilitywasassessedbytrypan In silicopathwayandfunctional analysis of Genes exhibiting significantly different expression on glutamine, 10%fetalcalfserum(FCS),100 2 altersmurinegenes g were anesthesized and primary hepatocytes hepatocytes g wereanesthesizedandprimary nM Na 5 cells/well were seeded on collagen-coated cells/wellwereseededoncollagen-coated 2 IU/mL penicillinG,100 inDMSO/30 2 IU/mL streptomycin, and 100 SeO 2 . After 3 3 withorwithout3,5-T g/L glucose, 1% glutamine, 0.5% g/L glucose,1%glutamine,0.5% Downloaded fromBioscientifica.com at09/24/202103:47:03PM mM HCl)for24 h and afterward incubated in incubatedin h andafterward h, the medium was renewed. h, the medium was renewed. IU/mL streptomycin, IU/mL streptomycin, 56 : 4 h. g/L glucose, g/L glucose, nM Na 2 (10 nM to nM to IU/mL IU/mL 313 2 SeO via freeaccess 3

Journal of Molecular Endocrinology 3,5-T significantly increased(bold)ordecreased (italic)mRNAlevelsfollowing Venn diagramillustratingtheoverlaps betweenhepaticgenesexhibiting Figure 2 by microarrayapproach. increased and30withdecreasedexpression(Fig.2). in HFDmice,withapartialoverlapof20genes 4-week treatment,thiswasonlythecasefor106genes genes exhibiteddifferenthepatictranscriptlevelsafterthe ). Interestingly, whileinNDanimals,221 (Fig. 2 4 weeks weekswerealsochangedafter already affectedafter2 treatment. Approximately, halfofthosetranscripts differentially expressedgenescomparedwiththe2-week (2.5 0.05. as P ≤ between treatmentgroupandcontrolwasdefined (version 5,GraphPadSoftware).Statisticalsignificance were done by GraphPad Prism and QuickCalcs software statistical analysesandtheidentificationofoutliers U test was used to compare between two groups. All Wallis test (Dunn’s Mann–Whitney post-test) otherwise groups andcontrolweredeterminedbyKruskal– Statistical differences between more than two treatment Impact of3,5-T Results Results generatedbyRT-qPCR areexpressedasmean ± Statistical analyses marked effectsasillustratedbyVenn diagram(Fig.2). hepatic transcriptome,whilea10-foldhigherdosecaused 0.25 time intheliverofHFDrats(deLangeetal.2011),thatis, that was described to be effective after the same treatment ramn fHDmc o 4 dayswiththe3,5-T treatment ofHFDmicefor14 Based ontheresultsoflivertranscriptomeanalysis, obese mice DOI: 10.1530/JME-15-0159 http://jme.endocrinology-journals.org Research 2 As expected,treatmentofHFDmicewith3,5-T µg/g bw) for 4 weeks resulted in ahigher number of µg/g bw) for4 weeks µg/g bw3,5-T treatmentinmicefedhigh-fat(HFD) ornormaldiet(ND)analyzed 2 onthelivertranscriptomeofleanand 2 , did not significantly change the murine , didnotsignificantlychangethemurine j

and others Printed inGreatBritain 2 dose dose s . e . m 2 .

3,5-T 3,5-T by RT-qPCR analyses(Fig.3). binding globulin.Theseexpressionprofileswerevalidated inhibitor, cladeA,member7,alsoknownasthyroxine- decreased thoseofSerpina7encodingserpinpeptidase responsive genes such as significantly increased the transcript levels of TH functions (Table 1).Moreover, 3,5-T ( those oftheCyp1,−2,−4,−17,−39,−46,and−51families coding forcytochromeP450enzymes(P450),especially family, polypeptide B37.Interestingly, 3,5-T family 5A,member1andUDP-glucuronosyltransferase 2 Ugt2b5, encodingtheconjugationenzymessulfotransferase the mRNAlevelsofSult5a1andUgt2b37,alsoknownas that treatmentofNDandHFDmicesignificantlyincreased ( of structurally diverse drugs (Staudinger catalyzing neutrallipidhydrolysisandbiotransformation carboxylesterases thatcompriseafamilyofproteins the transcriptlevelsofCes1 3 ND micealsoincreasedtheexpression ofPapss2encoding source insulfotransferasereactions.Thetranscriptome which catalyzesthesynthesis ofPAPS, theactivated sulfate indicated that3,5-T differentially expressedinHFDmice(Fig.4). and 2a,Sult1b1,Slc13a3,Slc39a4,Gpx6)wereexclusively (Cyp1a2, Cyp39a1, Cyp46a1, Cyp51, Cyp2d9, Ces1(f,g) the same profile in ND mice. However, 12 genes significant expressionchangesinHFDmiceexhibited weeks. Most DME-related genes with mice treated for 4 forND drug-metabolizing enzymes (DME)wasobserved Tableand zymosterolbiosynthesis(Supplementary 2). classified pathwaysinthecontextoffarnesyl,cholesterol, treatment inHFDmicespecificallyidentifiedgeneontology- weeksof analysis ofgenesdifferentiallyexpressedafter4 detoxification’ and‘nicotinedegradation’.Interestingly, the including theIPA pathways‘glutathione-mediated genes relatedtoxenobioticmetabolismanddetoxification, using ingenuitypathwayanalysis(IPA) mainlycomprised The significantlyassociatedcanonicalpathwaysidentified enzymes andtransporters Treatment ofHFDaswellNDmicewith3,5-T 3,5-T ), which are known to fulfill several physiological Fig. 4),whichareknowntofulfillseveralphysiological ). A more detailed look at Phase II enzymes revealed Fig. 4).AmoredetailedlookatPhaseIIenzymesrevealed ′ Published byBioscientifica Ltd. -phosphoadenosine 5′ With regardtoPhaseIenzymes,thetranscriptome data The greatestimpactonhepaticgenesexpressing 2 2 2 altersmurinegenes regulates geneexpression ofdrug-metabolizing exertseffects onTHresponsive genes 2 affects the expression of several genes affectstheexpressionofseveralgenes -phosphosulfate (PAPS) synthetase, Downloaded fromBioscientifica.com at09/24/202103:47:03PM Dio1 encoding deiodinase 1, and ( Ces1g) encoding , 2,and4encoding 56 2 also modulated alsomodulated : 4 2 treatment of treatmentof et al. 2010) 314 via freeaccess 2

Journal of Molecular Endocrinology data alsodemonstratedthat3,5-T of 3,5-T CAR asapotentialcandidate foranintermediatefactor 2 weeks andNDmicetreatedfor4 weeks (Fig.5A,B,andC). expression profilesobtainedforHFDmicetreated results obtained correlated well with the microarray-based II enzymesorPhaseIIItransportersviaRT-qPCR. The analyzed representativegenesencodingPhaseIand resistance-associated proteinMRP3(Fig.4). forAbcc3encodingthemultidrug HFD micewasobserved of geneexpressionafter3,5-T and HFDmice.Ontheotherhand,strongestdecrease anion transporter2(OAT2) wasstronglyinducedinND differentially affected:Slc22a7whichcodesfortheorganic genes (e.g.Gsta2andGsta4)(Fig.4). expression ofseveralglutathioneS-transferaseencoding metabolism, as treatment of ND and HFD mice reduced the encoded byNr1i3asatranscriptionalregulator ofDME Literature todatesuggests amajorrolefortheCAR http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0159 Research To confirmandvalidatethetranscriptome data,we Phase III(efflux)transporterencodinggeneswere 2 actionondrug-metabolizing enzymes 2 treatmentinbothNDand j

lietzow © 2016SocietyforEndocrinology 2 affects glutathione affectsglutathione andothers Printed inGreatBritain expressed after3,5-T data indicatedthatseveralofthosegenesdifferentially 3,5-T ( (Chen and ATP-binding cassette/solutecarrierfamilytransporters ( ( synthesis inrats,namely of Idi1,Sqle,Cyp51,Sc4mol known tobeinvolvedin farnesylandzymosterol the mRNA levels of several genes encoding enzymes primary hepatocytesdemonstratedthat3,5-T primary CAR (Table 2). Treatment ofHFDmicewith 3,5-T cholesterol,farnesyl, andbileacidsynthesis increased by3,5-T potential CARtarget,wasconcentration-dependently for 24 xenobiotic metabolism in murine hepatocytes exposed able to regulate the transcription of genes involved in 3,5-T Fig. 6). Nemoto Msmo1) Published byBioscientifica Ltd. The resultsobtainedfromtheinvitro modelof 2 2 altersmurinegenes enhancestheexpression ofgenesinvolvedin , a h. ThetranscriptconcentrationofSult1a1, et al.2012, , Nsdhl,andHsd17b7( et al.2013).Furthermore,enhanced Cyp39a1 0.001. 0.01, ***P < 0.05, **P < *P < different ascomparedwith thesalinegroup: comparisons ofRT-qPCR data.Significantly Mann–Whitney Utestwasusedforstatistical Expression ofTHresponsivegenesafter3,5-T Figure 3 (ND) treatedwith3,5-T of micefedhigh-fatdiet(HFD)ornormal RT-qPCR usingRNAsamplespreparedfromlivers thyroxine-binding globulin(B)wereassayedby deiodinase 1(A)andSerpina7encoding treatment: hepaticmRNAlevelsofDio1encoding expression inliverof3,5-T Corresponding significantfoldchangesofgene graph. Dataareexpressedasmean ± transcriptome analysiswereaddedtothe mice determinedbymicroarray-based 2 Xu inarangefrom10 2 treatmentarepotentialtargetsof et al.2005).Thetranscriptome Downloaded fromBioscientifica.com at09/24/202103:47:03PM Supplementary Table 2) Supplementary 2 for4 weeks increased 2 orsalineascontrol. 56 2 -treated HFDorND : 4 nM to10 s . e 2 . m isalso . 315 2 µM via freeaccess

Journal of Molecular Endocrinology given inblack,thelowestwhite. 3 pergroup).Eachcellintheheatmapindicatesexpressionlevelof thecorrespondinggeneforoneanimal.Thehighestexpressionlevelsare (n = by 3,5-T suggesting apotentialmodulation ofbileacidsynthesis inHFDbutnotNDmice,exclusively observed expression andreduced Cyp46a1 expressionwas The influenceof3,5-T Figure 4 0.05) comparedwiththecorrespondingcontrolgroups(NDorHFDcontrol) after3,5-T significantly differentially expressedgenes(*P < drug-metabolizing enzymes(DME)includingPhaseIandII aswellPhaseIIItransporters.Theheatmapontheright-handsideshows treatment for2and4 weeksinmicefedhigh-fatdiet(HFD)or inmicefednormaldiet(ND)areshown.Geneproductswereclassified DOI: 10.1530/JME-15-0159 http://jme.endocrinology-journals.org Research 2 underHFDconditions(Table 1 ). 2 treatmentontheexpressionofgenesencodingdrug-metabolizingenzymes (DME).Genesdifferentially expressedafter3,5-T j

and others Printed inGreatBritain Figure 7 shows two keyenzymesofsteroid synthesis(Fig. 7AandB). hydroxysteroid (17-)dehydrogenase 7,respectively, Sqle andHsd17b7encodingsqualeneepoxidase and the validationofmicroarray dataviaRT-qPCR for 3,5-T Published byBioscientifica Ltd. 2 altersmurinegenes Downloaded fromBioscientifica.com at09/24/202103:47:03PM 56 : 4 2 treatment 316 2 via freeaccess Journal of Molecular Endocrinology Cyp51 Cyp46a Cyp39a Cyp17a Cyp4a Cyp2u Cyp2d Cyp2c Cyp2b profiles ofmostgenesmodulatedby3,5-T analysis of3,5-T hepatic geneexpression.Theresultsofthecomparative (2.5 important rolefor3,5-T and exertdifferentimportant physiologicalfunctions comprise heme-containing microsomal mono-oxygenases encoding cytochrome P450 enzymes (P450), which or high-fatdiet. This studydemonstratesthattreatmentwith3,5-T Discussion Cyp1a P450 family Table 1 3,5-T to protecttheorganism(Xuetal.2005). metabolizes andeliminatesxenobioticsundendobiotics transporters, therebyrepresentingadefensesystemthat Phase I and Phase II enzymes as well as Phase III (efflux) organ expressing genes coding for DME including primary hormone aswelldrugmetabolism.Theliveristhe genes encoding enzymes involved in thyroid and steroid ( processing inanimalmodelsonHFDorcaloricrestriction hepatic expressionprofilesofgenesinvolvedinxenobiotic expressed genes. Recent studies have revealed different expression patternofDMEgenes,butfewerdifferentially mice willnotactadditivelyor synergisticallywithHFD. with thosealteredbyHFDandadministrationof3,5-T of exogenously added3,5-T Ghose factors andbiotransformationenzymes(Pascussietal.2003 , inflammation andcrosstalkbetweenproinflammatory liver duetopronouncedoxidativestressand/orhepatic of genesencodingnuclearreceptorsandDMEinfatty might be explained by an already modified expression Ghose http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0159 Research Compared with ND mice, the hepatic transcriptome of Compared withNDmice,thehepatictranscriptome of 3,5-T µg/g bw)inNDandHFDmicestronglyaffectsthe 2 -treated HFD mice demonstrated a specific and broad -treated HFDmicedemonstratedaspecificandbroad ), which might also affect the elimination et al.2011),whichmightalsoaffecttheelimination et al.2011, Putative functionsofproteinsencodedbyP450genesdifferentially expressedafter3,5-T 2 alteredthetranscriptconcentration ofgenes Isoforms modulatedby 29, 37,38,39,44 2 3,5-T -treated vssaline-treatedmicesuggestan ). Such differences Fu &Klaassen2014).Suchdifferences 2 12a, b treatment 9, 13 1 1 1 1 9 2 2 inmodulatingtheexpressionof 2 . Alternatively, expression j

lietzow © 2016SocietyforEndocrinology Bile acidsynthesis Cholesterol andsteroidbiosynthesis Bile acidsynthesis Steroid biosynthesis ω-hydroxylation offattyacidsandrelatedcompounds ω-hydroxylation offattyacids Steroid biosynthesis Metabolism ofsteroidsandacute-phaseresponse Oxidation ofxenobiotics Oxidation ofxenobiotics Putative hepaticfunction andothers 2 Printed inGreatBritain might overlap mightoverlap 2 to to 2

O’Leary function oxidasesystem(Ram&Waxman 1991, regulate numerousenzymesassociatedwiththemixed ). PreviousstudieshaveshownthatclassicalTH (Table 1 including steroid, lipid, and xenobiotic metabolism known forT control ofT and enterohepaticrecyclingaswelllocalprereceptor physiological importanceiniodothyronineconjugation studies haveshownthatSultandUgtisoformsareof sulfotransferase andUDP-glucuronosyltransferase.Previous enzymes includingSult5a1andUgt2b37,whichencode increased expressionofcertaingenesspecifyingPhaseII findings suggest a potential role of 3,5-T (Beckett content andreducedenzyme activitiesofGSTandGPx hyperthyroid ratsshowedadecline inhepaticglutathione in ND as well as HFD mice (Fig. 4). Previous studies on &Hines2002)weredownregulated glutathione (McCarver the conjugationofawidevarietyxenobioticswith Moreover, various Gst-encodedisoenzymescatalyzing is involvedinthedetoxificationofhydrogenperoxide. expression ofGpx6encodingglutathioneperoxidase6that resulting inasubsequentinactivationof3,5-T increased theexpressionofSult1a1(Fig.6),potentially (Visser 1996, hepatocytes that3,5-T (Silvestri differences betweenNDandHFDratsweredetected such effectswerereportedfor3,5-T with 0.25 of SULT1A1 in the liver of HFD rats treated over 30 days nochangeintheproteinlevels Silvestri etal.observed 3,5-T Published byBioscientifica Ltd. In addition,thetranscriptomeanalysesrevealed Treatment of HFD mice with 3,5-T 2 altersmurinegenes et al. 1997, et al.1988; µg/g bw3,5-T et al.2010).We demonstratedinprimary 3 3 (Andersonetal.1995). availabilityforTRbindingandactivation Wu

et al.2005, Ortiz de Montellano 2005), while no Chattopadhyay Downloaded fromBioscientifica.com at09/24/202103:47:03PM 2 comparedwithsham,though (Lund et al.2003,Lorbek (Debeljak et al.2003) (Li-Hawkins et al.2000) (Liu et al.2005) (Sue Masters&Marohnic2006) (Chuang et al.2004) (Jarukamjorn et al.2006) (Corton et al.1998) (Wang &Negishi2003) (Tolson &Wang 2010) Reference 2 concentration-dependently 2012) 2 treatmentinmicefednormal Runge-Morris 2 . 56 et al.2007).Our : 4 2 reducedthe 2 in altering et al.2013). 2 asitis 1992, 317 via freeaccess Journal of Molecular Endocrinology after 3,5-T the strongestalterationsintransportergeneexpression metabolites from hepatocytes to blood or bile. We detected canalicular membranestransferringbileacids,drugs,and hyperthyroid statusoftheliver. and turnoverofglutathionepossiblybymimicking a hepatic cellular redox status and influencing synthesis blood, whileOAT2 isinvolved inthetransportoforganic xenobiotics, andglucuronide metabolitesintosinusoidal is responsibleforthebasolateral exportofbileacids, the organic anion transporter OAT2, respectively. MRP3 the multidrug resistance-associated protein MRP3 and DOI: 10.1530/JME-15-0159 http://jme.endocrinology-journals.org Research Efflux transporters are localized to basolateral and Efflux transportersarelocalizedtobasolateraland 2 treatment for Abcc3 and j

encoding Slc22a7 encoding and others Printed inGreatBritain , both encoding transcript levelsofSlc25a30and Slc25a45, bothencoding 3,5-T clearance ofthecorresponding metabolites.Interestingly, expression ofeffluxtransporter genesresultinginahigher anions (Maher 2008, is alteredbyage,sex,anddietcomposition(Cheng Burckhardt 2012).HepaticexpressionofAbcc3andSlc22a7 was increased.We speculatethat3,5-T levels weresignificantlydecreased,whileAbcc3expression pattern of in liverofhypothyroidanimalsareversedexpression 2014). Besidethis,Dong 3,5-T Published byBioscientifica Ltd. 2 2 altersmurinegenes treatment of ND as well as HFD mice enhanced the treatmentofNDaswellHFD miceenhancedthe Ghose Slc22a7 and et al. 2011, et al.2005, 0.001. 0.01, ***P < 0.05, **P < *P < different ascomparedwith thesalinegroup: comparisons ofRT-qPCR data.Significantly Mann–Whitney Utestwasusedforstatistical metabolism after3,5-T proteins involvedinhepaticxenobiotic Differential expressionof genesencoding Figure 5 3,5-T diet (HFD)orfednormal(ND)treatedwith RNA preparedfromliversofmicefedhigh-fat transporters (C)wereassayedbyRT-qPCR using I enzymes(A),PhaseII(B),andIII the hepaticmRNAlevelsofgenesencodingPhase carrier family. Dataareexpressedasmean ± Abc: ATP-binding cassettetransporter, Slc:solute glucuronosyltransferase, Sult:sulfotransferase, cytochrome P450,Ugt:UDP- analysis wereaddedtothegraph.Cyp: determined bymicroarray-basedtranscriptome liver of3,5-T significant foldchangesofgeneexpressionin 2 orsalinefor4 weeks.Corresponding Downloaded fromBioscientifica.com at09/24/202103:47:03PM Abcc3, where Fu Zamek-Gliszczynski 2 demonstrated et al.(2007)demonstrated -treated HFDorNDmice et al. 2012, 2 treatment.Inparticular, 56 transcript Slc22a7 transcript : 2 4 modulates the modulatesthe Fu & Klaassen Fu & Klaassen et al.2006, 318 et al. s . e . m via freeaccess .

Journal of Molecular Endocrinology 2009; which isinlinewithpronouncedeffectsof3,5-T mitochondrial carrierproteins(Haitina genes encoding DMEandmodulated by3,5-T et al.2005, different DMEencodinggenessuchasSult5a1orMrp3( cross talkbetweenhepatictranscriptionfactorsregulating 2014). Previousstudiesdemonstratedastrongoverlapand 2005, enhanced THclearance(Maglich mechanisms through induced liver DME synthesis and T it isimportanttoconsiderthatactivationofCARby3,5- are knowntoexertstronginfluenceonTHmetabolism, the promoter region (Breuker responsive geneThrspthroughbindingtoTREsequencesof addition, itwasshownthatCARitselfcanregulatetheTH the PBREMregion(Ooe genes possiblybybindingoftheactivatedTHreceptorto that THmayregulatetheexpressionofspecificCARtarget smallhepatocyteshaveshown cell lineandcryopreserved linked tohepaticlipidandglucosemetabolism(Wada 2007). Beyondthis,studieselucidatedthatCARisalso promoter regions of genes encoding DME (Timsit & Negishi binding toxenobioticresponsiveelements(PBREM)inthe forming heterodimerswithretinoidXreceptorand xenobiotic sensorthatmodulatesgeneexpressionby to beregulatedbyCAR.CARisanuclearreceptorand transporter inliverisstillunknown(Paquette though thephysiologicalfunctionofencoded responsive elementinthepromoterregionofSlc25a45, et al.1998).Additionally, Paquette mitochondrial compartment(Arnold factors (Ahr, Pxr, andNrf2). mechanism possiblyinvolving furthertranscription additionally controlledvia an indirectorindependent Abcc3 Slc22a7 Gsta2 Ugt2b37 Sult5a1 Ces2a Cyp39a1 Cyp2c29 Cyp2b9 Cyp1a2 Annotated genes Table 2 this study. 2 http://jme.endocrinology-journals.org DOI: 10.1530/JME-15-0159 Research might alter hepatic TH homeostasis via secondary mightalterhepaticTHhomeostasisviasecondary Many oftheDMEgenesaffectedby3,5-T Radovic Xiao Known CARtargetgenesidentifiedtobedifferentially expressedafter3,5-T et al.2013). et al.2010, ). Therefore, Aleksunes &Klaassen2012 ). Therefore, Modulation by3,5-T down up down up up down up down up down In vitro datafromthehepaticHuh7 Roques et al.2009, et al. 2010 ). Since xenobiotics et al. 2013, et al.2004, j

lietzow © 2016SocietyforEndocrinology reported a TH et al.reportedaTH et al.1998; Bing andothers 2 Printed inGreatBritain ). In al.2014).In et Schraplau Qatanani ), et al.2006), et al. 2011). 2 are known areknown Lombardi 2 may be may be Drug-metabolizing enzymes Phase IIItransporter Phase IIItransporter Phase IIenzyme Phase IIenzyme Phase IIenzyme Phase Ienzyme Phase Ienzyme Phase Ienzyme Phase Ienzyme Phase Ienzyme 2 on the onthe et al. et al. et al. Xu

might beexplainedbypotentialbindingof3,5-T modulated by3,5-T involved infarnesyl,cholesterol,andbileacidsynthesisare Navarrete-Ramirez etal.2014). 3,5-T precursor ofcholesterol(Nemoto for enzymesinvolvedinthebiosynthesisofzymosterol,a but notinNDmice.Inducedgeneexpressionwasobserved (Hayashi of genes (Serpina7, Dio1) known to be TH responsive minor similaritieswithregard totheexpressionprofiles mitochondrial andfattyacid metabolism;however, only 2009). Regardingthepotentialeffectsof3,5-T Cyp39a1) involvedinbileacidsynthesis(Beilke demonstrated that3,5-T liver cholesterol levels (Rezen bile acidsynthesesaswellreducedtotalserumand induction ofhepaticgenesinvolvedincholesteroland with theCARactivatorTCPOBOPexhibitedacomparable activation, itisinterestingtonotethatHFDmicetreated ability of3,5-T be inlinewithpublisheddatashowingtheantisteatotic for cholesteroleliminationfromthebody. Thiswould cholesterol, themainroute increased secretionofbiliary stimulate hepaticcholesterolsynthesisaccompaniedby were treatedwith3,5-T treated with3,5-T analysis oflivertissuefromeuthyroidleanandobesemice 2009, Grassellietal.2012). and in tilapia (Koerner from recentstudieson3,5-T target genes.Thiswouldbeinagreementwithconclusions hepatic TH receptor novel 3,5-T 3,5-T Published byBioscientifica Ltd. Our transcriptome analysis reveals that several genes Aside fromDME,thetranscriptomeanalysis To ourknowledge,thisisthefirst transcriptome 2 2 altersmurinegenes modulatedthetranscriptlevelsofgenes(Cyp46a1, et al. 1993, 2 analogue,showedstrongeffects onhepatic 2 applicationinHFDrats(Mollica 2 2 treatmentbytranscriptomeanalysisin . ProteomicapproachesinHFD ratsthat 2 treatment for 4 weeks inHFDmice, treatmentfor4 weeks Reference (Staudinger et al.2013) (Jigorel et al.2006) (Roques et al.2013) (Richardson et al.2008) (Aleksunes &Klaassen2012) (Zhang et al.2012,Jones2013) (Beilke et al.2009) (Jackson et al.2004) (Rivera-Rivera et al.2003) (Tolson &Wang 2010) β1 mediating the transcription of Zhang 2 2 et al. 1975, (0.25 isalsomodulatingtheexpression Downloaded fromBioscientifica.com at09/24/202103:47:03PM 2 actioninrathepaticnuclei et al. 1998). These findings µg/g bw)orTRC150094,a et al. 2009). 3,5-T et al.2013).Inaddition, Mendoza 56 : 4 et al. 2013, 2 onCAR 2 2 might tothe 319 et al. et al. via freeaccess Journal of Molecular Endocrinology such as the observed inductionofdrug-metabolizing such astheobserved are possiblyaccompanied by toxichepaticsideeffects, be explained in part by short- vs long-term 3,5-T in vivoorbyusingbiomaterialsoriginatingfrom3,5-T murine effectsof3,5-T Detection of3,5-T Figure 6 Silvestri etal.2012). (Silvestri reported inthisstudywereobserved classical T metabolites thatmayaffectgeneexpressionevenviathe generates decarboxylatedand/ordeiodinatedfurtherTH et al.2015).ItmightbethatTHmetabolisminliver exhibit pronounceddeiodinationcapacity(Rathmann locally activehepaticTHmetabolites.Hepatocytes T of thyroid-stimulatinghormonebetaaswellserum transcriptaffected the HPT axis by lowering pituitary expression pattern.However, thesamedosealready dose (0.25 marked transcriptionaleffectsinliver. Atenfoldlower resulting froma3,5-T might indirectlyaffectthehepaticgeneexpression 0.001. ***P < Significantly different as comparedwiththecontrolgroup(c): Kruskal–Wallis test(Dunn’s post-test)wasusedforstatisticalcomparisons. or 10 µM3,5-T cultured for24 hintheabsence(c)orpresenceof10 nM;100 nM,1 µM, expression byRT-qPCR usingRNApreparedfromprimaryhepatocytes to studytheeffectsof3,5-T treatment ofhypothyroidvseuthyroidanimalmodels uptake, metabolism,action,andelimination. due tospecies with theHPTaxis(Goglia2005, as changesinmetabolicparameterswithoutinterfering exhibited alterationsinhepaticgeneexpressionaswell are surprisingsincestudiesinratsusingthesamedose 3 DOI: 10.1530/JME-15-0159 http://jme.endocrinology-journals.org Research concentration(Jonas Therefore, itshouldbetaken intoaccountthat Treatment ofmicewith2.5 The differencesbetweenpublished3,5-T 3 receptor. µg/g bw)didnotchangethehepaticgene 2 . Dataareexpressedasmean ± 2 concentration-dependentdifferential Sult1a1 differences in hepatic and pituitary 3,5-T differences inhepaticandpituitary 2 -induced alteredbalanceoffurther 2 onenergyandTHmetabolism et al.2014).Thesefindings 2

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3,5-T 0.001. 0.01, ***P < 0.05, **P < compared withthesalinegroup:*P < used forstatisticalcomparisonsofRT-qPCR data.Significantlydifferent as enzymes. We cannotexcludethattheeffectsof3,5-T Expression ofgenesinvolvedinsteroidbiosynthesisafter3,5-T Figure 7 01GI0922). and theGermanMinistryofEducation andResearch(BMBF, DZD, grant program ThyroidTransAct KO922/17-1to JK,WA1328/5-1 toGH andUV, This workwassupportedbyDFGgrants KFO218/2TP3;theDFGpriority Funding perceived asprejudicingtheimpartialityofresearchreported. The authorsdeclarethatthereisnoconflictofinterestcould be Declaration ofinterest JME-15-0159. This islinkedtotheonlineversionofpaperathttp://dx.doi.org/10.1530/ Supplementary data in liver. Therefore,thepotentialuseof3,5-T responsive genes and activation of xenobiotic metabolism hepatic effects demonstrated by altered expression of TH described clinicalassociationsbetweenhuman3,5-T scene, shouldbeviewedwithcaution.Anyhow, recently commentaries of the internet body-building and wellness antiobesity drug,asitissuggestedinseveralpublicationsor analysis. Dataareexpressedasmean ± (B) were determinedbyqPCRandmicroarraybasedtranscriptome (A) and Hsd17b7encodinghydroxysteroid(17-β)dehydrogenase7 treatment: hepaticmRNAlevelsofSqleencodingsqualeneepoxidase agt genes. target with theidentificationofthese andfurtherhepatic3,5-T et al. 2015) might find prospectivemolecular explanations such asTSH,fastingglucose,andleptin(Pietzner serum concentration and serum metabolic parameters include modulation of classical T not beencharacterizedindetailyet.Suchactionmight are mediatedbyvariousdifferentreceptorswhichhave 3,5-T Published byBioscientifica Ltd. Overall, administrationof3,5-T 2 2 altersmurinegenes concentrations. Downloaded fromBioscientifica.com at09/24/202103:47:03PM s . e . m 3 . Mann–WhitneyUtestwas receptors by high local 2 might exert adverse mightexertadverse 56 : 4 2

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Cheng Q, Aleksunes LM, Manautou JE, Cherrington NJ, Scheffer GL, Scheffer GL, Cherrington NJ, Manautou JE, Aleksunes LM, Cheng Q, Chuang SS, Helvig C, Taimi M, Ramshaw HA, Collop AH, Amad M, Amad M, Collop AH, Taimi M, Ramshaw HA, Helvig C, Chuang SS, Fu ZD, Csanaky IL & Klaassen CD 2012EffectsofagingonmRNA &Klaassen CD Csanaky IL Fu ZD, &Wade MG Douglas GR Yauk CL, Lee A, Williams A, Dong H, 2003Manyfacetsofmammalian &Rozman D Fink M Debeljak N, De de LangeP,Cioffi F, Silvestri E, Lombardi A, Moreno M, Senese R, Mode A Anderson SP, Cattley RC, Brown S, Bocos C, Fan LQ, Corton JC, 2014 &Goglia F Cioffi F, Silvestri E Moreno M, Glinni D, Coppola M, Goglia F 2005Biologicaleffectsof3,5-diiodothyronine(T(2)). Goglia F Shah P, Carpenter KC, Gandhi A, Strohacker K, Omoluabi O, Ghose R, 2007 &Orozco A Valverde RC Nunez J, Lopez-Bojorquez L, Garcia GC, 2014Short-termcalorierestrictionfeminizesthe &Klaassen CD Fu ZD Haitina T, Lindblom J, Renstrom T & Fredriksson R 2006Fourteennovel &Fredriksson R Renstrom T Haitina T, Lindblom J, DeMatteisR,Goglia F, Lanni A, Canesi L, Demori I, Voci A, Grasselli E, & Scanlan TS Yu S, Nagareddy PR, Huggins LA, Huang LS, Goldberg IJ, 2014Theeffectsof3,5-diiodothyronineonenergybalance. Goglia F 3,5-T Published byBioscientifica Ltd. Molecular Pharmacology transporter expressioninamousemodelofdiabetesandobesity. 2008Drug-metabolizingenzymeand Yamasaki H &Slitt AL Pharmacology drug-metabolizing enzymesanddrugtransporters.Biochemical Biological Chemistry of omega- and(omega-1)-hydroxylationoffattyacids.Journal human thymus-andbrain-specificcytochromeP450,catalyzes 2004CYP2U1,anovel &Korczak B Jones G Petkovich M, White JA, 1216–1225. MetabolismandDisposition male andfemalemice.Drug profiles fordrug-metabolizingenzymesandtransportersinliversof en.2007-0452) responsive element.Endocrinology characterizationofanovelnegativethyroid- juvenile mice: geneexpressionchangesinhypothyroid 2007 Hepatic Biophysics cytochrome P450familyCYP51.Archives and ofBiochemistry lanosterol 14alpha-demethylasefromtheevolutionarilyconserved thyronine inrats.Diabetes prevention ofdiet-inducedinsulinresistanceby3,5-diiodo-L- Mollica MP,Matteis R,Lionetti L, Goglia F, etal.2011Nonthyrotoxic 463–473. by peroxisomeproliferatorchemicals.MolecularPharmacology family membersandpositiveacute-phaseresponsegeneexpression 1998Down-regulationofcytochromeP4502C & Gustafsson JA World ofHepatology Journal Thyroid hormoneanaloguesandderivatives:actionsinfattyliver. Biochemistry Sciences expression ofdrugmetabolizingenzymesandtransporters.Life 2011Roleofhigh-fatdietinregulation gene &Guo T McFarlin B R877–R883. Physiology: Regulatory, IntegrativeandComparativePhysiology of hormone receptorbeta1inthekillifish.AmericanJournal type 2iodothyroninedeiodinase,growthhormone,andthyroid 3,5-Diiodothyronine invivomaintainseuthyroidalexpressionof (doi:10.1016/j.taap.2013.11.003) livers ofmice.Toxicology andAppliedPharmacology mRNA profilesofdrugmetabolizingenzymesandtransportersin 779–790. system.Genomics widely expressedinthecentralnervous human membersofmitochondrial solute carrierfamily25(SLC25) ofEndocrinology Journal expression ofgeneslipidmetabolism inaratmodeloffattyliver. 20123,5-Diiodo-L-thyroninemodulatesthe &Vergani L Gallo G (doi:10.1210/en.2012-1572) LDL receptor-mediatedpathway. Endocrinology 2012Thyroidhormonereducescholesterolviaanon- Ehrenkranz JR Frontiers inPhysiology 2 altersmurinegenes 89 57–64.(doi:10.1016/j.lfs.2011.05.005) (doi:10.1016/j.ygeno.2006.06.016) 409 159–171.(doi:10.1016/s0003-9861(02)00418-6) (doi:10.1124/dmd.111.044461) (doi:10.1152/ajpregu.00101.2007) 70 164–172.(doi:10.1007/s10541-005-0097-0) 83 1112–1126.(doi:10.1016/j.bcp.2012.01.030) 279 6305–6314.(doi:10.1074/jbc.M311830200) 5 528.(doi:10.3389/fphys.2014.00528) 5 77–91.(doi:10.1021/mp700114j) 212 149–158.(doi:10.1530/JOE-11-0288) 60 2730–2739.(doi:10.2337/db11-0207) 6 114–129.(doi:10.4254/wjh.v6.i3.114) Downloaded fromBioscientifica.com at09/24/202103:47:03PM 148 3932–3940.(doi:10.1210/ 56 153 5143–5149. : 4 274 137–146. 40 88

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