Modulation of thyroid hormone receptors, TRα and TRβ, by using different doses of triiodothyronine (T3) at different times

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1 originl rticle Modultion of thyroid hormone receptors, TRα nd TRβ, y using different doses of triiodothyronine (T3) t different times Modulção dos receptores de hormônio tireoidino, TRα e TRβ, utilizndo diferentes doses de triiodotironin (T3) em diferentes tempos Mirine de Oliveir, Rent de Azevedo Melo Luvizotto, Regine Mrques stro Olimpio, Mri Teres de Siio, rolin Biz Rodrigues Silv, Sndro José onde, rlos Roerto Pdovni 3, éli Regin Nogueir Deprtment of Internl linic, Botuctu Medicine School, São Pulo Stte University (Unesp), Botuctu, SP, Brzil Deprtment of Physiology, São Pulo Federl University (Unifesp), São Pulo, SP, Brzil 3 Deprtment of Biosttistics, Biosciences Institute, Unesp, Botuctu, SP, Brzil orrespondence to: Mirine de Oliveir Universidde Estdul Pulist, Fculdde de Medicin Distrito de Ruião Jr s/n 868- Botuctu, SP, Brzil mirine.deoliveir@yhoo.com.r Received on Dec/7/ Accepted on Jn//3 ABSTRAT Ojective: To exmine the effect of different doses of triiodothyronine (T3) on mrna levels of thyroid hormone receptors, TRα nd TRβ, t different times. Mterils nd methods: 3T3-L dipocytes were incuted with T3 (physiologicl dose: F; suprphysiologicl doses: SI or SII), or without T3 (control, ) for.5,, 6, or 4h. TRα nd TRβ mrna ws detected using rel-time polymerse chin rection. Results: F incresed TRβ mrna levels t.5h. After h, TRα levels incresed with F nd SI nd TRβ levels decresed with SII compred with, F, nd SI. After 6h, oth genes were suppressed t ll concentrtions. In 4h, TRα nd TRβ levels were similr to those of group. onclusions: T3 ction with F egn t h for TRα nd t.5h for TRβ. These results suggest the importnce of knowing the times nd doses tht ctivte T3 receptors in dipocytes. Arq Brs Endocrinol Met. 3;57(5): Keywords TRα; TRβ; triiodothyronine; dipocytes RESUMO Ojetivo: Exminr o efeito de diferentes doses de triiodotironin (T3) sore expressão gênic dos receptores TRα e TRβ em diferentes tempos. Mteriis e métodos: Adipócitos, 3T3-L, form incudos com T3 ns doses fisiológic (F, nm) e suprfisiológics (SI, nm ou SII, nm) ou veículo (controle, ) durnte,5,, 6 ou 4h. mrna dos TRs form detectdos utilizndo PR em tempo rel. Resultdos: Níveis de TRβ umentrm em F em,5h. Após h, níveis de TRα umentrm em F e SI comprdo o, enqunto TRβ diminuiu no SII comprdo com, F, e SI. Após 6h, mos os genes form suprimidos em tods concentrções. Em 4h, níveis de TRα e TRβ retornrm os do. onclusões: Ação do T3 em F iniciou-se em h pr TRα e,5h pr TRβ. Esses resultdos são importntes pr determinr tempo inicil e dose de T3 em que os receptores de HT são tivdos em dipócitos. Arq Brs Endocrinol Met. 3;57(5): Descritores TRα; TRβ; triiodotironin; dipócitos opyright ABE&M todos os direitos reservdos. INTRODUTION Thyroid hormones (TH) influence the metolism nd development of dipose tissue y modulting the prolifertion nd differentition of dipocytes (). TH ction is medited minly y the modifiction of gene expression y nucler receptors, which re trnscriptionl fctors regulted y lignds tht dhere to the chromtin (). TH receptors (TR) re proteins tht 368 Arq Brs Endocrinol Met. 3;57/5

2 T3 modultion of TRα nd TRβ elong to the nucler hormone receptor superfmily. These receptors originte from the genes for TR lph (TRα) nd TR et (TRβ) (3,4) tht, in humns, re locted on chromosomes 7 nd 3, respectively. According to Yen (5) nd Oregon (6), dipose tissue represents n importnt trget for TH ecuse it expresses TRα nd TRβ. THs re regrded s importnt fctors in the regultion of the development nd function of dipose tissue (5,7,8). It hs een shown tht TRs re expressed in oth white nd rown dipose tissue. However, there hve een no studies on the modultion of TRs y different doses of triiodothyronine (T3) t different times. In this study, we evluted the effects of different doses nd times of T3 on the expression levels of TRs. We oserved tht TRβ ws modulted y T3 strting t.5h, nd TRα ws modulted lter, strting t h. MATERIALS AND METHODS ell culture nd differentition The experimentl protocol ws pproved y the Ethics ommittee on Animl Experiments of the Botuctu School of Medicine-UNESP (protocol nº 75). For the in vitro study, the 3T3-L cell line ws used. These cells were cquired from the ell Bnk of the Rio de Jneiro Federl University (UFRJ) nd were cultured in Dulecco s Modified Egle s Medium (DMEM) (Gico, Life Technologies orportion, Grnd Islnd, NY, USA) ws supplemented with % fetl ovine serum (FBS) (Gico, Life Technologies orportion), % ntiiotic/ntimycotic (Sigm-Aldrich o. LL, St. Louis, MO, USA) gent, under n tmosphere of 5% O t 37. ells were mintined in these culture conditions until they reched pproximtely % confluency, nd were susequently trnsferred to 6-well pltes. After reching % confluency in the wells, cells were sumitted to the differentition process, during which they remined for 3 dys in DMEM contining % FBS, mm -methyl-3-isoutilxnthine (IBMX) (Sigm-Aldrich o. LL), mm dexmethsone (Sigm-Aldrich o. LL), nd 5 mg/l insulin (Sigm-Aldrich o. LL). After this period, cells were mintined for 7 dys in DMEM contining % FBS nd 5 mg/ml insulin. Following the period of cell differentition, dipocytes were sujected to TH depletion for 36 h in DMEM supplemented with chrcol-stripped fetl serum (Sigm-Aldrich o. LL). Susequently, cells were treted with physiologicl dose of T3 ( nm, which is herefter referred to s F) or suprphysiologicl doses of T3 ( nm nd nm, which re herefter clled SI nd SII, respectively) for.5,, 6, or 4h. A non-treted group, only.% NOH (diluent T3), ws used s control (). Oil red O stining After dys of differentition, the culture medium ws removed, nd the cells were wshed twice with phosphte-uffered sline. Susequently, ml of formldehyde ws dded, nd the cells were incuted for 3 min t room temperture, nd then wshed 3 times with phosphte-uffered sline. Therefter, 3 μl of Oil Red O dye (Sigm-Aldrich o. LL) ws dded, nd the cells were incuted for h t 37. After tht period, cells were wshed 3 times with distilled wter nd plced in n incutor to dry. ells were oserved under microscope in order to verify differentition y red coloring of the dipose cells. Gene expression Totl RNA ws extrcted from the 3T3-L cells with the TRIzol regent (Life Technologies orportion) ccording to the mnufcturer s instructions. The High pcity cdna Reverse Trnscription Kit for RT-PR (Life Technologies orportion) ws used to synthesize μl of cdna from ng of totl RNA. TRα nd TRβ mrna levels (Tle ) were determined y rel-time polymerse chin rections (RT-qP- R). Quntittive mesurements were performed with Applied Biosystems StepOne Plus detection system with the TqMn qpr commercil kit (Life Technologies orportion) ccording to the mnufcturer s instructions. The mplifiction conditions were s follows: enzyme ctivtion t 5 for min, denturtion t 95 for min. The cdna products were mplified y 4 cycles of denturtion t 95 for 5 s, nd nneling/extension t 6 for min. All ssys were performed in triplicte. Levels of gene expression Tle. Assys used t RT-qPR Gene TRβ TRα yclophilin Assy Mm43744_m Mm6755_m Mm434759_m TRβ: thyroid hormone receptor et; TRα: thyroid hormone receptor lph. TRβ nd TRα ssys correspond to homologue sequence of isoforms nd. opyright ABE&M todos os direitos reservdos. Arq Brs Endocrinol Met. 3;57/5 369

T3 modultion of TRα nd TRβ were quntified reltive to group vlue, fter normliztion with the internl control (cyclophilin) y the -ΔΔt method, s descried elsewhere (9).

3 T3 modultion of TRα nd TRβ were quntified reltive to group vlue, fter normliztion with the internl control (cyclophilin) y the -ΔΔt method, s descried elsewhere (9). Sttisticl nlysis The differences etween TRα nd TRβ mrna levels for ll experiments were ssessed y Student s t- -test. Differences of TRα nd for different T3 doses t ech moment were ssessed y one-wy nlyses of vrince (ANOVA) followed y Tukey s test. Dt re expressed s men ± stndrd devition. Significnce level ws set t 5%. ments (.5,, 6, or 4h), s mesured y RT-qPR. The different concentrtions of T3 did not ffect the levels of mrna expression of the TRα gene in the.5- h tretment period (Figure A). TRα expression levels were higher t h of tretment in the SI nd F groups in comprison with the nd SII groups (Figure B). At the 6-h time point, levels decresed in groups F, SI, nd SII in comprison with group (Figure ). TRα levels were incresed in tretment group F t 4h compred with. However, there ws decrese in the SI nd SII groups in comprison with the increse tht ws oserved in F (Figure D). RESULTS Figure A shows 3T3-L cells prior to differentition. In the presence of the differentition cocktil (insulin, dexmethsone, nd IBMX), cells differentited from predipocytes into dipocytes tht hd the morphology of mture dipocytes, chrcterized y lrge quntity of lipid droplets in the cytoplsm (Figures B nd ). These droplets ecome more evident with the oil red stining (Figure ), s the lipids get stined in red. Differentil expression of TRα nd TRβ in dipocytes Dt otined in the current study show differentil expression of TRα nd TRβ levels in dipocytes. TRα expression levels were higher in comprison with TRβ for ll groups (Tle ). Different concentrtions of T3 modulted TRα mrna levels t different time periods Figure shows the modultion of TRα mrna levels in the 3T3-L dipocytes in the sence ( group) or presence of T3 (F, SI, nd SII groups) t different mo- Tle. Differentil expression of TRβ nd TRα mrna levels Group TRβ TRα p.5h h 6h 4h. ±.4 3. ± 68. <. F.63 ± ± 75.5 =. SI.89 ± ± <. SII.88 ± ± 54.8 <.. ± ± 7. <. F. ± ± 7.9 <. SI.99 ±. 76. ± =. SII.4 ± ±.4 <.. ± ± 7.79 <. F. ± ± 7.98 =. SI.4 ± ±.97 <. SII.4 ± ±.3 <.. ± ± 44.7 =. F.76 ± ±.69 <. SI.8 ±.4.8 ±.3 <. SII.79 ± ± 7.4 <. 3T3-L dipocytes were treted with T3 t physiologicl dose (F, nm) nd suprphysiologicl doses (SI, nm or SII, nm) or without T3 (, control) for.5h, h, 6h, or 4h. Totl RNA ws extrcted nd nlyzed y RT-qPR. Dt re expressed s mens ± stndrd devitions. omprison etween TRβ nd TRα mrna levels were nlyzed using Student s t test. All ssys were performed in triplicte (n = 3 for ech tretment). A B opyright ABE&M todos os direitos reservdos. Figure. 3T3-L cells. (A) Undifferentited cells. (B) ells fter dys of differentition. () ells stined with oil red fter dys of differentition. The rrows indicte n dipocyte with lipid droplets in the cytoplsm. 37 Arq Brs Endocrinol Met. 3;57/5

4 T3 modultion of TRα nd TRβ A (reltive vlue).5.5 B (reltive vlue) (reltive vlue).5.5 D (reltive vlue).5.5 ontrol () nm T3 (F) nm T3 (SI) nmt3 (SII) Figure. Dose-dependent response for the effects of T3 on TRα mrna levels t different incution times. The 3T3-L dipocytes were treted with T3 t physiologicl dose (F, nm) or suprphysiologicl doses (SI, nm or SII,, nm), or without T3 (, control) for.5h, h, 6h, or 4h. Totl RNA ws extrcted nd nlyzed y RT-qPR. (A) Effects of T3 doses on TRα mrna levels t.5h. (B) Effects of T3 doses on TRα mrna levels t h. () Effects of T3 doses on TRα mrna levels t 6h. (D) Effects of T3 doses on TRα mrna levels t 4h. Dt re expressed s mens ± stndrd devitions. Dt were nlyzed using one-wy ANOVA complemented with Tukey s test. Similr letters indicte tht there ws no sttisticl difference, nd different letters indicte tht there ws sttisticl deference t p <.5. Assys were performed in triplicte (n = 3 for ech tretment). Different concentrtions of T3 modulted TRβ mrna levels t different time periods Figure 3 shows the modultion of TRβ mrna levels in the 3T3-L dipocytes in the sence ( group) or presence of T3 (F, SI, nd SII groups) t different periods (.5,, 6, or 4h), s mesured y RT-qPR. There ws n increse in TRβ mrna levels in F group when compred with those of, t.5h (Figure 3A). At h, suppression in the levels of ws evident in SII compred with those of groups, F, nd SI (Figure 3B). At 6h, decrese in TRβ mrna levels ws oserved in groups F, SI, nd SII compred with those of group (Figure 3). At 4h, TRβ levels decresed in F group when compred with (Figure 3D). DISUSSION Lipid metolism is closely ssocited with numer of helth prolems; the regultion of dipocytes represents n emerging re of interest. The dipose tissue, which is trget of TH, shows the expression of thyroid receptors tht re considered importnt fctors in the regultion of the development nd function of this tissue (5,7,8). The present study ws designed to elucidte the effects of T3 on TRα nd TRβ mrna expression levels in dipose tissue, without the interference of systemic fctors. As experimentl models, we used the 3T3-L cell line, emryonic cells from Mus musculus (Figure A), nd differentited in vitro dipocytes (Figures B nd ) ecuse they represent well-estlished models for dipogenesis (). Furthermore, we quntified TRα nd TRβ mrna levels y RT-qPR. The TRα gene is widely expressed in the erly stges of development, while the TRβ gene is restricted to the lte stges of emryogenesis when it is induced in the rin, pituitry glnd, nd other tissues (). We oserved tht TRα mrna levels were much higher thn TRβ mrna levels in 3T3-L dipocytes (Tle ), in opyright ABE&M todos os direitos reservdos. Arq Brs Endocrinol Met. 3;57/5 37

5 T3 modultion of TRα nd TRβ A (reltive vlue) B (reltive vlue) D.5 ontrol () nm T3 (F) nm T3 (SI) nmt3 (SII) (reltive vlue).5 (reltive vlue).5 Figure 3. Dose-dependent response for the effects of T3 on TRβ mrna levels t different incution times. The 3T3-L dipocytes were treted with T3 t physiologicl dose (F, nm) or suprphysiologicl doses (SI, nm or SII,, nm), or without T3 (, control) for.5h, h, 6h, or 4h. Totl RNA ws extrcted nd nlyzed y RT-qPR. (A) Effects of T3 doses on TRβ mrna levels t.5h. (B) Effects of T3 doses on TRβ mrna levels t h. () Effects of T3 doses on TRβ mrna levels t 6h. (D) Effects of T3 doses on TRβ mrna levels t 4h. Dt re expressed s mens ± stndrd devitions. Dt were nlyzed using one-wy ANOVA complemented with Tukey s test. Similr letters indicte tht there ws no sttisticl difference, nd different letters indicte tht there ws sttisticl deference of p <.5. Assys were performed in triplicte (n = 3 for ech tretment). opyright ABE&M todos os direitos reservdos. greement with other uthors (). The differentil expression of TR genes suggests tht they my medite distinct functions (3). It hs een shown tht TRα is criticl in thermoregultion vi regultion t the level of locl, vi centrl regultion, or oth, wheres TRβ seems to e more involved in the regultion of lipid metolic pthwys (4). Moreover, lthough TRα nd TRβ re similr in the DNA nd lignd domins (5), there re fundmentl differences in the ligndinding domin tht, nowdys, hve enled the design of lignds tht specificlly interct with TRα or TRβ, nd these hve een importnt tools of isoformspecific ctions (6). We oserved tht the modultion of the levels of TRα y different doses of T3 ws initited fter h of tretment when the physiologicl dose of nm (7) tht ws given to group F incresed the levels of TRα compred with those of groups nd SI. The TRα expression levels in the SII group remined unchnged compred with group (Figure A). We cn speculte tht this consistency in the TRα expression levels in the SII group ws due to downregultion of this receptor ecuse this group showed decrese in TRα levels compred with groups F nd SI. The downregultion process occurred due to the exposure of the cells to high doses of TH (8). Monden nd cols. (9) oserved decrese in TRα mrna levels in humn medullolstom cells (HTB- 85) t dose of nm, which ws the sme dose used in the SI group for 4h. In the present study, we lso oserved reduction in TRα in this group compred with the T3 physiologicl levels tht were dministrted to group F. However, group F hd incresed TRα levels compred with group, nd group SII ws decresed reltive to F (Figure D). Knmori e Brown () showed tht T3 modulted the TRα mrna expression levels in tdpoles, ut it hd no effect on the epithelil cell culture of emryonic Xenopus levis (XL-77) incuted with T3 for 4h t concentrtion of 5 nm. These findings do not cor- 37 Arq Brs Endocrinol Met. 3;57/5

6 T3 modultion of TRα nd TRβ roorte our results, which my e due to the use of different experimentl model nd of T3 dose tht ws lower thn tht used in our experiments. Jing nd cols. () nlyzed the TRα expression levels during the differentition period of 3T3-L cells in the sence or presence of T3 t concentrtion of nm. Although the treted group hd higher ccumultion of triglycerides, the TRα expression levels were similr for oth treted nd untreted cells, different from our results, nd proly ecuse mrna quntifiction y semi-quntittive PR is less sensitive thn tht done with RT-qPR. We oserved n increse in TRβ mrna levels in group F fter.5-h incution, ut not for TRα (Figure 3A). This led us to speculte tht t.5h, T3 ound to its receptor α, which is more undnt in dipocytes, resulting in n incresed trnscription of TRβ. After the -h incution, we oserved decrese in TRβ levels in group SII reltive to the other groups (Figure 3B), which indicted decrese of thyroid receptors in the presence of high doses of T3. Incresing the incution period to 6h, we oserved suppression of TRβ in the F, SI, nd SII groups compred with those in the group, which ws the sme oserved for TRα (Figures 3 nd ). If the trnscription of TRβ depends on the inding of T3 to TRα, tht would explin the decresed levels of TRβ t this incution time. At 4h, only the F group mintined TRβ levels elow group (Figure 3D). There ws n increse in the expression of TRβ t.5h, which ws followed y progressive reduction until 6h; TRβ levels tended to return to normlity in 4h. This suggested tht defense or dpttion mechnism occurred in the cells in response to tretment with TH. Previous studies from our group hve shown tht physiologicl doses of T3 decresed levels in the dipose tissue of oese nimls tht were sujected to food restriction (), corroorting the results otined in the present study t 6 nd 4h. Moreover, Liu nd cols. () found tht levels remin unchnged in the myocrdium of hyperthyroid rts compred with the myocrdium of euthyroid rts, suggesting tht the ctions of T3 in its receptors re different for different tissues nd experimentl models. In summry, the physiologicl nd suprphysiologicl doses of T3 modulted TR mrna expression levels y showing tht, t 6h, suppression of oth genes occurred in the presence of different doses of T3, nd tht, fter 4h, the expression levels tended to return to normlity. Since the interction with the T3 nucler receptors leds to the ctivtion or inhiition of trget expression of genes, implying the stimultion or locking of the synthesis of specific proteins, mechnism y which TH exerts its iologicl effects on cells, the results presented re importnt for n understnding in which T3 dose (physiologicl or suprphysiologicl), t different incution times, T3 ctivtes thyroid hormone receptors in dipocytes. Acknowledgements: we re grteful to Sueli A. lr, José. Georgete, Mário B. Bruno, nd Keize Ngmti Junior for their technicl support. We lso would like to thnk Editge for the trnsltion of the mnuscript into English. pes nd Fpesp (# /69-4) provided us with finncil support. Disclosure: no potentil conflict of interest relevnt to this rticle ws reported. REFERENES. Drimont, Gillrd D, Ailhud G, Negrel R. Terminl differentition of mouse predipocyte cells: dipogenic nd ntimitogenic role of triiodothyronine. Mol ell Endocrinol. 993;98(): heng SY, Leonrd JL, Dvis PJ. Moleculr spects of thyroid hormone ctions. Endocrin Rev. ;3(): Willims GR. loning nd chrcteriztion of two novel thyroid hormone receptor et isoforms. Mol ell Biol. ;(): Mcchi PE, Tkeuchi Y, Kwi T, u K, Guthier K, hssnde O, et l. Incresed sensitivity to thyroid hormone in mice with complete deficiency of thyroid hormone receptor lph. Proc Ntl Acd Sci USA. ;98(): Yen PM. Physiologicl nd moleculr sis of thyroid hormone ction. Physiol Rev. ;8(3): Oregon MJ. Thyroid hormone nd dipocyte differentition. Thyroid. 8;8(): Flores-Delgdo G, Mrsch-Moreno M, Kuri-Hrcuch W. Thyroid hormone stimultes dipocyte differentition of 3T3 cells. Mol ell Biochem. 987;76(): hwl A, Lzr MA. Induction of Rev-ErA lph, n orphn receptor encoded on the opposite strnd of the lph-thyroid hormone receptor gene, during dipocyte differentition. J Biol hem. 993;68(): Livk KJ, Schmittgen TD. Anlysis of reltive gene expression dt using rel-time quntittive PR nd the (-Delt Delt (T)) Method. Methods. ;5(4):4-8.. Green H, Meuth M. An estlished pre-dipose cell line nd its differentition in culture. ell. 974;3(): Brdley DJ, Towle H, Young WS, 3rd. Sptil nd temporl expression of lph- nd et-thyroid hormone receptor mrnas, including the et -sutype, in the developing mmmlin nervous system. J Neurosci. 99;(6): Jing W, Miymoto T, Kkizw T, Skum T, Nishio S, Tked T, et l. Expression of thyroid hormone receptor lph in 3T3- L dipocytes; triiodothyronine increses the expression of lipogenic enzyme nd triglyceride ccumultion. J Endocrinol. 4;8():95-3. opyright ABE&M todos os direitos reservdos. Arq Brs Endocrinol Met. 3;57/5 373

7 T3 modultion of TRα nd TRβ 3. Forrest D, Hneuth E, Smeyne RJ, Everds N, Stewrt L, Wehner JM, et l. Recessive resistnce to thyroid hormone in mice lcking thyroid hormone receptor et: evidence for tissue-specific modultion of receptor function. EMBO J. 996;5(): Zhu X, heng SY. New insights into regultion of lipid metolism y thyroid hormone. urr Opin Endocrinol Dietes Oes. ;7(5): Liu YY, Schultz JJ, Brent GA. A thyroid hormone receptor lph gene muttion (P398H) is ssocited with viscerl diposity nd impired ctecholmine-stimulted lipolysis in mice. J Biol hem. 3;78(4): Figueir A, Sidemerg DM, Souz P, Mrtinez L, Scnln TS, Bxter JD, et l. Anlysis of gonist nd ntgonist effects on thyroid hormone receptor conformtion y hydrogen/deuterium exchnge. Mol Endocrinol. ;5(): Yoshid T, Monkw T, Hyshi M, Srut T. Regultion of expression of leptin mrna nd secretion of leptin y thyroid hormone in 3T3-L dipocytes. Biochem Biophys Res omm. 997;3(3): Frnklyn JA, Rmsden DB, Shepprd M. Down-regultion of nucler T3 receptors y thyroid hormones in the rt nterior pituitry. Mol ell Endocrinol. 985;4(-3): Monden T, Nkjim Y, Hshid T, Ishii S, Tomru T, Shiusw N, et l. Expression of thyroid hormone receptor isoforms downregulted y thyroid hormone in humn medullolstom cells. Endocr J. 6;53():8-7.. Knmori A, Brown DD. The regultion of thyroid hormone receptor et genes y thyroid hormone in Xenopus levis. J Biol hem. 99; 67(): Luvizotto RA, onde SJ, Siio MT, Nscimento AF, Lim-Leopoldo AP, Leopoldo AS, et l. Administrtion of physiologic levels of triiodothyronine increses leptin expression in clorie-restricted oese rts, ut does not influence weight loss. Metolism. ;59():-6.. Liu R, Li LY, Shi F, Zng XY, Liu YM, Sun Y, et l. Effects of hyper- nd hypothyroid on expression of thyroid hormone receptor mrna in rt myocrdium. J Endocrinol. 7;95(3): opyright ABE&M todos os direitos reservdos. 374 Arq Brs Endocrinol Met. 3;57/5

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