Research Article Experimental Hyperthyroidism Decreases Gene Expression and Serum Levels of Adipokines in Obesity

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1 The Scientific World Journl Volume 22, Article ID 7889, 7 pges doi:./22/7889 The cientificworldjournal Reserch Article Experimentl Hyperthyroidism Decreses Gene Expression nd Serum Levels of Adipokines in Oesity Rent de Azevedo Melo Luvizotto, AndréFerreirdoNscimento, Mri Teres de Síio, Regine Mrques Cstro Olímpio, Sndro JoséConde, An Pul Lim-Leopoldo, 2 AndréSoresLeopoldo,, 2 Antonio Crlos Cicogn, nd Céli Regin Nogueir Deprtment of Internl Medicine, Botuctu School of Medicine, University of São Pulo Stte, 868- Botuctu, SP, Brzil 2 Deprtment of Sports, Center of Physicl Eduction nd Sports, Federl University of Espirito Snto (UFES), Vitóri, ES, Brzil Correspondence should e ddressed to Rent de Azevedo Melo Luvizotto, reluvizotto@yhoo.com Received 29 Octoer 2; Accepted 27 Decemer 2 Acdemic Editors: S. Bernsconi, R. E. Felerum, nd A. Ferlin Copyright 22 Rent de Azevedo Melo Luvizotto et l. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Aims. To nlyze the influence of hyperthyroidism on the gene expression nd serum concentrtion of leptin, resistin, nd diponectin in oese nimls. Min Methods. Mle Wistr rts were rndomly divided into two groups: control (C) fed with commercil chow d liitum nd oese (OB) fed with hypercloric diet. After group chrcteriztion, the OB rts continued receiving hypercloric diet nd were rndomized into two groups: oese nimls (OB) nd oese with 25 μg triiodothyronine (T 3 )/ BW (OT). The T 3 dose ws dministered every dy for the lst 2 weeks of the study. After 3 weeks the nimls were euthnized. Smples of lood nd dipose tissue were collected for iochemicl nd hormonl nlyses s well s gene expression of leptin, resistin, nd diponectin. Results. T 3 tretment ws effective, incresing ft 3 levels nd decresing ft 4 nd TSH serum concentrtion. Administrtion of T 3 promotes weight loss, decreses ll ft deposits, nd diminishes serum levels of leptin, resistin, nd diponectin y reducing their gene expression. Conclusions. Our results suggest tht T 3 modulte serum nd gene expression levels of leptin, resistin, nd diponectin in experimentl model of oesity, providing new insights regrding the reltionship etween T 3 nd dipokines in oesity.. Introduction The thyroid hormones influence energetic metolism [] nd perform centrl role in the regultion of dipose tissue metolism [2]. Disturnces of these hormones re ssocited with ltertions of ody weight nd energy expenditure [3]. While it is well known tht hyperthyroidism leds to weight loss nd hypothyroidism is ssocited with weight gin, the chnges of thyroid function re discussed controversilly in oesity [4]. Oesity, pulic helth prolem ssocited with innumerle incpcitting nd chronic diseses [5], is defined s n excessive or norml ccumultion of dipose tissue thtcne detrimentl to helth [6]. The dipose tissue, previously considered to e the lrgest, lthough inert, energy store in the ody, ctively produces vriety of iologicl sustnces. These sustnces re denominted dipokines nd cn influence the function nd structurl integrity of other tissues [7, 8]. Inoesity, the relese of dipokines such s leptin, resistin, nd diponectin cn e ltered [9]. Leptin cts s signl of stiety in the hypothlmus nd thus controls the ody weight not only y diminishing the ingestion of foods ut lso y incresing energetic expenditure []. The levels of leptin in the circultory system re elevted fter mels; this increse is due to direct stimultion of expression of the o gene nd/or secretion of leptin from dipose tissue y glucose nd insulin []. Resistin is secreted y monocytes nd dipocytes, nd present proinflmmtory properties such s TNF-α nd IL-6 [2]. Despite eing expressed nd secreted in thin individuls, elevted levels re ssocited

2 2 The Scientific World Journl with oesity oth in humns nd in experimentl niml models [3]. However, the detiled functions of resistin re still not understood; it hs een pprecited tht resistin cn cuse heptic insulin resistnce nd tht it my, long with its closely relted homologs, interct with immune cells s well[4]. Adiponectin is expressed exclusively in differentited dipocytes [5], regultes lipid nd glucose metolism, suppresses gluconeogenesis, increses insulin sensitivity, stimultes ftty cid oxidtion, protects ginst chronic inflmmtion, nd regultes food intke nd ody weight [6]. Its reduction is ssocited with decrese in lipid oxidtion, incresed triglycerides, nd suppression of insulin-dependent signling in the liver nd muscle, ll of which cn contriute to insulin resistnce nd oesity [7]. Thyroid hormones re involved in the regultion of dipose tissue wheres the hormones produced y dipose tissue such s resistin, diponectin, nd leptin re involved in regultion of the energetic lnce [8]; however, the reltionship etween these hormones in oesity is controversil nd scrcely ddressed. In this context, our ojective in this study ws to nlyze the influence of suprphysiologicl dose of T 3 on the gene expression nd serum concentrtion of leptin, resistin, nd diponectin in oese nimls. 2. Mterils nd Methods 2.. Animls nd Experimentl Protocol. This study utilized mle Wistr rts, weighing pproximtely 5 g, supplied y the Animl Center of the Experimentl Lortory for Clinicl Medicine t the Júlio de Mesquit Filho Pulist Stte University in Botuctu, So Pulo, Brzil. The nimls were initilly divided into two groups: control (C) fed with commercil chow d liitum nd oese (OB) fed with hypercloric diet, s previously descried [9], in order to induce oesity. After the oesity induction, the OB nimls were rndomized into two groups: oese nimls (OB, n = ), nd oese nimls dministered suprphysiologicl dose of T 3 (OT, n = ) t concentrtion of 25 μg/ g ody weight (BW) [2]. The T 3 ws dministered y sucutneous injections, once dy, during the finl 2 weeks [2, 22]. Approprite volumes of sline were dministered, y sucutneous injections, to the OB nd C groups. The nimls were housed in individul cges under controlled mient temperture (22 26 C) nd lighting (2 h lightdrk cycle). Dietry consumption ws controlled dily, nd weight ws ssessed weekly. The experimentl protocol ws pproved y the Commission for Ethics in Animl Experimenttion t the Botuctu UNESP School of Medicine, nd followed the Guidelines for the Cre nd Use of Experimentl Animls Totl Body Ft. The totl ody ft ws mesured s the sum of epididyml, retroperitonel, nd viscerl ft deposits [23]. This dt point ws utilized to confirm oesity in the nimls. In ddition, the diposity index (totl ody ft divided y finl ody weight multiplied y, dpted from Boustny et l. [24]) ws clculted Biochemicl Anlysis of Serum. Thenimlswerefsted for 2 to 5 hours, nesthetized with sodium pentoritl, 5 mg/kg/ip, nd scrificed y decpittion. The lood ws collected in dry tues nd centrifuged t 3 rpm for minutes. The serum ws stored t 8 C. Serum concentrtions of glucose nd tricylglycerol (TG) were ssyed using specific kits (CELM, São Pulo, Brzil). Free ftty cids (FFAs) were determined using commercil kit (WAKO, WAKO Pure Chemicl Industries Ltd., Osk, Jpn). Dosing ws nlyzed y the utomted colorimetric enzyme method (Technicon, RA-XT System, Glol Medicl Instrumenttion, Minessot, USA) Hormonl Mesurements. Serum concentrtions of insulin, leptin, resistin, diponectin, free T 3,freeT 4,nd TSH were mesured in ll nimls. The mesurements were performed y immunossy, mesured with microplte reder (Spectr Mx 9 Moleculr Devices, Sunnyvle, CA, USA). Commercil kits were utilized for the mesurement of leptin, insulin, diponectin (ELISA kit-millipore, St. Chrles, MO, USA), resistin (ELISA kit-b-bridge Interntionl Inc., Mountin View, CA, USA), nd thyroid hormones (ELISA kit-uscn Life Science&Technology Compny, Wuhn, Chin) Gene Expression. Whole RNA ws extrcted from retroperitonel dipose tissue using the regent Trizol (Invitrogen, So Pulo, Brzil), ccording to the mnufcturer s instructions. The SuperScript II First-Strnd Synthesis System for RT-PCR (Invitrogen, So Pulo, Brzil) kit ws utilized for the synthesis of 2 μl of complementry DNA (cdna) from ng of whole RNA. The mrna levels of leptin (ssy Rn ml Applied Biosystems), resistin (ssy Rn ml AppliedBiosystems), nddiponectin (ssy Rn ml Applied Biosystems) were determined y rel-time PCR. Quntittive mesurements were mde with the commercil kit TqMn qpcr (Applied Biosystems), ccording to the mnufcturer s instructions, in the detection system Applied Biosystems StepOne Plus. Cycling conditions were s follows: enzyme ctivtion t 5 C for 2 min, denturtion t 95 C for min, the cdna products were mplified for 4 cycles of denturtion t 95 C for 5 s, nd nneling/extension t 6 C for min. Gene expression ws quntified in reltion to the vlues of the C group fter normliztion y n internl control (cyclophilin-ssy Rn ml Applied Biosystems) y the method 2 ΔΔCT, s previously descried [25] Sttisticl Anlysis. Chnges in ody weight were evluted y confidence intervl of 95%. Gene expression, iochemistry, nd hormone dt were nlyzed using nlysis of vrince (ANOVA) complemented y Bonferroni s test. Thedtreexpressedsmen± stndrd devition. A 5% significnce level ws dopted. 3. Results 3.. Evolution of Body Weight. All the nimls hd similr BW t the eginning of the study. By week 3, the OB nimls

3 The Scientific World Journl Similrly, T 3 dministrtion decresed totl ody ft nd diposity index (Tle ) Biochemicl Anlysis. Tle 2 presents the vlues for glucose, TG, nd FFA. The hypercloric diet did not lter the iochemicl prmeters when compred to the C group. There ws n increse in glucose nd FFA levels, ut there ws no sttisticl difference in TG levels in the OT group in comprison to the OB group. C OB OT Figure : Weekly evolution of ody weight in the control group (C, n = ), oese group (OB, n = ), nd oese with 25 μg T 3 / g BW (OT, n = ). Dt re expressed s mens with 95% confidence intervl Hormonl Mesurements. The hypercloric diet did not chnge the thyroid profile; however, n increse in leptin nd resistin levels nd decrese in diponectin serum concentrtion ws oserved. In the OT group, T 3 levels were elevted, yet the serum concentrtions of free T 4 nd TSH were diminished (Tle 3). Administrtion of T 3 reduced the serum levels of dipokines, evluted leptin (Figure 2()), resistin (Figure 2()), nd diponectin (Figure 2(c)). Tle : Composition of ody ft: retroperitonel, epididyml, viscerl, nd totl ft deposits, nd diposity index. Vrile Groups Epid. ft (g) 8.65 ± ± ±.5 Retro ft (g) 9.68 ± ± ± 3.9 Visc. ft (g) 6.37 ± ± ±.8 Totl ft (g) 24.7 ± ± ± 6.8 Adipos. I. 5.6 ±. 7.6 ±. 4.2 ±.4 Epid. Ft: epididyml ft; Retro. Ft: retroperitonel ft; Visc. Ft: viscerl ft; totl Ft: totl ody ft; Adipos. I: diposity index; C: control; OB: oese; OT: oese with 25 μg T 3 / g BW. Dt expressed s men ± stndrd devition. ANOVA ws utilized, complemented y Bonferroni s test. Use of sme letters represent P>.5; different letters represent P<.5. Tle 2: Biochemicl nlysis: glucose, triglycerides, nd free ftty cids. Vrile Groups Glucose (mg/dl) 95. ± ± ± 9.7 TG (mg/dl) 82.5 ± ± ± 4.8 FFA (mmol/l).5 ±..54 ±..72 ±. TG:triglycerides;FFA:freefttycids;C:control;OB:oese;OT:oese with 25 μg T 3 / g BW. Dt expressed s men ± stndrd devition. ANOVA ws utilized, complemented y Bonferroni s test. Use of sme letters represent P>.5; different letters represent P<.5. were hevier thn the C group. After 3 weeks of experiment, the BW of the OB group (64 g ± 36 g) ws sttisticlly higher thn tht of the C group (488 g ± g). The BW of the OB +T 3 group (489 g ± 6 g) ws significntly lower thn the OB group, ut not significntly different from the C group (Figure ) Totl Body Ft. The hypercloric diet incresed ft deposits nd the diposity index. Administrtion of T 3 decresed retroperitonel, viscerl, nd epididyml ft deposits Gene Expression. Using rel-time PCR, gene expression ws nlyzed using 6 nimls per group. The smples were normlized y n internl control (cyclophilin), nd the C group ws normlized y. The gene expression of leptin (Figure 3()) nd resistin (Figure 3()) ws incresed in the OB group, ut diminished in the OT group. Compred to the C group, diponectin gene expression (Figure 3(c)) ws decresed y hypercloric diet. Compred to the OB group, T 3 dministrtion decresed diponectin expression levels. 4. Discussion Oesity is condition tht hs reched epidemic levels in recent yers [6]. It is complex disese, where lifestyle intercts with genetic susceptiility to produce the oese phenotype. Nowdys, the sustntil rise in oesity indices ppers to e due to the lifestyle of the popultion, prticulrly due to inpproprite diets nd the lck of physicl ctivity [4]. Lifestyle is highly recognized s plying centrl role in the etiology of chronic diseses [26]. Furthermore, oesity is ssocited with severl chronic diseses including coronry rteril disese, hypertension, dietes mellitus type 2, nd some forms of cncer [5]. The homogenous ehvior of the nimls is not ssured in experimentl studies, even when they re mintined under lortory conditions. In this context, rts, given normocloric or hypercloric rtions in models of dietinduced oesity, cn present different responses with common chrcteristics [27]. Thus, clssifiction errors my occur, such tht nimls sumitted to normocloric diet cn e clssified s controls, when in fct they exhiit responses similr to nimls tht ecme oese vi hypercloric diet, or vice vers. For this reson, it ecomes necessry to estlish criterion tht would enle the seprtion of nimls into control or oese. A study in our lortory showed tht the est indictor of oesity is odily diposity, ut this index is otined fter the niml is euthnized [27]. However, BW, evluted in vivo, presents good correltion with the diposity index [27]. In this

4 4 The Scientific World Journl Leptin serum concentrtion (ng/ml) Resistin serum concentrtion (ng/dl) () () Adiponectin serum concentrtion (ng/dl) 3 2 c (c) Figure 2: Influence of different doses of T 3 on serum concentrtion of leptin (), resistin (), nd diponectin (c). C: control (n = ); OB: oese (n = ); OT: oese with 25 μg T 3 / g BW (n = ). Dt expressed s men ± stndrd devition. ANOVA ws utilized, complemented y Bonferroni s test. Use of sme letters represent P>.5; different letters represent P<.5. context, the control nd oese groups were constituted y pplying BW s the clssifiction criterion of the study. Administrtion of suprphysiologicl doses of T 3 ugmented the serum concentrtion of T 3 [28]. In the OT nimls, free T 4 nd TSH levels were diminished compred to OB nimls, not surprisingly s exogenous T 3 suppresses the endogenous secretion of TSH nd T 4 y the thyroid [29], showing the effectiveness of tretment. Hypercloric diets induce ccentuted weight gin nd diposity. Consumption of diets rich in ft does not ugment lipid oxidtion in the sme proportion, which leds to the elevtion of ody weight due to the deposition of tricylglycerol in dipose tissue [3, 3]. The suprphysiologicl doses of T 3 diminished the weight of OB nimls nd reduced the diposity index (Figure, Tle ). Hormones nd cytokines induce distinct metolic responses in different ft deposits [32], nd this study shows similr moiliztion of ll ft deposits in the OT group (Tle ). Thyroid hormones regulte the metolism of lipids [33] nd tht their excess, mong other effects, ugments lipolysis, the plsmtic concentrtion of intermedite lipids nd lipid peroxidtion [34]. Concentrtions of free ftty cids re used to indicte the moiliztion of ft [35]. In the present study, T 3 dministrtion elevtes lipolysis in the OT group ut did not lter TG levels (Tle 2). These dt re in greement with other reports of TG levels not eing influenced y thyroid hormone [36]. Excess of thyroid hormones ugments plsm glucose levels [37]. The dministrtion of T 3 produced significnt diminution of plsm insulin levels in the nimls treted Tle 3: Hormonl mesurement of free triiodothyronine (T 3 ), free thyroxine (T 4 ), nd thyroid-stimulting hormone (TSH). Vrile Groups Free T 3 (pmol/l).3 ±.6.2 ±.3.22 ±.4 Free T 4 (pmol/l) 45.8 ± ± ±.8 TSH (miu/l) 3.9 ± ±.8. ±.2 TSH: thyroid stimulting hormone; T 3 triiodothyronine; T 4 thyroxine; C: control; OB: oese; OT: oese with 25 μg T 3 / g BW. Dt expressed s men ± stndrd devition. ANOVA ws utilized, complemented y Bonferroni s test. Use of sme letters represent P >.5; different letters represent P<.5. (dt not shown). Nevertheless, the exct influence of thyroid hormones on insulin sensitivity nd glucose metolism remins controversil [38]. However, one importnt fctor to highlight in this study is tht the thyroid hormones induce weight loss in mnner relted to improving of resistnce to insulin (dt not shown) [39]. Leptin cn influence the interction etween genes nd environmentl fctors. Diets rich in ft rise leptin levels cn differentilly ffect ody composition even with similr diets. However, the rise in leptin levels is etter explined y increse in ody ft [3]. Experimentl studies suggest tht sensitivity to leptin cn e controlled y hormonl nd nutritionl fctors [4]. The literture shows cler positive correltion etween dipose tissue nd leptin expression. Diet-induced oesity elevtes the gene expression of leptin

5 The Scientific World Journl 5 Leptin gene expression (reltive vlue) 3 2 Resistin gene expression (reltive vlue) 2 () () Adiponectin gene expression (reltive vlue).5.5 c (c) Figure 3: Influence of different T 3 doses on the gene expression of leptin (), resistin (), nd diponectin (c). C: control (n = 6); OB: oese (n = 6); OT: oese with 25 μgt 3 / g BW (n = 6). Dt expressed s men ± stndrd devition. ANOVA ws utilized, complemented y Bonferroni s test. Use of sme letters represent P>.5; different letters represent P<.5. [4, 42]. However, the effects of T 3 on the gene expression of leptin present inconsistent results; despite the in vitro dt showing tht T 3 produces dose-dependent rise in leptin expression [43], our dt revel tht, following in vivo hyperthyroidism, leptin gene expression ws reduced. In concordnce, Pinkney et l. [2] nd Zrock et l. [28] oserved diminution of leptin expression in response to tretment with T 3. The physiologicl role of diponectin hs not yet een completely elucidted. Experimentl dt suggest tht diponectin ugments sensitivity to insulin nd cn present ntitherogenic nd ntiinflmmtory properties [44]. It is well estlished tht diponectin levels re inversely proportionl to the degree of diposity [45], nd weight loss elevtes the endogenous production of diponectin [46]. Here we demonstrte tht the OB group hd decresed diponectin serum levels when compred to the C group, nd the dministrtion of T3, interestingly, even diminishing the ody ft mss, presented lower levels of diponectin (Figures 2(c) nd 3(c)). Confirming this dt Cnels et l. [47] show reduced diponectin gene expression in inguinl explnts of norml rts; lso, we hve demonstrte, recently tht diponectin levels re decresed in clorie-restricted oese rts [48]. However, in contrst, n experimentl study of rts with hyperthyroidism showed n importnt rise in serum diponectin [49]. Our dt show tht suprphysiologicl T 3 doses lter diponectin expression in oesity, suggesting tht T 3 cuses undesirle effects on dipose tissue. Resistin prejudices glucose homeostsis nd insulin ction in mice [5, 5]. Thus, resistin my perform n intermediry role etween oesity nd insulin resistnce in rodents lthough this role is still questioned in humns [52]. In this study, hypercloric diet increses the serum levels of resistin, while T 3 tretment decreses it. The significnt diminution of serum resistin in the OT group corroortes the first study performed on humns, in which ptients with hyperthyroidism exhiited low serum resistin concentrtions [53]. However, these initil findings contrst with susequent studies tht report high resistin levels in hyperthyroidism ptients [54, 55], showing divergence in the dt. Nevertheless, resistin gene expression is lmost undetectle in rts with hyperthyroidism [56]. Our results show tht dministering suprphysiologicl doses of T 3 decrese resistin expression to the C group levels (Figure 3()). 5. Conclusion The exogenous tretment with T 3 is effective in ugmenting serum levels of free T 3 nd diminishing concentrtions of free T 4 nd TSH. Administrtion of T 3 promotes weight loss nd decreses diposity. Following dministrtion of T 3, there ws decrese in serum concentrtion of leptin, resistin, nd diponectin, s well s reduction in the gene expression. Our dt demonstrte tht T 3 cts, directly or indirectly, on dipose tissue-derived dipokines which cn influence whole-ody homeostsis. This report provides new

6 6 The Scientific World Journl insights regrding the reltionship etween T 3 nd dipokines in oesity model. Acknowledgments The uthors thnk Sndr Fáio, José Georgete, Mário Bruno, nd Sueli Clr for their technicl ssistnce. They re grteful to Dijon HS Cmpos nd Ricrdo L Dmtto for their contriution in the nimls euthnsi. They lso thnk FAPESP for the finncil support (processes: 6/5877- nd 7/54-4). This mnuscript hs een proofred nd edited y ntive English spekers with relted ckground in BioMed proofreding. All uthors declre tht there re no conflict of interests. References [] M. Krotkiewski, Thyroid hormones in the pthogenesis nd tretment of oesity, Europen Journl of Phrmcology, vol. 44, no. 2-3, pp , 22. [2] J. H. Pinkney, S. J. Goodrick, J. Ktz et l., Leptin nd the pituitry-thyroid xis: comprtive study in len, oese, hypothyroid nd hyperthyroid sujects, Clinicl Endocrinology, vol. 49, no. 5, pp , 998. [3] U. Feldt-Rsmussen, Thyroid nd leptin, Thyroid, vol. 7, no. 5, pp , 27. [4] T. 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