31 Thyroxine and tri-iodothyronine differently affect uncoupling protein-1 content and antioxidant enzyme activities in rat interscapular

N Petrovic´, G Cvijic´and V Davidovic´ Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Yugoslavia (Requests for offprints should be addressed to N Petrovic´ ; Email: [email protected]) (N Petrovic´ is currently at Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden)

Abstract The activity of the antioxidant enzymes copper–zinc were significantly decreased. Thyroxine treatment signifi- superoxide dismutase (CuZnSOD), manganese super- cantly decreased IBAT CAT activity while MDA levels oxide dismutase (MnSOD) and catalase (CAT), as well markedly increased. Cold exposure induced a significant as mitochondrial glycerol-3-phosphate dehydrogenase augmentation of UCP1 content and MnSOD and (mGPDH) activity, uncoupling protein-1 (UCP1) con- mGPDH activities only in animals that were rendered tent, catecholamine degrading enzyme monoamine oxi- hyperthyroid by T4 treatment. In T3-treated animals dase (MAO) activity and malonyl dialdehyde (MDA) acutely exposed to cold stress, MDA concentration, an concentration were studied in rat interscapular brown indicator of lipid peroxidation, was significantly higher adipose tissue (IBAT). Rats were treated with either compared with that of T3-treated animals housed at room thyroxine (T4) or tri-iodothyronine (T3) for five days and temperature. However, in T4-treated animals, MDA con- then exposed to cold (4 C, 24 h) or housed at room centrations were markedly lower. These results show that  ff ff temperature (22 C). Under basal conditions, T3 treatment T4 and T3 di erently a ect IBAT parameters studied not significantly increased UCP1 content and MnSOD only under basal but also under cold-stimulated conditions. activity whereas CuZnSOD, CAT and MAO activities Journal of Endocrinology (2003) 176, 31–38

Introduction noradrenaline (NA) (Silva & Larsen 1983) have led to a better understanding of the active role of Brown adipose tissue (BAT) is an organ highly specialized in BAT . Nevertheless, the action of thyroid for heat production by a mechanism referred to as ‘non- hormones on BAT is complex and is dependent on shivering’ thermogenesis (Nicholls & Locke 1984). BAT tri-iodothyronine (T3) content (Bianco & Silva 1987), a mitochondria contain uncoupling protein-1 (UCP1) physiologically active thyroid hormone. Since T3 concen- which allows protons to re-enter the tration in BAT is strongly influenced by D2 that is without coupling to ATP synthesis (Cannon & Lindberg inhibited by its substrate thyroxine (T4) (Silva & Larsen 1979). UCP1 transforms electrochemical energy into heat 1986), it might be argued that BAT of T4-hyperthyroid (Nicholls & Locke 1984), enabling small mammals to rats may lack T3 (Ablenda & Puerta 1992). Otherwise, tolerate cold exposure (Nedergaard et al. 1999). nuclear and mitochondrial thyroid receptors in BAT of The thermogenic activity of BAT and UCP1 gene T3-hyperthyroid rats can be occupied fully by excessive expression are mainly regulated by the sympathetic ner- T3. Given that response elements for thyroid hormone vous system (SNS), which innervates the tissue (Cassard- receptors have been identified in the UCP1 gene pro- Doulcier et al. 1993). Although it is known that thyroid moter (Silva & Rabelo 1997), it is not surprising that hormones are involved in cold-induced thermogenesis, alterations in BAT nuclear thyroid receptor occupancy their role in BAT thermogenesis is not yet clear. Thyroid levels produce substantial changes in UCP1 concentration. hormones increase obligatory thermogenesis and play a Thus, hyperthyroidism produced by T3 treatment signifi- role in the facultative thermogenesis interacting with the cantly increased UCP1 mRNA levels (Masaki et al. 1997), SNS or may affect BAT activity directly (Silva 1995). The UCP1 content (Branco et al. 1999), and guanosine 5- presence of type 2 thyroxine 5-deiodinase (D2) in BAT diphosphate (GDP) binding to brown-adipocyte mito- (Leonard et al. 1983) and its manifold stimulation by chondria (Woodward & Saggerson 1989). On the other

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ff   hand, the e ects of T4 on BAT UCP1 levels are still Rats were kept at room temperature (22 1 C)  controversial. It is reported that T4 treatment may either (mean S.E.M), in a 12 h light:12 h darkness cycle and had increase GDP binding to brown-adipocyte mitochondria free access to commercial rat food (Subotica, Yugoslavia) (Nedregaard et al. 1997) or maintain it at the same level and tap water. (Sundin 1981). Animals were randomly assigned to one of three groups: However, Echtay et al. (2002) recently postulated that euthyroid (treated with 9 mM NaOH, which is the UCP1 evolved a thermogenic role in mammals as a side solvent for T4 and T3, in a volume of 1 ml/kg body pathway of an original, more general function of protection weight, i.p. for five days), T4-treated (300 µg/kg against the cold-induced production of reactive oxygen body weight, i.p. for five days) and T3-treated (200 µg/kg species (ROS). They substantiated this opinion by the fact body weight, i.p. over five days). After the last injection that a mild uncoupling decreases mitochondrial production the animals of all three groups were subdivided into two of ROS (Papa & Skulachev 1997, Brand 2000) and that groups: one group remained at room temperature for 24 h superoxide anion increases mitochondrial conductance and the other was exposed to cold (4 C) for 24 h. Animals through effects on uncoupling proteins (UCPs). In accord- were killed by decapitation with a guillotine (Harvard- ance with this presumption are the results of Nègre- Apparatus, Holliston, MA, USA). The IBAT, heart and Salvayre et al. (1997), who showed that the inhibition of liver were rapidly excised, dissected (4C), weighed and UCP1 by GDP induced a rise in mitochondrial membrane stored at 20 C prior to enzyme activity and UCP1 potential and hydrogen peroxide production. Since exten- concentration measurements. Experiments were approved sive changes in the BAT mitochondrial compartment by the local ethics committee for animal research. occur in response either to thyroid hormones or to cold exposure (Triandafillou et al. 1982), and since thyroid Analytical procedure hormones exert profound effects on the energy metabolism and influence BAT oxygen consumption (Saha et al. All chemicals used were purchased from Sigma (St Louis, 1998), it is likely that thyroid hormones alter ROS MO, USA) unless otherwise specified. production in BAT and the activity of its antioxidant protective system. Antioxidant enzymes assays in IBAT IBAT was The aim of the present study was to examine the homogenized (Sorwall omni-mixer, Norwall, CT, USA) content of UCP1 in terms of its thermogenic and possible inabuffer containing 0·25 M sucrose, 0·05 M Tris–HCl ROS protective function and its relationship with key and 1 mM EDTA, pH=7·4 and sonicated three times at antioxidant enzymes – cytosolic copper–zinc superoxide 100 W for 20 s with 10 s pause in a Bronson model B-12 dismutase (CuZnSOD), mitochondrial manganese super- sonicator to release MnSOD. The samples were then oxide dismutase (MnSOD) and peroxisomal catalase centrifuged at 20 000 r.p.m. in a Beckman centrifuge, (CAT) in interscapular brown adipose tissue (IBAT) of rats JA-20, for 120 min and used for CAT, CuZnSOD and rendered hyperthyroid either by T4 or T3 treatment under MnSOD determination. basal or cold-stimulated conditions. We have also studied SOD activity was determined by the method the activities of two mitochondrial enzymes involved of Misra and Fridovich (1972), based on the spectro- in the hydrogen peroxide production – mitochondrial photometrical measurement of the degree of adrenaline glycerol-3-phosphate dehydrogenase (mGPDH) and auto-oxidation inhibition by SOD, contained in the (MAO). MAO, a catecholamine examined samples. Total specific SOD activity and that degrading enzyme localized in the outer mitochondrial of MnSOD (after CuZnSOD inhibition with potassium membrane, produces hydrogen peroxide. Recent results of cyanide (KCN)) were measured, and then CuZnSOD Drahota et al. (2002) indicate that hydrogen peroxide is activity was calculated. CAT activity was measured spec- also produced directly by mGPDH, aFAD-linked, thyroid trophotometrically by the method of Beutler (1982), based hormone responsive enzyme, localized in the inner mito- on the rate of hydrogen peroxide degradation by the action chondrial membrane. Finally, to evaluate possible damage of CAT contained in the examined samples. of IBAT membranes as a consequence of altered ROS production and/or ROS degradation, we have measured Lipid peroxidation measurement The extent of the concentration of tissue lipid peroxides. the peroxidative reactions in IBAT was determined by measuring malonyl dialdehyde (MDA) concentration in IBAT homogenates (prepared in 50 mM Tris–HCl buffer, pH=7·4 in a ratio of 1:50) by the modified thiobarbituric Materials and Methods acid method without stimulation of peroxidative processes with Fe2+ and ascorbate (Rehncrona et al. 1980). Animals Male rats of the Wistar strain (Rattus norvegicus), three MAO activity MAO activity in IBAT was determined months old when killed, were used for experiments. by the method of Wurtman and Axelrod (1963), based on

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Table 1 Assessment of hyperthyroid state in terms of changes in body weight gain, relative heart weight and  the liver mGPDH activity of rats treated with either T4 or T3 and thereafter exposed to 4 C for 24 h or housed at 22 C. Values are meansS.E.M. of six animals

Body weight gain Heart weight/body weight1 Liver mGPDH activity (g) (mg/g) (A500/min/mg protein) Housed at 22 C Euthyroid 26·0920·7 2·930·08 0·0320·003    T4-treated 19·25 2·21* 3·36 0·08* 0·073 0·013*    T3-treated 4·91 2·15*** 3·96 0·18** 0·145 0·025** Exposed to 4 C for 24 h Euthyroid 15·502·58§ 3·420·08§§ 0·0350·003   §  T4-treated 16·67 6·04 3·84 0·11* 0·103 0·013***    T3-treated 4·77 3·62* 4·13 0·12** 0·167 0·007***

1Heart weight (mg)/body weight (g). *P<0·05, **P<0·01, ***P<0·001 versus the corresponding euthyroid group; §P<0·05, §§P<0·01 versus the group treated in the same way but housed at 22 C. the measurement of radioactivity of 14C-indol-3-acetic The obtained number of pixels for the control group acid, which is generated during incubation of 14C- (10 µg mitochondrial proteins) represents one arbitrary tryptamine bissucinate with the IBAT homogenate. unit. The UCP1 content of treated groups is expressed relative to control IBAT. Assay for mGPDH in IBAT and liver The assay of Protein content of the tissues was measured by the mGPDH is based on the ability of iodonitrotetrazolium to method of Lowry et al. (1951). directly accept electrons from the dehydrogenase (con- The results are expressed as meansS.E.M. Student’s tained in the solubilized mitochondrial fraction) with t-test was employed for comparing the two groups and the its reduction to iodoformazan, the concentration of level of significance was set at P<0·05. which was determined spectrophotometrically at 500 nm (Gardner 1974, Bianco & Silva 1987). Results UCP1 assay UCP1 protein levels were measured by ff Western blot analysis. Samples of solubilized mitochondrial The e ectiveness of both T4 and T3 administration in fraction (containing 10 µg of the IBAT mitochondrial producing hyperthyroidism, expressed in body weight protein) were added to an equal volume of buffer (con- gain and relative heart weight, as well as liver mGPDH sisting of 0·125 M Tris–HCl, 0·14 M SDS, 20% glycerol, activity reflecting T3 tissue availability during the treat- 0·2 mM dithiothreitol, 0·03 mM bromophenol blue, ment, is presented in Table 1.  pH=6·8). After denaturation by heating to 100 Cfor Both T4 and T3 treatment produced a marked IBAT 5 min, samples were separated on a 12·5% polyacrylamide hypertrophy in terms of significant increase in absolute and gel and electrotransferred to a PVDF membrane (pore size relative IBAT weight (Table 2). However, it is important 0·45 µm, LKB, Belgrade, Yugoslavia). After transfer of to point out that only T3 treatment produced a marked proteins, the membrane was soaked in Tris-buffered saline decrease in total IBAT protein concentration and signifi- (50 mM Tris–HCl, 150 mM sodium chloride, pH=7·5) cant increase in the rat rectal temperature regardless of twice for 5 min, followed by quenching of nonspecific ambient temperature. binding (1 h at room temperature in 5% nonfat dry milk, 0·2% Tween 20 in Tris-buffered saline). After quenching, UCP1 content and mGPDH activity in IBAT of T -or the membrane was incubated with a solution of rabbit 4 T -treated rats under basal or cold-stimulated conditions antibody against rat UCP1 (Alpha Diagnostic Inter- 3 national, San Antonio, TX, USA) overnight at 4 C. The Given that thyroid hormone treatment significantly primary antibody was detected with a secondary antibody changed IBAT weight and/or its protein concentration (horseradish peroxidase-conjugated goat anti-rabbit anti- (Table 2), UCP1 concentration is expressed as total UCP1 body (ICN, Belgrade, Yugoslavia)) and enhanced chemi- content (arbitrary units (AU)/tissue). Under basal con- luminescence (ECL, Amersham Life Science). The blots ditions, only T3 treatment significantly increased UCP1 were exposed to X-ray film for autoradiography. The content in brown fat mitochondria (Fig. 1). However, after intensity of signals was evaluated by the Image Quant 24 h of cold exposure, UCP1 content of T3-treated rats, in program (Molecular Dynamics, Amersham Biosciences). contrast to the control and T4-treated groups, was not www.endocrinology.org Journal of Endocrinology (2003) 176, 31–38

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Table 2 Effects of T4 and T3 on the rat rectal temperature, absolute and relative IBAT weight and its total and mitochondrial protein concentration under basal (22 C) or cold-stimulated conditions (4 C for 24 h). Values are meansS.E.M. of six animals

IBAT weight Protein concentration Rectal temperature Absolute Total Mitochondrial (C) (mg) Relative1 (g/mg tissue) (g/mg tissue) Housed at 22 C Euthyroid 37·90·1 179·97·8 0·800·03 121·319·53 5·690·18      T4-treated 38·0 0·0 223·5 13·3** 1·08 0·07** 107·16 6·91 5·62 0·27      T3-treated 38·5 0·2* 291·8 12·8*** 1·57 0·13*** 90·68 5·41* 4·88 0·60 Exposed to 4 C for 24 h Euthyroid 37·30·2§ 187·28·4 0·920·06 153·977·64§ 9·420·58§§     §§  §§ T4-treated 37·7 0·1 222·9 12·7* 1·07 0·07 135·19 6·87 9·81 0·34     §  T3-treated 38·2 0·2** 281·3 8·5*** 1·44 0·06*** 112·48 6·43*** 6·59 0·73*

1IBAT weight (mg)/body weight (g). *P<0·05, **P<0·01, ***P<0·001 versus the corresponding euthyroid group; §P<0·05, §§P<0·01 versus the group treated in the same way but housed at 22 C.

significantly enhanced relative to the basal content in T3-treated rats. Unlike the increment of the liver mGPDH activity (Table 1), hyperthyroidism, produced ff with either T4 or T3 treatment, had no e ect on its activity in IBAT of animals housed at room temperature. In our present experiment, acute cold exposure produced similar changes in IBAT mGPDH and UCP1. Thus, after a 24 h cold exposure, IBAT mGPDH activity and UCP1 content increased significantly only in the control and T4-treated groups.

MAO activity in IBAT of hyperthyroid animals under basal and cold-stimulated conditions The activity of the catecholamine degrading and hydrogen peroxide producing enzyme, MAO, decreased signifi- cantly following T3 treatment (Fig. 2) irrespective of the ambient temperature. Cold stress produced a significant increase in IBAT MAO activity in all groups studied, indicating the activation of the sympathoadrenal system.

Activity of antioxidant enzymes in IBAT of hyperthyroid rats under basal and cold-stimulated conditions Under basal conditions, SODs activities in IBAT were changed only in T3-treated animals (Fig. 3). Thus, CuZn- Figure 1 UCP1 content and mGPDH activity in rat IBAT. Rats SOD activity markedly decreased while that of MnSOD were made hyperthyroid by either T4 or T3 treatment and significantly increased in the IBAT of animals rendered  thereafter exposed to cold (4 C for 24 h) or housed at room hyperthyroid by T administration. However, the IBAT temperature (22 C). UCP1 content is expressed in arbitrary units 3 (AU)/tissue (10 g IBAT mitochondrial protein were used for the CAT activity was markedly decreased in both T4- and analysis and the obtained number of pixels for the control group T3-treated animals. After cold stress, each of the IBAT represents one arbitrary unit. UCP1 content of treated groups is antioxidant enzymes studied was affected differently. expressed relative to control IBAT). mGPDH activity is expressed Thus, CuZnSOD activity was increased in IBAT of both  3 as A500/min/mg wet tissue weight 10 . Values are T - and T -treated animals (no effect on control animals) meansS.E.M. of six animals. *P<0·05 versus the corresponding 4 3 euthyroid group; §P<0·05, §§P<0·01, §§§P<0·001 versus the group while that of CAT was significantly increased in all groups treated in the same way but housed at 22 C. studied relative to basal activities. After cold exposure,

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Figure 2 MAO activity in rat IBAT. Rats were made hyperthyroid  by either T4 or T3 treatment and thereafter exposed to cold (4 C for 24 h) or housed at room temperature (22 C). MAO activity is expressed as pmol/min/mg wet tissue weight. Values are meansS.E.M. of six animals. *P<0·05 versus the corresponding euthyroid group; §P<0·05, versus the group treated in the same way but housed at 22 C.

MnSOD activity changed in a manner similar to that of other mitochondrial parameters (UCP1 and mGPDH) studied (cold stress increased MnSOD activity in IBAT of control and T4-treated animals but not T3-treated animals).

Lipid peroxidation in IBAT of hyperthyroid rats under basal and cold-stimulated conditions Figure 4 clearly shows that the extent of peroxidative processes in IBAT of hyperthyroid animals depends on whether T4 or T3 is used for inducing hyperthyroidism. Thus, under basal conditions, T4 treatment, but not T3 treatment, produced a significant increase in IBAT MDA concentration. Interestingly, exposure to cold induced ff opposite e ects in MDA levels in IBAT of T4- and T3-treated rats as compared with animals exposed to 22 C. Cold stress produced a marked decrease in IBAT Figure 3 CuZnSOD, MnSOD and CAT activities in rat IBAT. Rats MDA concentration of T -treated rats and increased were made hyperthyroid by either T4 or T3 treatment and 4 thereafter exposed to cold (4 C for 24 h) or housed at room MDA concentration in T3-treated animals compared with temperature (22 C). Values are expressed as units of SOD or animals treated in the same way but housed at room CAT/mg wet tissue weight and represent the meansS.E.M. temperature. Cold exposure had no effect on MDA of six animals. *P<0·05, **P<0·01, ***P<0·001 versus the § < §§ < §§§ < concentrations in the control animal group. corresponding euthyroid group; P 0·05, P 0·01, P 0·001 versus the group treated in the same way but housed at 22 C.

Discussion thyroid hormone-induced heat production would partly Our present results clearly show that UCP1 content, lipid compensate for the need for extra heat production in the peroxide level, antioxidant enzyme activities and MAO cold. Thus, at any subthermoneutral temperature an and mGPDH activities in IBAT of hyperthyroid rats are animal would require less BAT-derived heat to counteract ff changed di erently depending on whether T4 or T3 is heat loss to the surroundings (Nedergaard et al. 1997). On used for inducing hyperthyroidism. To explain these the other hand, BAT itself is a target for thyroid hormones results one must bear in mind that in the hyperthyroid and has a large number of 1 and 1 thyroid hormone state, obligatory thermogenesis is elevated (Silva 1995) and receptors (Hernández & Obregón 1996). Since high SNS activity is significantly attenuated (Matsukawa et al. plasma levels of T4 inhibit D2 (Silva & Larsen 1986) it 1993). Hence, at subthermoneutral temperatures the could be argued that the BAT of T4-hyperthyroid rats is www.endocrinology.org Journal of Endocrinology (2003) 176, 31–38

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and T4-treated rats. It is noteworthy that mGPDH and MnSOD activities and IBAT mitochondrial protein con- centrations were changed in a similar manner. To theorize why IBAT mitochondrial parameters, investigated in this study, were not increased further after cold exposure of T3-treated rats, it is necessary to point out that (1) D2 activity in IBAT is increased by cold stimulation (Silva & Larsen 1983, 1986, Guerra et al. 1998) and (2) in addition to T4,T3, reverse T3 and other iodothyronines are substrates for deiodinase enzymes (Goglia et al. 1999). It is ff conceivable that some of the e ects observed following T4 or T3 administration might be due, at least in part, to some of their deiodinated products. Recently, Moreno et al. Figure 4 MDA concentration in rat IBAT. Rats were made (2002) showed that when T3 is injected into euthyroid hyperthyroid by either T4 or T3 treatment and thereafter exposed ff to cold (4 C for 24 h) or housed at room temperature (22 C). animals, not all the e ects on resting metabolic rate are MDA concentration is expressed as pmol of MDA formed/mg attributable to T3 itself but could be largely due to its  wet tissue weight and represents the means S.E.M. of six animals. in vivo deiodination to 3,5-di-iodothyronine (3,5-T2) *P<0·05 versus the corresponding euthyroid group; §P<0·05 (there were increased 3,5-T2 levels in serum and liver versus the group treated in the same way housed at 22 C. 12–24 h after T3 injection). Taking into account these facts, it could be rationalized that, after 24 h cold exposure, really lacking in T3 and, therefore, results obtained in T3 concentrations in IBAT of T4-treated animals increase T4-hyperthyroid rats cannot be attributed to an excess of due to an increased deiodination of T4, while T3 concen- T3 in BAT (Ablenda & Puerta 1992). Knowing that tration in IBAT of T3-treated animals probably decreases UCP1 was predominantly affected by the occupancy of due to T3 deiodination to di-iodothyronines. Since T3 BAT nuclear T3 receptors (Branco et al. 1999), it is nuclear receptors occupancy is dependent on IBAT T3 understandable why T4 treatment, under basal conditions, concentration, it is understandable why UCP1 content, does not enhance IBAT UCP1 content. This finding is not mGPDH and MnSOD activities as well as mitochondrial in agreement with the results reported by Branco et al. protein concentrations have not significantly increased (1999). This discrepancy may result from different doses after exposure of T3-treated rats to low ambient tempera- applied and different acclimation temperature. The maxi- ture. Measurement of D2 activity under the described mum T4 dose applied by Branco et al. (1999) was 12·5 experimental regimes would clarify this issue. times compared with 37·5 times as much as the physio- Despite the lower UCP1 content, rectal temperature of logical replacement daily dose in this study. Branco et al. T3-treated and cold-exposed rats was still significantly (1999) acclimated animals at 28 C, while 22 C was used higher than that of control cold-exposed animals. Since in this study. Since the SNS plays a much more significant di-iodothyronines, or at least 3,5-T2, are considered as role in nonshivering thermogenesis at any temperature active iodothyronines and not as simple products of T4 or below thermoneutral than at thermoneutral temperature, T3 metabolism (Goglia et al. 1999), it is likely that they it may be supposed that the decrement in the SNS activity can directly affect mitochondrial metabolism in IBAT. produced by T4 treatment influences UCP1 expression Di-iodothyronines can enhance the resting metabolic rate to a lesser degree at thermoneutral than at subthermo- independently of protein synthesis (Moreno et al. 1997) neutral temperature. On the other hand, in BAT, T3 and improve the cold tolerance of hypothyroid rats (Lanni has little ability, if any, of inhibiting D2 activity and even et al. 1998). 2-fold increases are reported (Silva & Larsen 1986). Mitochondrial UCPs are of interest for controlling the Tri-iodothyronine-induced hyperthyroidism increases D2 level of ROS. Nègre-Salvayre et al. (1997) demonstrated mRNA levels (Croteau et al. 1996) and cultured rat brown that UCP1 inhibition activated hydrogen peroxide gen- adipocytes require T3 for the adrenergic stimulation of D2 eration in brown fat mitochondria, and Echtay et al. (2002) mRNA expression and D2 activity (Martinez-deMena demonstrated that superoxide anion activates mito- et al. 2002). Hence, T3 application enables enough T3 to chondrial UCPs and suggested that superoxide–UCP directly occupy nuclear T3 receptors in IBAT so as to interaction may be a mechanism for decreasing ROS significantly increase UCP1 content. Similar results were concentrations inside mitochondria. This indicates that obtained by Branco et al. (1999), who showed augmented changes not only in oxidative metabolism, but also in UCP1 content with increasing T3 doses, and by Masaki UCP1 expression will provoke marked alterations in free et al. (1997) and Gong et al. (1997), who showed increased radical synthesis, and, consequently, in the control mecha- IBAT UCP1 mRNA levels after T3 treatment. nisms and limitations of these ions that have deleterious When SNS activity was stimulated by exposing animals effects on cellular processes. Our results correlate well with to 4 C, IBAT UCP1 content increased only in control this observation. In IBAT of rats made hyperthyroid by T4

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Downloaded from Bioscientifica.com at 09/24/2021 08:25:35PM via free access Effects of hyperthyroidism on rat brown adipose tissue · N PETROVICu and others 37 treatment, UCP1 content and SOD activity were not Bianco AC & Silva JE 1987 Optimal response of key enzymes and uncoupling protein to cold in BAT depends on local T generation. changed whereas MDA levels were significantly increased 3 253 as compared with the euthyroid group. In contrast, after American Journal of Physiology E255–E263. Branco M, Ribeiro M, NegrãoN&BiancoAC19993,5,3-Tri- T3 treatment both IBAT UCP1 content and MnSOD iodothyronine actively stimulates UCP in brown fat under minimal activity significantly increased but MDA concentrations sympathetic activity. American Journal of Physiology 276 E179–E187. were unaltered. Similarly, cold exposure of hyperthyroid Brand MD 2000 Uncoupling to survive? The role of mitochondrial rats induced different changes in MDA levels, depending inefficiency in ageing. Experimental Gerontology 35 811–820. on whether T or T was used for provoking hyper- CannonB&Lindberg O 1979 Mitochondria from brown adipose 4 3 55 thyroidism. Thus, in cold-exposed T -treated rats, IBAT tissue: isolation and properties. Methods in Enzymology 65–78. 4 Cassard-Doulcier AM, Gelly C, Fox N, Schrementi J, Raimbault S, UCP1 content as well as CuZnSOD, MnSOD and CAT Klaus S, Forest C, BouillaudF&Ricquier D 1993 Tissue-specific activities were markedly increased. Consequently, the and beta-adrenergic regulation of the mitochondrial uncoupling IBAT MDA concentration was significantly decreased in protein gene: control by cis-acting elements in the 5 flanking respect to that in animals treated in the same way but region Molecular Endocrinology 7 497–506. housed at room temperature (although MAO and Croteau W, Davey JC, Galton VA & St Germain DL 1996 Cloning of the mammalian type II iodothyronine deiodinase. 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Hence, it might be postulated that Bioenergetics and Biomembranes 34 105–113. both mitochondrial proteins, UCP1 – the proton conduct- Echtay KS, Roussel D, St-Pierre J, Jekabsons MB, Cadenas S, Stuart ance of which is activated by superoxide anion and JA, Harper JA, Roebuck SJ, Morrison A, Pickering S, Clapham JC which, by decreasing mitochondrial membrane potential, & Brand MD 2002 Superoxide activates mitochondrial uncoupling decreases superoxide anion production (Echtay et al. 2002) proteins. Nature 415 96–99. Gardner RS 1974 A sensitive colorimetric assay for mitochondrial – and MnSOD, the main scavenger of superoxide anion in -glycerophosphate dehydrogenase. Analytical Biochemistry 59 mitochondria, probably play a crucial role in the brown 272–276. adipocyte protection against ROS. Goglia F, Moreno M & Lanni A 1999 Action of thyroid hormones at the cellular level: the mitochondrial target. 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Our results clearly show that the ROS-stimulated damage HernándezA&Obregón MJ 1996 Presence and mRNA expression of IBAT membranes, evaluated by MDA concentration, ff of T3 receptors in di erentiating rat brown adipocytes. Molecular and depends not only on antioxidant enzyme activities but also Cellular Endocrinology 121 37–46. on UCP1 content. Lanni A, Moreno M, LombardiA&Goglia F 1998 3,5-Di-iodo-- thyronine and 3,5,3-tri-iodo--thyronine both improve the cold tolerance of hypothyroid rats, but possibly via different mechanisms. Acknowledgements Pflügers Archiv European Journal of Physiology 436 404–414. Leonard JL, Mellen SA & Larsen PR 1983 Thyroxine 5-deiodinase The Serbian Ministry of Science, Technologies and activity in brown adipose tissue. Endocrinology 112 1153–1155. Development Grant No 1550 supported this study. We Lowry OH, Rosebrough NJ, Farr AL & Randall R 1951 Protein measurement with the Folin phenol reagent. 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