3,5,3'-Triiodothyronine actively stimulates UCP in brown fat under minimal sympathetic activity

Marcelo Branco, Miriam Ribeiro, Nubio Negrão, and Antonio C. Bianco

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, 05508-900 São Paulo, Brazil

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To investigate the role of type II 5'-deiodinase (5'D-II) in the expression of mitochondrial uncoupling protein (UCP) in brown adipose tissue (BAT), we injected intact male rats with reverse (r) 3,5,3'-triiodothyronine (T3; 100 µg · 100 g body wt-1 · day-1), an inhibitor of 5'D-II, for 2-5 days. UCP decreased by ~20% in rats kept at 28°C and failed to increase during cold exposure (4°C). Next, thyroxine treatment (1-10 µg · 100 g body wt-1 · day-1) increased nuclear T3 in rats kept at 28 or 4°C. In these rats, nuclear T3 correlated positively with UCP. In addition, T3 (1-50 µg · 100 g body wt-1 · day-1) given to intact rats (5-15 days; 28°C) induced an approximately twofold increase in UCP. In these T3-treated animals, the interscapular BAT thermal response to norepinephrine infusion also correlated positively with T3 dose and UCP content. Treatment with propranolol or reserpine failed to block the T3 induction of UCP (~1.8- and ~2.3-fold). The results emphasize the importance of local 5'D-II and reveal an independent role of T3 in the expression of UCP.

thyroid hormones; 5'-deiodinase; norepinephrine; thermogenesis; cold acclimation; uncoupling protein

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

HEAT IS A UBIQUITOUS by-product of processes involved in the absorption, storage, and oxidation of biological substrates. Most heat production occurs during ATP synthesis due to the intrinsic thermodynamic inefficiency of the processes involved. Altogether, overall efficiency is only ~28%, and the remaining free energy eventually appears as heat. Mitochondria are the main site of ATP synthesis, which is based on the coupling between oxidative reactions and ADP phosphorylation. The energy from the oxidation of components of the respiratory chain is used to create an H+ electrochemical potential difference across the membrane where energy is transiently stored. Subsequently, as the mitochondrial ADP-to-ATP ratio increases, stored energy is used to drive a membrane-bound ATP synthase that phosphorylates ADP (see Ref. 27 for review).

Brown adipose tissue (BAT) contains a protein named uncoupling protein (UCP; see Ref. 20). UCP is organized in the inner mitochondrial membrane and functions to dissipate the H+ electrochemical potential, thereby uncoupling fuel oxidation from the phosphorylation of ADP (25). Consequently, stimulated BAT synthesizes relatively little ATP and liberates substantial heat that can serve physiological purposes, e.g., 1) warming the body during the arousal from hibernation or during cold exposure and 2) regulating body fat and, ultimately, body weight (10, 18, 34).

More recently, two new UCPs [named UCP-2 (13) and UCP-3 (8, 38) to differentiate them from the BAT UCP-1] have been identified also in the BAT mitochondria and in several other tissues where they have been implicated in the general imperfect coupling of mammalian mitochondria (14, 30). These proteins have a high degree of homology and similar molecular mass (32-34 kDa), and the net result of their function is also the exothermal movement of H+ through the mitochondrial membrane (8, 13, 25, 38). UCP-1 is exclusive to BAT where it is regulated mostly by norepinephrine (NE) and thyroid hormones (6, 19, 33). On the other hand, the mechanisms controlling UCP-2 and UCP-3 expression are the subject of intense ongoing investigation. UCP-2 is expressed in a number of tissues and was shown to be induced by a high-fat diet (13). The expression of UCP-3, however, is largely confined to the skeletal muscle, heart, and BAT, where it is also positively regulated by T3 (16, 21).

Thyroid hormones are deeply implicated in the regulation of energy expenditure (35). Even though these hormones have long been known to play a role in facultative thermogenesis, it was only recently that BAT was recognized as a major target for 3,5,3'-triiodothyronine (T3; see Refs. 4-7, 12). This tissue contains a substantial number of T3 receptors and also type II thyroxine (T4) 5'-deiodinase (5'D-II), which catalyzes the conversion of T4 to T3. As in the brain and pituitary gland, the overall BAT nuclear T3 receptor occupancy is >70% due to the contribution of locally generated T3, as opposed to the ~50% usually found in other tissues (5). In BAT, however, 5'D-II is markedly activated by sympathetic stimulation, resulting in a three- to fourfold increase in local concentration of T3 (36) and virtual saturation of nuclear T3 receptors (7). Consequently, processes that are primarily dependent on thyroid hormones are also activated. Therefore, the physiological changes that take place during cold-NE stimulation of BAT are actually a composite of interactions between NE- and T3-generated signals that eventually lead to heat liberation (1-3). As a result, mitochondrial UCP content increases rapidly, reaching a two- to threefold induction within ~5 days of cold stimulation, a process that involves approximately eightfold transcription stimulation of its gene. T3 potentiates the effects of NE on UCP gene transcription in vivo and in vitro (3). The molecular basis of this synergism relies on two functional thyroid hormone responsive elements (TRE) and other gene sequences (e.g., cAMP responsive element) that were recently identified in the UCP gene promoter at a location critical for gene control (37).

In addition to the transcriptional stimulation that is shut off by 12-24 h despite continuous adrenergic stimulation (31), UCP mRNA remains two- to threefold higher based on the approximately fourfold prolongation of its half-life (3, 31). This is also true in isolated brown adipocytes where T3 can increase the half-life of UCP mRNA even in the absence of NE (2). Altogether, these findings raise the possibility that the greater T3 impact on BAT resulting from the activation of local T4-to-T3 conversion is more important than previously recognized for UCP expression, particularly after the initial 12-24 h of cold exposure. In this case, as recently found for NE-induced BAT lipogenesis (1), an additional major role of NE-generated signals is to stimulate the local 5'D-II and to maintain a high BAT nuclear T3 receptor occupancy.

The aim of the present study was to evaluate the individual role of T3 in the UCP expression and to investigate the role of BAT 5'D-II in this process, particularly the possibility that, by influencing BAT T3 concentration in intact rats, it might bring about relevant changes in BAT UCP expression.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals and treatments. Unless specified otherwise, all drugs and reagents used in this section were purchased from Sigma Chemical (St. Louis, MO). Experiments were performed on male Wistar rats weighing 180-200 g, obtained from our breeding colony. Animals were maintained on a 12:12-h dark-light cycle either at 28-30°C or 3-5°C and had free accesses to food and water. When at 28°C, the animals were kept in collective cages (a maximum of 6 animals/cage) and were allowed to acclimate for at least 5 days before the experiments, to minimize BAT sympathetic activity. When at 4°C, the animals were kept in individual cages with bedding. Surgical thyroidectomy (Tx) was carried out under light ether anesthesia; on the same day, the animals were placed on 0.05% methimazole and then used at least 30 days later. Treatments included 1) two daily subcutaneous injections of one or more of the following: reverse (r) T3 (100 µg · 100 g body wt-1 · day-1), T4 (1-10 µg · 100 g body wt-1 · day-1), T3 (1-50 µg · 100 g body wt-1 · day-1), propranolol (20 mg · 100 g body wt-1 · day-1); 2) subcutaneous implant of a continuous-release pellet of T3 (0.6-2.4 µg · 100 g body wt-1 · day-1; Innovative Research of America, Sarasota, FL); or 3) addition of reserpine (~2.5 mg · kg body wt-1 · day-1) to the drinking water. Reserpine was dissolved in 5% acetic acid, and the iodothyronines were dissolved in 40 mM NaOH. Further dilutions were made in 0.9% NaCl before administration. Timing, length, and combination of the different treatments are described along with each experiment.

Interscapular BAT thermal response. All animals to be submitted to measurement of interscapular BAT (IBAT) temperature were anesthetized with urethan (1.2 g/kg ip) in the morning (9-10 h). A polyethylene (PE-50) cannula was inserted in the left jugular vein and later was used for NE infusion. IBAT temperatures (TIBAT) were measured using a precalibrated thermistor probe secured under the IBAT pad. The thermistor was connected individually to a multichannel amplifier that was then connected to a low-level DC 7PIK preamplifier, and the signals were recorded with a Grass model 7 polygraph (Grass Instruments, Braintree, MA). Based on pilot experiments, the whole system was calibrated to measure temperatures in the range of 34-40°C, with a gain of 0.2°C/mm. TIBAT was measured during a period of ~15 min to obtain a stable baseline, and then NE infusion was started. The NE infusion (6-8 µg · kg-1 · min-1) was performed with an infusion pump (Harvard model 2274) at a rate of 0.643 µl/min for 60 min. Longer NE infusion periods did not increase substantially the IBAT thermal response (data not shown). Raw data were plotted over time and expressed in terms of maximum Delta T (°C) or area under the Delta T (°C) vs. time (min) [area under the curve (°C · min)].

Sources of BAT nuclear T3 and T3 receptor occupancy. This procedure was carried out as described in detail elsewhere (5, 7). The tracers [131I]T3 (~3,300 µCi/µg) and [125I]T4 (~5,700 µCi/µg) were purchased from Du Pont NEN Research Products (Boston, MA). Injections consisted of either 20 µCi/100 g body wt of tracer T3 administered 2-3 h before killing or 100 µCi/100 g body wt of tracer T4 administered 6-8 h before killing. To determine the appropriate timing of the injections, we used Tm figures published for rats acclimated at room temperature or 4°C (5, 7). The Tm has been defined as that short interval of time when the influx and efflux of radioactive T3 to and from the nucleus are equal and is a critical variable to control in this type of quantitative in vivo analysis. During Tm, one can calculate the mass of nuclear T3 derived from either plasma T3 (T3[T3]) or locally from T4 (T3[T4]) from the specific activity of these iodothyronines in serum (5, 7). At the time of the injections, blood samples were obtained for measurement of T4 and T3 by RIA. Later, the animals were killed by exsanguination under ether anesthesia. IBAT was then removed, and nuclei were prepared as described previously (5, 7). DNA was measured by the method of Giles and Myers (15), and the recovery was 0.4-0.5 mg/g tissue, ~50%. Identification and quantification of iodothyronines in the nuclei and the serum were performed by paper chromatography for all labeled iodothyronines of interest (6), except for serum [125I]T3 derived from [125I]T4, which was measured by a combination of immunoaffinity and paper chromatography (6). Recovery of [131I]T3 added as internal standard was usually ~50%.

Analytical procedures. Rats were killed by perfusion with 0.9% NaCl over a period of 15 min, under chloral hydrate anesthesia (33%; 0.1 ml/100 g body wt ip). The IBAT was rapidly removed and processed as described for mitochondrial isolation (6) and UCP quantification (see below). Essentially, the BAT was homogenized in a Polytron (Omini Mixer, Waterbury, CT) with 4 ml of 0.01 M Tris base, pH 7.4, containing 0.32 M sucrose, 2 mM EDTA, and 5 mM 2-mercaptoethanol. The resulting homogenate was centrifuged at 10,000 g for 10 min, and the pellet was resuspended in 2 ml of the same buffer and centrifuged at 1,000 g for 10 min. The supernatant was recovered and centrifuged at 10,000 g for 10 min. This last step was repeated one time, and the final mitochondrial pellet was resuspended in the same buffer and stored at -20°C for further processing. Protein was determined by the method of Bradford (9).

UCP is a 32-kDa protein that can be identified easily and quantified by SDS-PAGE of BAT mitochondrial proteins (see Fig. 4). In such gels, the identity of this 32-kDa band was verified in previous publications by Western blots using rabbit antiserum against purified BAT UCP (6, 26), which did not cross-react with liver or skeletal muscle mitochondria (data not shown). However, in view of the recent identification of two additional UCPs in BAT with very similar molecular masses (33-34 kDa), it is likely that our quantification of an ~32-kDa band also included both new UCPs. However, because 1) no data are available in regard to the relative expression of these three proteins in BAT and 2) in previous publications the behavior of that ~32-kDa band mirrored the mRNA UCP-1 by Northern blotting (3, 4, 6), and it is likely that the detectable ~32-kDa band is mostly UCP-1. For the procedure, mitochondrial proteins were size fractionated by 12% SDS-PAGE as described (6, 26). The gel was then stained with Coomassie blue and scanned with a transmission densitometer at 595 µm (CS-9310PC; Shimadzu, Tokyo, Japan). Usually, 11.5 µg of total mitochondrial protein were loaded to each gel track. The precision error was ~6% when reading 450 ng of UCP. Sensitivity, as determined by loading different amounts of mitochondrial proteins, was ~250 ng UCP. The UCP concentration obtained for euthyroid rats acclimated at room temperature was ~40 ng/µg mitochondrial protein, which is in agreement with our previous data obtained by solid-phase immunoassay (6). Furthermore, in pilot experiments, we verified that the UCP BAT content increased by two- to threefold in a time-dependent manner when rats were exposed to cold for 2 wk, which is also in agreement with data from the literature (10, 18, 34).

BAT 5'D-II activity was measured as previously described (24), using BAT microsomal fraction and 2 nM 5'-[125I]rT3 (Du Pont NEN Research Products) as substrate in the presence of 1 mM propylthiouracil and 20 mM dithiothreitol. Mitochondrial alpha -glycerolphosphate dehydrogenase (alpha -GPD) was assayed as described previously (22).

Statistical analysis. Results are expressed as means ± SD throughout the text, Tables 1-4, and Figs. 1-4. Multiple comparisons were performed by one-way ANOVA followed by the Student-Newman-Keuls test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of rT3 administration on BAT UCP. To investigate the role of locally generated T3[T4] on UCP expression by intact rats, we took advantage of the rT3 capacity to inhibit 5'D-II (29). Treatment with rT3 lasted 2 or 5 days and was administered to rats acclimated at 28°C or exposed to cold for the same period of time. BAT 5'D-II was reduced to 15-20% in rT3-treated rats acclimated at 28°C (8.0-15 fmol I- · h-1 · mg protein) and to 25-30% in rats acclimated at 4°C (75-90 fmol I- · h-1 · mg protein) compared with vehicle-injected controls (75 or 300 fmol I- · h-1 · mg protein, respectively). Liver alpha -GPD activity was measured to monitor tissue T3 availability during treatment with rT3 and was found not to be significantly affected (6.9 ± 1.2 vs. 7.5 ± 1.3 Delta OD · min-1 · mg protein-1 · 10-2, found in controls).

The results obtained for BAT are presented in Fig. 1. Treatment with rT3 for 2 days resulted in a modest ~20% decrease of UCP concentration (P < 0.05 vs. vehicle group), an effect that was not present when treatment lasted for 5 days. Even though this had only minor consequences for UCP, it was a notable event because it occurred under conditions of minimal/stable sympathetic activity (animals were kept at 28°C), suggesting a small, but independent role of T3[T4] in UCP expression.


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Fig. 1.   Effects of reverse (r) 3,5,3'-triiodothyronine (T3) administration and cold exposure on brown adipose tissue (BAT) mitochondrial uncoupling protein (UCP) concentration. Intact rats were acclimated at thermoneutrality (28°C) and then were treated with rT3 (100 mg · 100 g body wt-1 · day-1 sc) and/or exposed to 4°C for 2 or 5 days. BAT mitochondrial UCP concentration was measured by densitometry in 10% SDS-PAGE loaded with 10 µg of total mitochondrial proteins. Statistical analysis was performed within each group, i.e., 2 or 5 days. * P < 0.05 vs. vehicle-injected rats acclimated at 28°C; ** P < 0.05 vs. vehicle-injected rats acclimated at 4°C. Entries are means ± SD of 3 rats. Degree of stimulation was calculated based on the UCP concentration of rats acclimated at 28°C.

As expected, cold exposure for 2 or 5 days increased mitochondrial UCP content by ~40% and ~2.8-fold, respectively (P < 0.05 vs. 28°C group). Now, when rT3 was injected in these stimulated animals, the mitochondrial UCP content did not rise, as observed in the vehicle-injected rats. In fact, by 2 days of cold exposure, UCP concentration did not change at all and, by 5 days, it only increased ~70% as opposed to the ~180% increase observed in the vehicle-injected rats.

Effects of T4 administration on sources of BAT nuclear T3, T3 receptor occupancy, and UCP. In the next set of experiments, BAT T3[T3] and T3[T4] were fine tuned by injecting progressively higher doses of T4 and subsequently measuring its impact on UCP concentration. T4 was administered for 5 days to rats acclimated at thermoneutrality or exposed to cold, at doses ranging from 1 to 10 µg · 100 g body wt-1 · day-1. In addition, the sources and amounts of nuclear T3 in BAT at the end of the treatment with T4 were measured by the dual-labeling technique.

The T3 sources and BAT nuclear T3 receptor occupancy of rats acclimated at 28°C are shown in Table 1. Treatment with T4 at doses up to 2.5 µg · 100 g body wt-1 · day-1 increased nuclear T3 receptor occupancy by 24% as a result of a greater contribution of T3[T4] (P < 0.05). With higher T4 doses, 5 and 7.5 µg · 100 g body wt-1 · day-1, nuclear T3 receptor occupancy stabilized or decreased slightly to rise by 34% when the dose of 10 µg · 100 g body wt-1 · day-1 was used (P < 0.05). At this dose, nuclear T3 receptor occupancy was basically sustained by plasma T3. This is probably the combination of decreased BAT 5'D-II activity and increased T4 deiodination via type I 5'-deiodinase (5'D-I), which is stimulated by thyroid hormones (35). In these same animals, UCP content increased significantly after treatment with T4 regardless of the dose used. When doses of 1, 2.5, and 5 µg · 100 g body wt-1 · day-1 were used, UCP concentration increased progressively up to 1.5-fold and then reached a plateau. A slightly higher dose of T4 (7.5 µg · 100 g body wt-1 · day-1) did not change UCP concentration any further. However, a dose of 10 µg · 100 g body wt-1 · day-1 of T4 caused a higher increase in BAT UCP concentration, up to 1.9-fold compared with vehicle-injected rats.

                              
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Table 1.   Effects of T4 administration on BAT mitochondrial UCP concentration and sources of BAT nuclear T3 and T3 receptor occupancy in rats acclimated at 28°C

In cold-exposed animals treated with the same doses of T4 (Table 1), a similar profile was observed in terms of nuclear T3 receptor occupancy and UCP concentration. However, here the changes were more marked, probably because BAT 5'D-II activity was increased to a higher level due to the sympathetic stimulation. One thing that is notable in these cold-exposed rats is that nuclear T3 receptor occupancy was close to virtual saturation at all times due to increased BAT 5'D-II activity (7). Treatment with T4 at all doses resulted in only small changes (5-6%) in BAT T3 receptor occupancy, which did not reach statistical significance. It is interesting, however, that as the T4 dose increased, the contribution of local T3 generation decreased (from 56 to 45%), and the contribution of plasma T3 increased accordingly (from 40 to 56%). In regard to the UCP content, the lowest dose of T4 (1 µg · 100 g body wt-1 · day-1) increased UCP concentration by 1.6-fold (P < 0.05). However, when the T4 dose was raised to 2.5 and 5 µg · 100 g body wt-1 · day-1, the increase in UCP concentration was less pronounced but still significant. Finally, when using T4 doses of 7.5 or 10 µg · 100 g body wt-1 · day-1, the UCP concentration increased by 1.5- or 1.8-fold, respectively (P < 0.05).

Next, data collected from these two experiments were used to plot nuclear T3 receptor occupancy vs. UCP mitochondrial content. The resulting exponential curve is presented in Fig. 2. It is clear that, regardless of the source, as T3 receptor occupancy increased the UCP concentration also increased exponentially. This was true for the animals acclimated at thermoneutrality and also for the animals exposed to cold. For a rise in nuclear T3 receptor occupancy from ~65 to ~90%, there was an approximately twofold increase in UCP concentration. In addition, when rats were placed in the cold, a rise from ~90 to ~100% resulted in another degree of stimulation of mitochondrial UCP, totaling almost threefold compared with basal levels.


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Fig. 2.   Relationship between BAT nuclear T3 receptor occupancy and BAT mitochondrial UCP concentration. Intact rats were acclimated at thermoneutrality (28°C) and then were treated with different doses of thyroxine (T4; 1, 2.5, 5, 7.5, or 10 µg · 100 g body wt-1 · day-1) and/or were exposed to 4°C for 5 days. UCP mitochondrial concentration was measured as described in the legend to Fig. 1. Nuclear T3 receptor occupancy was measured by the dual-labeling technique using [125I]T4 and [131I]T3 as described in MATERIALS AND METHODS. Animals were killed ~12 h after the last T4 injection. Individual values are presented in Table 1. Curve fits an exponential growth with R2 = 0.97 and rate constant of 0.026 ± 0.0035.

Effects of T3 administration on BAT mitochondrial UCP content of intact rats. The next set of experiments was designed to test whether plasma T3 could also affect UCP expression as we found with the locally generated T3. Here also the animals were kept at thermoneutrality. T3 administration lasted 5 days, and the different groups of animals received progressively higher doses of T3 (1, 5, 15, 25, and 50 µg · 100 g body wt-1 · day-1). Table 2 indicates that UCP concentration increased significantly (~35%; P < 0.05 vs. vehicle-injected group) even with the lowest dose of T3. Higher T3 doses resulted in proportionally greater increases in UCP concentration, reaching a 2.0 ± 0.03-fold increase (P < 0.05 vs. vehicle-injected group) when the highest (receptor-saturating) dose of T3 was used (50 µg · 100 g body wt-1 · day-1).

                              
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Table 2.   Effects of T3 on BAT mitochondrial UCP content

Effects of T3 treatment on the IBAT thermal response to NE infusion. To test further the effects of T3 alone on BAT UCP expression and IBAT thermal response, groups of Tx rats were treated with progressively higher doses of T3, except that this time treatment lasted 15 days and T3 was employed at a much lower dose range, i.e., 0.6, 1.2, or 2.4 µg · 100 g body wt-1 · day-1, and was delivered by means of a subcutaneously implanted release pellet. As shown in Fig. 3, in Tx untreated rats, UCP values were down to ~58% compared with euthyroid intact rats (~43 ng/µg mitochondrial protein). This was associated with a marked reduction in the IBAT thermal response compared with intact euthyroid rats. A T3 dose of 0.6 µg · 100 g body wt-1 · day-1, approximately twofold greater than the physiological T3 replacement dose, did not fully restore BAT UCP content, which was still ~30% below that of intact rats. However, even with this lower UCP concentration, the thermal response of the IBAT to NE infusion was fully restored and did not differ from intact controls. Indeed, it was notable that, with the next dose of T3 (1.2 µg · 100 g body wt-1 · day-1), the IBAT thermal response was already above normal, whereas the UCP concentration was still below the levels found in intact rats. In fact, UCP concentration was only normalized when Tx rats were treated with a dose of T3 that was approximately eightfold higher than the physiological replacement dose (2.4 µg · 100 g body wt-1 · day-1). At this level, the IBAT thermal response was two to three times greater than in intact controls. Remarkably, Tx rats treated with a relatively high dose of T3, ~16 times the physiological replacement dose (4.8 µg · 100 g body wt-1 · day-1), presented much higher UCP levels, ~2.5-fold above the intact rats. However, these animals did not present a proportionally greater thermal response, which tended to plateau. This might be explained by 1) a greater capacity of the IBAT to equilibrate its temperature with blood as the thermal gradient increases and/or 2) the existence of one or more limiting factors of the IBAT thermal response that are not proportionally stimulated by treatment with T3.


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Fig. 3.   Relationship between BAT thermal response, BAT mitochondrial UCP content, and dose of T3 administered. Thyroidectomized rats were acclimated at thermoneutrality (28°C) and then were implanted subcutaneously with a continuous T3 release pellet (0.6, 1.2, and 2.4 µg · 100 g body wt-1 · day-1) for 15 days. Before death, all animals were anesthetized with urethan and subjected to an in situ temperature measurement of the interscapular BAT (IBAT) during a 60-min iv infusion of NE (6-8 µg · kg-1 · min-1) in one of the jugular veins. Raw data were plotted over time and were expressed in terms of maximum change in temperature (Delta T) (°C; open circle ) or area under the Delta T (°C) vs. time curve (AUC; ). UCP mitochondrial concentration was measured as described in the legend to Fig. 1. Shaded areas enclosed by lines represent normal IBAT thermal response (horizontal) or the normal BAT UCP concentration (vertical) observed in intact control rats. Entries are means ± SD of 3-5 rats. BW, body wt.

Effects of adrenergic antagonists on the T3 induction of BAT UCP. To investigate the individual contributions of T3 or sympathetic nervous system (SNS) to the expression of BAT UCP, animals kept at 28°C were subjected to pharmacological adrenergic blockade and were treated with receptor-saturating doses of T3. Animals were treated with propranolol, a selective beta -adrenergic receptor blocker, or reserpine, a depletive of the cathecolamines stored in vesicles at the nerve end terminals, with or without T3. The results are presented in Table 3. As expected from the above results, T3 alone increased BAT UCP content by a factor of ~1.9 after 3 days (P < 0.05 vs. vehicle group). In the propranolol-treated rats, BAT UCP content decreased by 20% (P < 0.05 vs. vehicle group), indicating the contribution of the adrenergic input to basal UCP expression, even in these thermoneutrality-acclimated rats. It is noteworthy that, when the animals were treated with propranolol and T3, the BAT UCP increased by a factor of 1.8, not different from the animals that received T3 alone. Reserpine (2.5 mg · kg body wt-1 · day-1) was shown to be a much more potent adrenergic blocker. Alone, it decreased BAT UCP to ~60% of the control values. This effect is dose dependent, and higher reserpine doses (5-10 mg · kg body wt-1 · day-1) resulted in a more pronounced UCP drop down to 40% (data not shown). Nevertheless, T3 administration to these reserpine-treated rats increased BAT UCP by a factor of ~2.3 (P < 0.05 vs. reserpine group). This is one more line of evidence indicating that T3 by itself can induce UCP expression independent of the SNS.

                              
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Table 3.   Effects of sympathetic blockade by propranolol or reserpine on BAT mitochondrial UCP content

Comparison of T3- and cold-induced changes in BAT UCP. The next set of experiments was designed to carefully monitor the relationship between these two stimuli and the relative magnitude of the effects involved in BAT UCP expression. With that in mind, rats kept at 28°C were treated with receptor-saturating doses of T3 or were exposed to cold (4°C). Both treatments lasted exactly 5 days. As shown in Table 4, cold exposure was associated with an ~2.5-fold induction of UCP. The results obtained in the T3-treated rats are presented in greater detail in Fig. 4 and are also included in Table 4 for easier comparison. Treatment with T3 induced the UCP expression by a factor of ~1.9, ~30% lower than the induction observed in the cold-exposed rats.

                              
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Table 4.   Comparison of T3- and cold-induced changes in BAT UCP


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Fig. 4.   BAT UCP levels of T3-treated intact rats. Intact rats acclimated at room temperature were treated with T3 (20 µg · 100 g body wt-1 · day-1) for 5 days. This is an SDS-PAGE (10%) of total BAT mitochondrial protein. UCP was quantified by transmission densitometry. First track from the left contains 1 µg carbonic anhydrase (30 kDa). Each sample track was loaded with 13.1 µg of total mitochondrial protein. From left to right each sample track contained (T3-) 628, 655, 629 (637 ± 15) and (T3+) 1,323, 1,179, 1,310 (1,271 ± 80) ng of UCP, P < 0.05.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A general well-accepted concept derived from previous publications is that BAT stimulation by NE-cold exposure induces a severalfold increase in the local 5'D-II activity, which in turn increases the BAT T3 concentration by a factor of approximately three and results in virtual saturation of BAT nuclear T3 receptors. As a consequence, NE-generated signals interact synergistically with T3-generated signals to stimulate the expression of the UCP gene (2-7). However, these conclusions, although important, were obtained largely in Tx rats and converge on the role of the local 5'D-II in reducing the effects of hypothyroxinemia on BAT nuclear T3 receptor occupancy (12) or on the rapid revival of BAT thermogenic capacity and UCP gene expression after T4 administration (6).

In the present investigation, treatment of intact rats with rT3, a potent inhibitor of 5'D-II, blunted the typical cold-induced BAT UCP buildup, confirming the need, even in the euthyroid rat, for the activation of the local 5'D-II and the resulting increase in BAT T3. Accordingly, iopanoic acid, another potent inhibitor of 5'D-II activity, also limited the BAT mRNA[UCP] accumulation in cold-exposed Djungarian hamsters (32). These findings are in agreement with and strongly support the idea of synergism between T3- and NE-generated signals in the regulation of BAT UCP. However, and most important, a clue that T3 alone might play a role in the regulation of UCP was obtained in rats kept at 28°C that were treated with rT3. In such nonstimulated animals, there was a brief 20% decline in BAT UCP, which could be attributed to the rT3 blockade of the 5'D-II. These findings in rats acclimated at thermoneutrality, in which BAT sympathetic activity is supposedly minimal and stable, open room for the possibility that the higher occupation of nuclear T3 receptors normally found in BAT greatly contributes to the basal levels of UCP and, together with the results for cold-exposed rats, suggest that NE-induced further T3 buildup in the BAT of intact rats is more important than previously recognized for the cold-induced UCP expression.

To explore both possibilities, we promoted small changes of nuclear T3 receptor occupancy, particularly at the exponential portion of the nuclear T3 vs. UCP curve (occupation >70%), where the effects on UCP concentration would be detected more easily (4). The strategy was to treat intact rats with progressively higher doses of T4 (1- to 10-fold the physiological replacement daily dose). We anticipated, however, that, for each dose of T4, the resulting nuclear T3 would be a composite of three different effects, namely 1) decrease in BAT 5'D-II half-life and hence enzyme activity (23), 2) increase in plasma T4 and hence substrate availability to 5'D-II, and 3) increase in T3[T3] via 5'D-I present in other tissues. Therefore, we took advantage of the dual-labeling technique to measure the sources and amounts of nuclear T3 in BAT at the end of each treatment with T4. At thermoneutrality, the overall BAT nuclear T3 receptor occupation increased significantly with some T4 doses, from ~65 to ~90%. During cold exposure, BAT nuclear T3 receptor occupation remained high (90-100%), not significantly affected by T4 administration. However, when nuclear T3 was plotted against UCP, the result was a highly converging exponential curve with an R2 = 0.97. Most important, the shape of the nuclear T3 vs. UCP curve was not affected by the temperature at which the rats were kept. In fact, UCP was predominantly affected by the occupation of BAT nuclear T3 receptors. To test the potency of the T3 inducibility of UCP, rats kept at 28°C were treated with receptor-saturating doses of T3 in an attempt to trigger a maximal UCP response. Indeed, a comparison of T3 vs. cold induction of UCP for 5 days revealed that the T3 effect on UCP (~2-fold) was only ~33% lower than the cold effect on UCP (~2.5-fold), which is by itself a new and remarkable finding.

Next, we attempted to minimize even further the BAT sympathetic activity using high doses of adrenergic antagonists such as propranolol or reserpine. In fact, the administration of either drug caused the BAT UCP to decrease by 20-40%, confirming the continuing BAT sympathetic activity at thermoneutrality. However, it is important to note that, by reducing BAT sympathetic activity, we also reduced the T3-generating signals originated from the local 5'D-II, which is primarily regulated by the SNS. Nevertheless, although complete sympathectomy is hard to attain, it is notable that the BAT of these adrenergic antagonist-treated rats retained the full capacity to respond to the treatment with receptor-saturating doses of T3. Indeed, T3 induction of BAT UCP increased approximately twofold over basal levels in rats treated with either one of the adrenergic antagonists, not different from when T3 was injected in control rats.

These results provide new insights into the mechanisms involved in the maintenance of high levels of UCP during cold exposure and the relative importance of NE and/or T3. Indeed, because 1) BAT nuclear T3 receptors are fully saturated shortly after cold exposure is initiated (7) and 2) in view of the fact that T3 alone is capable of powerfully stimulating UCP expression, close to the cold-induced level, under conditions of stable and minimal sympathetic activity, it is tempting to speculate that this greater T3 impact on BAT might make an important contribution to the cold-induced UCP buildup.

As to the mechanism of T3 induction of BAT UCP, at least two pathways might be involved, namely, an increase in the rate of transcription initiation and/or in the mRNA[UCP] half-life (31). In fact, Obregón et al. (28) have shown in fetal BAT that increases in UCP mRNA correlate well with increases in BAT T3 concentrations. Based on the magnitude of the T3-induced changes in UCP found in the present investigation and on the presence of two fully characterized TREs in the UCP-1 gene (37), it would be fair to assume that both mechanisms might be activated during treatment with T3. At least in fetal rat brown adipocyte primary cultures, T3 induces the transcription of the UCP gene and stabilizes its mRNA (17). However, because in vivo the UCP-1 transcriptional regulation is highly dependent on the synergism between T3 and NE (3), it is conceivable that the T3 induction of UCP might be explained better by stabilization of mature mRNA UCP-1. This is by itself a powerful mechanism that does not seem to require such synergism to be activated (2, 17).

A final important element to be discussed is that the induction of BAT UCP by T3 does not necessarily mean BAT heat liberation nor does it contribute to the basal metabolic rate as recently suggested for the T3 induction of UCP-3 in skeletal muscle (16, 21). In BAT, the current theory claims that mitochondrial respiration remains coupled until the GDP, bound to the UCP, is displaced by cytosolic fatty acids upon NE stimulation. To evaluate this aspect in the present investigation, we measured the IBAT pad thermal response during NE infusion. In all cases, regardless of the T3 dose or size of the UCP pool, heat liberation was only detected minutes after NE infusion was started. Furthermore, the thermal response was proportional to the T3 dose and to the BAT UCP concentration. This is one more line of evidence indicating that the independent T3 induction of BAT UCP focuses on facultative thermogenesis and certainly is more important than previously recognized for the physiological BAT response to cold exposure. However, a new and exciting finding was that, in T3-treated Tx rats, the IBAT thermal response was normalized when its UCP content was only one-half of that found in intact euthyroid rats. This is an indication that BAT heat liberation is also strongly influenced by other UCP-independent mechanisms, possibly an amplification at the cAMP/cAMP-dependent protein kinase A signaling level (11) or at the formation of the mitochondrial electrochemical potential, which might be activated during hypothyroidism (hypothyroxinemia) to compensate for lower UCP levels.

In conclusion, the present investigation produced hard evidence that minor alterations of the BAT nuclear T3 receptor occupancy, in intact rats, produce substantial changes in UCP concentration, even in the nonstimulated BAT. Changes of 5'D-II activity are rapidly translated into adjustments of BAT nuclear T3, in turn an important determinant of UCP expression. In addition, the results expand the common knowledge that T3- and NE-generated signals act in synergism to stimulate BAT expression of UCP. Our data also indicate that T3 alone vigorously stimulates UCP expression under minimal interference of the SNS and might be responsible for up to 60% of the increase in BAT UCP during cold exposure.

    ACKNOWLEDGEMENTS

We are grateful to José Luiz dos Santos and Alessandra Crescenzi for technical assistance.

    FOOTNOTES

This work was supported partially by a research grant provided by Fundação de Amparo à Pesquisa do Estado de São Paulo. M. Branco is the recipient of a scholarship grant from Conselho Nacional de Pesquisa.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: A. C. Bianco, Rm. 560, Harvard Institutes of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115. E-mail: acbianco{at}usp.br.

Received 21 January 1998; accepted in final form 3 September 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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