0013-7227/91/1284-2149$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 4 Printed in U.S.A.
Central Role of Brown Adipose Tissue Thyroxine 5'-Deiodinase on Thyroid Hormone-Dependent Thermogenic Response to Cold* SUZY D. CARVALHO, EDNA T. KIMURA, ANTONIO C. BIANCO, AND J. ENRIQUE SILVA Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, 05508 Sao Paulo, Brazil; and the Thyroid Unit, Division of Endocrinology, Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215
ABSTRACT. As judged by the response of uncoupling protein and key enzymes, brown adipose tissue (BAT) is highly dependent upon the local generation of T3 catalyzed by the tissue type II T4 5'-deiodinase (5'D-II). In hypothyroid rats treated with T3 or T4, the capacity to withstand cold seems better correlated with the normalization of BAT responses than with the liver thyroid status. 5'D-II is activated by cold via sympathetic nervous system (SNS) stimulation, and the activation generates enough T3 to nearly saturate BAT nuclear T3 receptors (NTR) in euthyroid rats. In hypothyroidism, 5'D-II is highly stimulated by the SNS and hypothyroxinemia. In the present studies we have taken advantage of this situation to test 1) the capacity of 5'D-II to maintain nuclear T3 in rats with various degrees of hypothyroxinemia, and 2) the hypothesis that thyroid hormonedependent BAT-facultative thermogenesis, rather than the effect of thyroid hormone on obligatory thermogenesis (basal metabolic rate), is the basic mechanism by which thyroid hormone confers protection against acute cold exposure. We treated methimazole-blocked rats (undetectable plasma T4 and T3) for a week with either subreplacement doses of T4 (0.5, 1, 2, and 4 Mg/kgday) or replacement doses of T4 or T3 (8 or 3 ^g/kgday, respectively). Sources and content of BAT nuclear T3 were studied at 25 C and after 48 h at 4 C by labeling the plasmaborne T3 (T3[T3]) with [131I]T3 and the locally generated T3 (T3[T4]) with [125I]T4. Neither the kinetics of nuclear-plasma exchange of T3[T3], the time of appearance of T3[T4] in BAT nuclei, nor NTR maximal binding capacity (0.71 ng T3/mg DNA)
H
YPOTHYROIDISM is associated with multiple alterations in virtually every tissue of the body, including retardation or arrest of differentiation and growth and decreased basal metabolic rate. Among the most striking shortcomings of hypothyroid animals is the inability to adapt to cold environments (1). We have recently shown that the inability to maintain body temReceived October 22,1990. Address all correspondence and requests for reprints to: J. Enrique Silva, M.D., Thyroid Unit, Department of Medicine, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215. * This work was supported by Grants 88/3645-9 from Fundacao de Amparo a Pesquisa (Foundation for Research Support) of Sao Paulo and ROl-DK-42431 from NIK.
was affected by hypothyroidism. Kinetic analyses indicated a maximal BAT NTR occupancy of 40% at euthyroid serum T3 concentrations if T4 is not present. Replacement with T4 normalized both serum T4 and T3, while replacement with T3 normalized serum T3; for all other doses of T4, serum T4 and T3 concentrations were predictably related to the dose. 5'D-II activity decreased with increasing doses of T4, but for each dose of T4, this activity was 2-4 times greater at 4 C than at 25 C. BAT NTR occupancy normalized with 2 fig T4/kg in rats maintained at 25 C and with 4 fig T4/kg in cold-exposed rats, although in neither condition were serum T4 and T3 normalized nor more than 30% of NTR occupied by plasma T3. The thermogenic response to cold, measured as an increase in oxygen consumption (QO2), was significantly greater in T4-replaced than in T3-replaced rats. Two micrograms of T4/kg/day sufficed to normalize the response of QO2 to cold. Prazosin, which blunts the adrenergic-induced stimulation of 5'D-II, caused a marked reduction in T3[T4] and blunted the T4-dependent increase in QO2. We conclude that the protective effect of thyroid hormone against coldinduced hypothermia is largely the result of the stimulation of facultative thermogenesis in BAT, stimulation in which the adrenergic activation of BAT 5'D-II plays an essential role. This enzyme is, furthermore, of adaptive value in hypothyroidism by allowing a normal thermogenic response to cold in the face of substantial reductions in plasma T4. (Endocrinology 128: 21492159,1991)
perature in severe cold can be better correlated with a defective response of brown adipose tissue (BAT) to acute cold exposure than with the thyroid status of the liver, as reflected by a-glycerophosphate activity. These findings suggest that the inability of hypothyroid animals to adapt to cold results from a defective facultative thermogenesis rather than from a depressed obligatory thermogenesis (basal metabolic rate) (2, 3). A major, probably the main, site of facultative thermogenesis in small mammals, including the human newborn, is BAT (4). BAT thermogenesis can be activated by cold or overfeeding (diet-induced thermogenesis) via the sympathetic nervous system (SNS) (5). The adrenergic input
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BROWN FAT, THYROID HORMONE, AND RESPONSE TO COLD
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into the tissue activates and stimulates the synthesis of a unique protein, uncoupling protein (UCP), which, by uncoupling oxidative phosphorylation, boosts BAT respiration and dissipates the resulting energy as heat (5, 6). Thyroid hormones and SNS interact synergistically in BAT. Mitochondrial UCP expression is decreased in hypothyroid rats acclimated to room temperature and fails to increase after cold exposure (2, 3, 7, 8). T 3 increases the rate of synthesis of this protein by amplifying the induction of the gene by norepinephrine (9). On the other hand, the sympathetic nervous system stimulates the type II T4 5'-deiodinase (5'D-II) present in the tissue (10), producing enough T 3 to nearly saturate the nuclear receptors (NTR) for this hormone (11). This high level of NTR occupancy has been found to be essential for a maximal response of UCP to acute cold exposure (2, 3). In the present studies we aimed to obtain further evidence to support the growing concept, emerging from our previous studies (2, 3, 11), that the adaptive role of thyroid hormone in temperature homeostasis is largely mediated by its stimulation of facultative thermogenesis in BAT. For this, we took advantage of the central role of 5'D-II in providing T 3 for BAT. By treating hypothyroid rats with replacement and subreplacement, i.e. suboptimal, doses of T4, or replacement doses of T3, we generated various levels of NTR occupancy. BAT NTR occupancy this time was related to a more direct measure of the thermogenic response to cold, namely oxygen consumption (QO2). Our results support the hypothesis that the protective effect of thyroid hormone from coldinduced hypothermia results from its stimulation of facultative thermogenesis in BAT, and that in this physiological function of thyroidal secretion, BAT 5'D-II plays a pivotal role; our results also indicate that BAT 5'D-II can effectively protect the thermoregulatory mechanisms in hypothyroidism of significant magnitude. Materials and Methods Animals and treatments Sprague-Dawley male rats, weighing 145-165 g, were used throughout. Hypothyroidism was induced by adding 0.02% methimazole (MMI) to the drinking water during at least 30 days [in 1 week, serum T4 and T 3 become undetectable, and in 3 weeks there is complete arrest of growth (12)]. Water and regular rat chow were provided ad libitum. Rats were maintained on a 14-h light, 10-h dark cycle and were acclimated at 25 C. For cold exposure, rats were individually housed in plastic cages with wood shaving bedding and placed at 4 C for up to 48 h as indicated, without changing the light-dark cycle. T4 or T3 was given daily for a week by the sc route in two divided doses, with the total daily dose being equal to (replacement dose) or less than the daily production rates (13, 14). Unless
Endo • 1991 Vol 128 • No 4
indicated otherwise, serum concentrations were measured approximately 12 h after the last dose. Prazosin in injectable formulation (Minipress, Pfizer Laboratories, New York, NY) at a concentration of 50 mg/ml was given ip in an initial dose of 4 mg/kg, followed by doses of 2 mg/kg at approximately 6-h intervals (10) while the animals were exposed to cold. These experimental protocols have been approved by the Harvard University Standing Committee on Animals and Beth Israel Hospital Committee on Animal Research (no. 191.128.02). Tracer kinetics Tracers [125I]T3 or [131I]T3 (SA, -3000 /xCi/Mg), and [125I]T4 (SA, ~4200 /iCi/jug) were prepared in our laboratory as described previously (15). Radioactive T 3 was over 95% pure, with the main contaminant being iodide, and [125I]T4 contained less than 0.5% [125I]T3, measured as outlined below. The tracers were injected with or without a load of unlabeled T 3 to assess the nonspecific binding. Injections consisted of either 20 /uCi/ 100 g BW tracer T3 or 100 MCi/100 g BW tracer T4. They were mixed with unlabeled hormone, when appropriate, and injected in one of the external jugular veins under light ether anesthesia in 0.1-0.2 ml vehicle. The latter was 10% normal rat serum in 0.9% saline containing 0.1% Nal (16). At the time of injections, blood samples were obtained for measurement of serum T4 and T 3 by RIA (17, 18). The animals were killed by aortic exsanguination under ether anesthesia, and tissue samples were handled as described previously and summarized below (16). The model used for the studies to be described is based on that developed by Pearson et al. (19) to study the distribution kinetics of albumin in humans. This model was validated by Oppenheimer et al. (20) to study nuclear plasma exchange of T 3 and by us to assess the contribution of intracellularly generated T 3 from T4 to nuclear T 3 (21); specific aspects relevant to BAT have been discussed in detail in previous publications (11,16). In essence the method consists of pulse labeling plasma T 3 or plasma T4 and allowing nuclear tracer T 3 to reach equilibrium with the plasma source, i.e. T 3 or T4. Equilibrium is only transient owing to the relatively rapid fall of plasma tracer iodothyronines. During this short interval of time, called equilibrium time point or Tm (19-21), one can calculate the mass of nuclear T 3 derived from either plasma T 3 (T;t[T3]) or locally from T4 (T3[T4]) from the specific activity of these iodothyronines in the serum (16, 19-21). In the calculation of locally produced T3[T41, one has to deduct from the observed nuclear [125I]T3[T4] the contribution of [125I]T3 present in plasma. This latter largely derives from T4 to T 3 conversion in other tissues and, to a lesser extent, from the small amounts of [125I]T3 contaminating [125I]T4. For this correction, the product of the equilibrium nuclear to serum ratio of [131I]T3 by the serum concentration of [125I]T3 is subtracted from the observed nuclear [125I]T3 (16, 21). The local nuclear [125I]T3[T4] so obtained is multiplied by 2 to correct for the halving of the specific activity inherent to 5'-deiodination. The number is further multiplied by 651/777, to correct for the change in mol wt, when the mass of T3[T4] is calculated. Tissue processing and analysis The whole interscapular BAT pad (270-350 mg) was processed for preparation of cell nuclei (16). All procedures were
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BROWN FAT, THYROID HORMONE, AND RESPONSE TO COLD carried out at 0-4 C to minimize dissociation and reassociation of tracers to subcellular fractions (16). Briefly, tissue samples were weighed and immediately placed in 0.25 M sucrose and subsequently minced, blotted, and homogenized with a motordriven Teflon-glass homogenizer in 20 vol ice-cold 0.32 M sucrose, 1 mM MgCl2. The 1000 x g pellet was resuspended in 2.3 M sucrose containing 1 mM MgCl2 and centrifuged at 84,000 x g for 30 min. This high speed pellet was resuspended in 0.32 M sucrose, 1 mM MgCl2, and 0.5% Triton X-100, buffered with 10 mM Tris at pH 7.4, and centrifuged again at 1000 X g for 10 min; this step minimized the nonspecific binding of radioactivity to the nuclei. The final nuclear pellets were then rapidly counted and extracted in butanol saturated with 2 N HC1 for chromatographic analysis of the iodothyronines. DNA was measured by the method of Giles and Myers (22). The recovery of DNA was 60-70%. Identification and quantitative analysis of radioactive iodothyronines in the nuclei and serum were performed by paper chromatography for all iodothyronines of interest (23), except for serum [125I]T3 derived from [125I]T4 injection. The latter as well as the [125I]T3 contaminating the [125I]T4 were measured by a combination of immunoaffinity and paper chromatography, described previously (24). The immunoaffinity chromatography results in up to 1000-fold enrichment of T 3 over T4. Recovery of [l31I]T3, either from injection or added to the tubes, was used as internal standard, and it usually ran in the 45-55% range. BAT type II 5'D activity was measured as previously described (25), using 2 nM 5'-[125I]rT3 as substrate in the presence of 1 mM propylthiouracil and 20 mM dithiothreitol. Although T4 is a better substrate for 5'D-II, the physiological responses of the enzyme are equally reflected with either substrate (25, 26), and [125I]rT3 has a longer shelf life than [125I]T4. Indirect calorimetry was used to estimate thermogenesis. QO2 was measured in a Beckman gas analyzer (Palo Alto, CA) that measures both O2 and CO2 during periods of 10 min. Rats are placed in individual closed chambers, and the air of each chamber is analyzed for period of 10 min in a successive fashion, so that in 2 h each rat in an experimental group of four is analyzed three or four times. Results shown are the average of these readings in intervals of 2 h. Studies were performed at 25 or 4 C, as indicated. During the period of recording, rats were kept at the specified temperature with free access to food and water and subjected to the same light-dark cycle. Measurements were performed during the daytime hours, usually between 0900-1200 h. Readings of QO2 were corrected by barometric pressure, temperature, humidity, and CO2 and expressed as milliliters of O2 per min~7kg~075; this latter represents a weight-based estimate of the surface area. Since all rats were fed the same diet, and respiratory quotients were similar, results are merely presented as QO2.
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Results are reported as the mean ± SEM. For simplicity, mention of statistical significance is only made when relevant to an argument.
Results Effect of ambient temperature on the kinetics of nuclearplasma exchange and nuclear binding of T3 in hypothyroid rats Since the treatments of the rats (hypothyroidism and cold exposure) and their interaction may shift the Tm, this must be determined under the precise experimental conditions under which estimates of the mass of nuclear T 3 will be made. The results of these studies are shown in Figs. 1 and 2. In the experiment illustrated by Fig. 1, rats were injected with [125I]T3. Ambient temperature (25 C or 48 h at 4 C) did not affect either the clearance of plasma T 3 or the time and magnitude of nuclear binding. Tm occurred between 2 and 3 h after the tracer injections regardless of the ambient temperature, which was not different from the Tm determined for euthyroid rats (16). After the Tm, nuclear tracer T 3 fell, slowly approaching the rate of plasma disappearance. The nuclear serum ratio (N/S) of [125I]T3 at 2.5 h was approximately 0.6 ml/mg DNA for both groups of rats. Figure 2 shows the accumulation of locally produced nuclear [125I]T3 and plasma disappearance of [125I]T4 in hypothyroid rats similarly treated. In rats acclimated at 25 C, maximal nuclear [125I]T3[T4] uptake occurred at 6 h, whereas in the cold-exposed animals, it continued to increase until 8 h (P < 0.001). Both, the higher nuclear
Serum 25°C Serum 4°C
°i
?.
\
.
—m- Nuclei25°C Nuclei 4°C
1 -
8
Statistical analyses Data were analyzed by one (AOV)- and two-way analyses of variance (TWAOV), followed by multiple comparisons using the Neuman-Keuls test. Nuclear T 3 binding data were linearized by plotting nuclear to serum ratio of tracer T 3 us. the calculated mass of T 3 specifically bound to the nuclei and analyzed by linear regression, as previously described (11, 16).
Time after
125
10
I - T3 injection (hr)
FIG. 1. Time course of serum and nuclear concentrations of [125I]T3 in hypothyroid rats injected iv at time zero with 20 /uCi/lOO g BW [125I] T3. Rats were maintained at either 25 C or housed at 4 C for 48 h, as indicated. Serum and nuclear [125I]T3 were measured as described in Materials and Methods. Each point represents the mean ± SEM of four rats. TWAOV showed no significant effect of ambient temperature.
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BROWN FAT, THYROID HORMONE, AND RESPONSE TO COLD
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Endo • 1991 Voll28«No4
/mi;
Serum 25°C 0) V)
o
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Serum 4°C
Q O)
E
2-
Nuclei 25°C
|
Nuclei 4°C
B 400
(A
o •o n N
'"3E Q)
CO
ro a> o 3
Z
T3 Nuclear/Serum Ratio (ml/mg DNA) 10
15
20
O Time after 125, l-T 4 injection (hr) 125 FIG. 2. Time course of serum [ I]T4 and nuclear [125I]T3[T4] in hypothyroid rats injected iv at time zero with 100 ^Ci/100 g BW [125I]T4. Rats were maintained either at 25 C or housed at 4 C for 48 h, as indicated. Serum and nuclear radioactive iodothyronines were measured as described in Materials and Methods. Nuclear [125I]T3[T4] corresponds to the corrected, locally produced [125I]T3, as the contribution made by plasma [I25I]T3 has been subtracted, and the resulting value has been multiplied by 2 to correct by the 50% drop in specific activity derived from the outer ring deiodination. Each point represents the mean ± SEM of four rats. TWAOV shows no significant effect of cold on serum [125I]T4, and a marginal effect of cold on nuclear [125I]T3[T4] (P < 0.05) due to the 8 h point, which was significantly higher at 4 than at 25 C (P < 0.001).
[125I]T3[T4] concentrations and the slight delay for [125I] T3[T4] to peak in the nuclei observed in the cold-exposed animals reflect the enhanced fractional T 4 to T 3 conversion induced by cold in BAT, as previously reported (11) and further documented below by a higher 5'D-II activity in the cold-exposed rats. As depicted in Fig. 2, this short cold exposure did not significantly affect serum [125I]T4 levels. As a consequence, the local nuclear [125I]T3[T4] to serum [125I]T4 ratio was about 60% higher in the coldexposed rats ([4.6 ± 0.18 x 10~2] vs. [2.8 ± 0.26] x 10"2; P < 0.005). Maximum binding capacity (MBC) and apparent affinity of BAT NTR in hypothyroid rats maintained at room temperature For forthcoming calculations of NTR occupancy, we estimated the MBC of BAT T 3 receptors by in vivo saturation analysis, as previously described for room temperature-acclimated or cold-exposed euthyroid rats (11, 16). Several groups of three rats each were injected with [125I]T3 and various doses of unlabeled T3, and killed at the Tm (2.5 h after the injection). Results are presented in Fig. 3. Data plotted as indicated fitted a straight line with coefficient of correlation of -0.985 (P < 0.001; F = 417 for the linear correlation, while attempts to fit
FlG. 3. In vivo saturation analysis of BAT nuclear receptor affinity
and MBC in hypothyroid rats. Data have been linearized as shown; the inset represents the nontransformed data. In the linearized plot, each dot represents one rat, and the dose of cold T3, in micrograms per 100 g, has been indicated by different symbols; in the inset, each dot represents the mean nuclear T 3 ± SEM for each dose of cold T3. See Materials and Methods for details regarding conceptual background and techniques. Regression analysis of the linearized data showed a coefficient of correlation of -0.985 (P < 0.0001). The F value was 417.31 for the linear equation; attempts to fit to a curvilinear model resulted in smaller F values. The calculated MBC was 713 pg/mg DNA, with 95% confidence limits of 683 and 743. The slope of the curve represents the serum T 3 level required to occupy 50% of the MBC; the value was 0.95 ng/ml, with 95% confidence limits of 0.85 and 1.05 ng/ml.
the data to curvilinear function gave lower F values). The calculated MBC was 0.71 ng/mg DNA, with 95% confidence limits of 0.683 and 0.743 ng/mg DNA. The MBC was not significantly different from the one observed in euthyroid rats [95% confidence limits, 0.730.69 ng/mg DNA (11)]. The slope of this line represents the plasma concentration of T 3 to occupy 50% of NTR ([T3]50) and was 0.95 ng/ml, with 95% confidence limits of 0.85 and 1.05 ng/ml. This means that at euthyroid plasma T 3 levels (~0.55 ng/ml) nuclear occupancy would not be greater than 40%, and that in order to reach the levels of 95% occupancy or more seen in cold-exposed rats (11), plasma T 3 should be 18 ng/ml or greater, that is about 40-fold the euthyroid level. It is also worth noting here that [T3]50 in hypothyroid rats is significantly lower than that in euthyroid rats at room temperature (1.26 ng/ml) (16) or exposed to cold for 3 weeks (3.12 ng/ml) (11). This progressive increase in [T3]50 as the local BAT T 3 production from T4 increases reflects the dilution of plasma T 3 by the T 3 generated within the brown adipocytes. NTR occupancy and sources of nuclear T3 in rats with varying degrees of hypothyroidism: effects of cold exposure Rats made hypothyroid with MMI were given graded doses of T4 for a week, and the sources and quantity of
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BROWN FAT, THYROID HORMONE, AND RESPONSE TO COLD
exposed rats with regard to the contribution of plasma T 3 to BAT nuclear T 3 (F = 3.86; P = NS), in accord with the lack of effect of cold on the N/S ratio of [131I]T3; in both groups, nuclear T3[T3] was solely a function of T4 dose-dependent plasma T 3 (F = 88.89; P < 0.0001). There was also a T4 dose-dependent increase in nuclear T3[T4] (F = 70.64; P < 0.0001), but in this case cold exposure caused a significant approximately 2-fold increase in nuclear T3[T4] for each T4 dose (F = 104.97; P < 0.0001). Consequently, and because of the significance of the contribution of T3[T4], total BAT nuclear T 3 was also T4 dose-dependent (F = 174.5; P < 0.0001) and significantly greater in the cold-exposed rats (F = 69.24; P < 0.0001). With regard to NTR occupancy, in the rats acclimated at 25 C it plateaued at about 60% with the dose of 2 /ig T4/kg-day, a rate of occupancy that is not different from the value of approximately 70% found in intact rats acclimated to 23 C (16). It should be noted that with this dose of T4, serum T4 was about 30% and serum T 3 about 60% of the corresponding values in fully replaced rats (Table 2) or intact euthyroid rats (2, 3, 16). In coldexposed rats, multiple comparison analysis indicated that nuclear occupancy plateaued at about 85% with the dose of 4 /ug/kg-day, although with this dose of T4, serum T4 and T 3 were significantly lower than in the fully replaced rats (Table 2); most importantly, the contribution of T3[T4] to nuclear T 3 was identical to that of fully replaced rats in spite of a 45% lower serum T4 in the rats given4)iigT4/kg-day. To assess the effect of adrenergic activation of 5'D-II on the maintenance of BAT nuclear T 3 content, groups of animals identically treated and exposed to 4 C for 48
nuclear BAT T 3 were assessed as described in Materials and Methods. Studies were performed in rats acclimated at 25 C and in rats exposed to 4 C for 48 h. Radioisotopic data are presented in Table 1, and the calculated mass of BAT nuclear T 3 in the same rats is presented in Table 2. Table 1 shows the serum concentration and nuclear content of [131I]T3, the serum concentration of [125I]T4, the nuclear content of [125I]T3[T4], and the corresponding N/S ratios. All values were obtained at the respective Tm for which [131I]T3 was injected 2.5 h before killing the rats and [125I]T4 either 6 or 8 h (cold-exposed rats) before killing the rats. TWAOV shows no effect of the dose or ambient temperature on the serum concentrations of [131I]T3 and [125I]T4. The nuclear content and N/S ratio of [131I]T3 decreased significantly with the dose of T4, so that in the rats treated with the replacement dose of T4, they are about 50% the values seen in untreated rats. Likewise, nuclear [125I]T3[T4] and the nuclear [125I]T3[T4] to serum T4 ratio decreased with T4 in a dose-dependent fashion, but in this case the drop with the full replacement dose of T4 was more than 50%. Cold exposure did not affect the T4-induced fall in nuclear [131I]T3 and the corresponding N/S or the T4-induced fall in nuclear [125I]T3[T4] and the ratio of this to serum [125I]T4; however, for each dose of T4, nuclear [125I]T3[T4] and its ratio to serum [125I]T4 were higher in the coldexposed rats by factors of about 1.5 and 2, respectively (P< 0.001). The results in Table 2 show a T4 dose-dependent increase in both serum T4 and T3, but cold did not significantly affect either value by TWAOV. There was no difference between room temperature- and coldTABLE 1. Serum and BAT nuclear concentrations of [l31I]T3) [125I]T4) and with [131I]T3 and [125I]T4 and housed at different ambient temperatures 1
Dose of T4 (^g/kg-day)
Serum (%/ml X 10)
I. Acclimated at 25 C 0 0.5 1 2 4 8 II. After 48 h at 4 C 0 0.5 1 2 4 8
1.2 1.2 1.2 1.1 1.1 1.2
±0.1 ±0.1 ±0.1 ±0.1 ± 0.1 ± 0.1
1.2 ± 1.1 ± 1.1 ± 1.2 ± 1.2 ± 1.2 ±
0.1 0.1 0.1 0.1 0.2 0.1
5
I]T3[T4] at the equilibrium time point in hypothyroid rats injected 5
I]T3
Nuclei (%/mg DNA x 10)
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N/S
Serum T4 (%/ml)
I]T4 or [125I]T3
Nuclear T3 (%/mg DNA x 10)
0.75 ± 0.63 ± 0.59 ± 0.53 ± 0.46 ± 0.45 ±
0.1 0.1 0.1 0.1 0.1 0.1
0.65 ± 0.56 ± 0.50 ± 0.46 ± 0.42 ± 0.38 ±
0.02 0.01 0.01 0.02 0.02 0.01
1.8 ± 1.9 ± 2.1 ± 2.1 ± 2.3 ± 2.6 ±
0.1 0.1 0.1 0.1 0.1 0.2
0.53 ± 0.50 ± 0.50 ± 0.42 ± 0.25 ± 0.19 ±
0.02 0.03 0.01 0.03 0.01 0.01
0.82 ± 0.62 ± 0.53 ± 0.54 ± 0.49 ± 0.44 ±
0.1 0.1 0.1 0.1 0.1 0.1
0.68 ± 0.56 ± 0.48 ± 0.45 ± 0.41 ± 0.37 ±
0.01 0.01 0.01 0.01 0.00 0.01
1.3 ± 0.1 1.6 ± 0.1 1.6 ± 0.1 1.7 ± 0.1 1.9 ± 0.1 2.1 ± 0.1
0.76 ± 0.71 ± 0.68 ± 0.57 ± 0.38 ± 0.31 ±
0.03 0.01 0.04 0.05 0.02 0.01
N-T3/S-T4 x 100 3.0 ± 0.1 2.7 ± 0.1 2.5 ± 0.1 2.0 ± 0.2 1.1 ±0.1 0.74 ± 0.1 6.1 ± 5.1 ± 4.2 ± 3.3 ± 2.0 ± 1.5 ±
0.1 0.2 0.3 0.2 0.1 0.1
All values are the mean ± SEM of four rats. [125I]T4 was injected 6 or 8 h (cold exposed) and [131I]T3 2.5 h before killing the animals. After subtracting from the total observed nuclear [125I]T3[T4] the contribution made by plasma [125I]T3[T4], the corrected values were multiplied by 2 to correct for random deiodination of [125I]T4. TWAOV showed no effect of T4 on serum [131I]T3 and no effect of cold on nuclear [131I]T3) [131I]T3 N/S ratio, and serum [125I]T4. Nuclear [125I]T3[T4] and N-[125I]T3[T4]/S-[125I]T4 were significantly higher in cold-exposed rats (P < 0.001). See text for more details.
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BROWN FAT, THYROID HORMONE, AND RESPONSE TO COLD
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Endo • 1991 Vol 128 • No 4
TABLE 2. Serum concentrations of T4 and T3, and sources and content of BAT nuclear T 3 in hypothyroid rats treated with various doses of T4 and housed at 25 C or exposed to 4 C for 48 h Serum cone (ng/ml)
Dose of T 4
(Mg/kg-day)
Nuclear T 3 (pg/mg DNA)
Nuclear occupancy
T4
T3
T3[T3]
T3[T4]
Total
%
Vol. 128, No. 4 Printed in U.S.A.
Central Role of Brown Adipose Tissue Thyroxine 5'-Deiodinase on Thyroid Hormone-Dependent Thermogenic Response to Cold* SUZY D. CARVALHO, EDNA T. KIMURA, ANTONIO C. BIANCO, AND J. ENRIQUE SILVA Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, 05508 Sao Paulo, Brazil; and the Thyroid Unit, Division of Endocrinology, Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215
ABSTRACT. As judged by the response of uncoupling protein and key enzymes, brown adipose tissue (BAT) is highly dependent upon the local generation of T3 catalyzed by the tissue type II T4 5'-deiodinase (5'D-II). In hypothyroid rats treated with T3 or T4, the capacity to withstand cold seems better correlated with the normalization of BAT responses than with the liver thyroid status. 5'D-II is activated by cold via sympathetic nervous system (SNS) stimulation, and the activation generates enough T3 to nearly saturate BAT nuclear T3 receptors (NTR) in euthyroid rats. In hypothyroidism, 5'D-II is highly stimulated by the SNS and hypothyroxinemia. In the present studies we have taken advantage of this situation to test 1) the capacity of 5'D-II to maintain nuclear T3 in rats with various degrees of hypothyroxinemia, and 2) the hypothesis that thyroid hormonedependent BAT-facultative thermogenesis, rather than the effect of thyroid hormone on obligatory thermogenesis (basal metabolic rate), is the basic mechanism by which thyroid hormone confers protection against acute cold exposure. We treated methimazole-blocked rats (undetectable plasma T4 and T3) for a week with either subreplacement doses of T4 (0.5, 1, 2, and 4 Mg/kgday) or replacement doses of T4 or T3 (8 or 3 ^g/kgday, respectively). Sources and content of BAT nuclear T3 were studied at 25 C and after 48 h at 4 C by labeling the plasmaborne T3 (T3[T3]) with [131I]T3 and the locally generated T3 (T3[T4]) with [125I]T4. Neither the kinetics of nuclear-plasma exchange of T3[T3], the time of appearance of T3[T4] in BAT nuclei, nor NTR maximal binding capacity (0.71 ng T3/mg DNA)
H
YPOTHYROIDISM is associated with multiple alterations in virtually every tissue of the body, including retardation or arrest of differentiation and growth and decreased basal metabolic rate. Among the most striking shortcomings of hypothyroid animals is the inability to adapt to cold environments (1). We have recently shown that the inability to maintain body temReceived October 22,1990. Address all correspondence and requests for reprints to: J. Enrique Silva, M.D., Thyroid Unit, Department of Medicine, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215. * This work was supported by Grants 88/3645-9 from Fundacao de Amparo a Pesquisa (Foundation for Research Support) of Sao Paulo and ROl-DK-42431 from NIK.
was affected by hypothyroidism. Kinetic analyses indicated a maximal BAT NTR occupancy of 40% at euthyroid serum T3 concentrations if T4 is not present. Replacement with T4 normalized both serum T4 and T3, while replacement with T3 normalized serum T3; for all other doses of T4, serum T4 and T3 concentrations were predictably related to the dose. 5'D-II activity decreased with increasing doses of T4, but for each dose of T4, this activity was 2-4 times greater at 4 C than at 25 C. BAT NTR occupancy normalized with 2 fig T4/kg in rats maintained at 25 C and with 4 fig T4/kg in cold-exposed rats, although in neither condition were serum T4 and T3 normalized nor more than 30% of NTR occupied by plasma T3. The thermogenic response to cold, measured as an increase in oxygen consumption (QO2), was significantly greater in T4-replaced than in T3-replaced rats. Two micrograms of T4/kg/day sufficed to normalize the response of QO2 to cold. Prazosin, which blunts the adrenergic-induced stimulation of 5'D-II, caused a marked reduction in T3[T4] and blunted the T4-dependent increase in QO2. We conclude that the protective effect of thyroid hormone against coldinduced hypothermia is largely the result of the stimulation of facultative thermogenesis in BAT, stimulation in which the adrenergic activation of BAT 5'D-II plays an essential role. This enzyme is, furthermore, of adaptive value in hypothyroidism by allowing a normal thermogenic response to cold in the face of substantial reductions in plasma T4. (Endocrinology 128: 21492159,1991)
perature in severe cold can be better correlated with a defective response of brown adipose tissue (BAT) to acute cold exposure than with the thyroid status of the liver, as reflected by a-glycerophosphate activity. These findings suggest that the inability of hypothyroid animals to adapt to cold results from a defective facultative thermogenesis rather than from a depressed obligatory thermogenesis (basal metabolic rate) (2, 3). A major, probably the main, site of facultative thermogenesis in small mammals, including the human newborn, is BAT (4). BAT thermogenesis can be activated by cold or overfeeding (diet-induced thermogenesis) via the sympathetic nervous system (SNS) (5). The adrenergic input
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BROWN FAT, THYROID HORMONE, AND RESPONSE TO COLD
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into the tissue activates and stimulates the synthesis of a unique protein, uncoupling protein (UCP), which, by uncoupling oxidative phosphorylation, boosts BAT respiration and dissipates the resulting energy as heat (5, 6). Thyroid hormones and SNS interact synergistically in BAT. Mitochondrial UCP expression is decreased in hypothyroid rats acclimated to room temperature and fails to increase after cold exposure (2, 3, 7, 8). T 3 increases the rate of synthesis of this protein by amplifying the induction of the gene by norepinephrine (9). On the other hand, the sympathetic nervous system stimulates the type II T4 5'-deiodinase (5'D-II) present in the tissue (10), producing enough T 3 to nearly saturate the nuclear receptors (NTR) for this hormone (11). This high level of NTR occupancy has been found to be essential for a maximal response of UCP to acute cold exposure (2, 3). In the present studies we aimed to obtain further evidence to support the growing concept, emerging from our previous studies (2, 3, 11), that the adaptive role of thyroid hormone in temperature homeostasis is largely mediated by its stimulation of facultative thermogenesis in BAT. For this, we took advantage of the central role of 5'D-II in providing T 3 for BAT. By treating hypothyroid rats with replacement and subreplacement, i.e. suboptimal, doses of T4, or replacement doses of T3, we generated various levels of NTR occupancy. BAT NTR occupancy this time was related to a more direct measure of the thermogenic response to cold, namely oxygen consumption (QO2). Our results support the hypothesis that the protective effect of thyroid hormone from coldinduced hypothermia results from its stimulation of facultative thermogenesis in BAT, and that in this physiological function of thyroidal secretion, BAT 5'D-II plays a pivotal role; our results also indicate that BAT 5'D-II can effectively protect the thermoregulatory mechanisms in hypothyroidism of significant magnitude. Materials and Methods Animals and treatments Sprague-Dawley male rats, weighing 145-165 g, were used throughout. Hypothyroidism was induced by adding 0.02% methimazole (MMI) to the drinking water during at least 30 days [in 1 week, serum T4 and T 3 become undetectable, and in 3 weeks there is complete arrest of growth (12)]. Water and regular rat chow were provided ad libitum. Rats were maintained on a 14-h light, 10-h dark cycle and were acclimated at 25 C. For cold exposure, rats were individually housed in plastic cages with wood shaving bedding and placed at 4 C for up to 48 h as indicated, without changing the light-dark cycle. T4 or T3 was given daily for a week by the sc route in two divided doses, with the total daily dose being equal to (replacement dose) or less than the daily production rates (13, 14). Unless
Endo • 1991 Vol 128 • No 4
indicated otherwise, serum concentrations were measured approximately 12 h after the last dose. Prazosin in injectable formulation (Minipress, Pfizer Laboratories, New York, NY) at a concentration of 50 mg/ml was given ip in an initial dose of 4 mg/kg, followed by doses of 2 mg/kg at approximately 6-h intervals (10) while the animals were exposed to cold. These experimental protocols have been approved by the Harvard University Standing Committee on Animals and Beth Israel Hospital Committee on Animal Research (no. 191.128.02). Tracer kinetics Tracers [125I]T3 or [131I]T3 (SA, -3000 /xCi/Mg), and [125I]T4 (SA, ~4200 /iCi/jug) were prepared in our laboratory as described previously (15). Radioactive T 3 was over 95% pure, with the main contaminant being iodide, and [125I]T4 contained less than 0.5% [125I]T3, measured as outlined below. The tracers were injected with or without a load of unlabeled T 3 to assess the nonspecific binding. Injections consisted of either 20 /uCi/ 100 g BW tracer T3 or 100 MCi/100 g BW tracer T4. They were mixed with unlabeled hormone, when appropriate, and injected in one of the external jugular veins under light ether anesthesia in 0.1-0.2 ml vehicle. The latter was 10% normal rat serum in 0.9% saline containing 0.1% Nal (16). At the time of injections, blood samples were obtained for measurement of serum T4 and T 3 by RIA (17, 18). The animals were killed by aortic exsanguination under ether anesthesia, and tissue samples were handled as described previously and summarized below (16). The model used for the studies to be described is based on that developed by Pearson et al. (19) to study the distribution kinetics of albumin in humans. This model was validated by Oppenheimer et al. (20) to study nuclear plasma exchange of T 3 and by us to assess the contribution of intracellularly generated T 3 from T4 to nuclear T 3 (21); specific aspects relevant to BAT have been discussed in detail in previous publications (11,16). In essence the method consists of pulse labeling plasma T 3 or plasma T4 and allowing nuclear tracer T 3 to reach equilibrium with the plasma source, i.e. T 3 or T4. Equilibrium is only transient owing to the relatively rapid fall of plasma tracer iodothyronines. During this short interval of time, called equilibrium time point or Tm (19-21), one can calculate the mass of nuclear T 3 derived from either plasma T 3 (T;t[T3]) or locally from T4 (T3[T4]) from the specific activity of these iodothyronines in the serum (16, 19-21). In the calculation of locally produced T3[T41, one has to deduct from the observed nuclear [125I]T3[T4] the contribution of [125I]T3 present in plasma. This latter largely derives from T4 to T 3 conversion in other tissues and, to a lesser extent, from the small amounts of [125I]T3 contaminating [125I]T4. For this correction, the product of the equilibrium nuclear to serum ratio of [131I]T3 by the serum concentration of [125I]T3 is subtracted from the observed nuclear [125I]T3 (16, 21). The local nuclear [125I]T3[T4] so obtained is multiplied by 2 to correct for the halving of the specific activity inherent to 5'-deiodination. The number is further multiplied by 651/777, to correct for the change in mol wt, when the mass of T3[T4] is calculated. Tissue processing and analysis The whole interscapular BAT pad (270-350 mg) was processed for preparation of cell nuclei (16). All procedures were
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BROWN FAT, THYROID HORMONE, AND RESPONSE TO COLD carried out at 0-4 C to minimize dissociation and reassociation of tracers to subcellular fractions (16). Briefly, tissue samples were weighed and immediately placed in 0.25 M sucrose and subsequently minced, blotted, and homogenized with a motordriven Teflon-glass homogenizer in 20 vol ice-cold 0.32 M sucrose, 1 mM MgCl2. The 1000 x g pellet was resuspended in 2.3 M sucrose containing 1 mM MgCl2 and centrifuged at 84,000 x g for 30 min. This high speed pellet was resuspended in 0.32 M sucrose, 1 mM MgCl2, and 0.5% Triton X-100, buffered with 10 mM Tris at pH 7.4, and centrifuged again at 1000 X g for 10 min; this step minimized the nonspecific binding of radioactivity to the nuclei. The final nuclear pellets were then rapidly counted and extracted in butanol saturated with 2 N HC1 for chromatographic analysis of the iodothyronines. DNA was measured by the method of Giles and Myers (22). The recovery of DNA was 60-70%. Identification and quantitative analysis of radioactive iodothyronines in the nuclei and serum were performed by paper chromatography for all iodothyronines of interest (23), except for serum [125I]T3 derived from [125I]T4 injection. The latter as well as the [125I]T3 contaminating the [125I]T4 were measured by a combination of immunoaffinity and paper chromatography, described previously (24). The immunoaffinity chromatography results in up to 1000-fold enrichment of T 3 over T4. Recovery of [l31I]T3, either from injection or added to the tubes, was used as internal standard, and it usually ran in the 45-55% range. BAT type II 5'D activity was measured as previously described (25), using 2 nM 5'-[125I]rT3 as substrate in the presence of 1 mM propylthiouracil and 20 mM dithiothreitol. Although T4 is a better substrate for 5'D-II, the physiological responses of the enzyme are equally reflected with either substrate (25, 26), and [125I]rT3 has a longer shelf life than [125I]T4. Indirect calorimetry was used to estimate thermogenesis. QO2 was measured in a Beckman gas analyzer (Palo Alto, CA) that measures both O2 and CO2 during periods of 10 min. Rats are placed in individual closed chambers, and the air of each chamber is analyzed for period of 10 min in a successive fashion, so that in 2 h each rat in an experimental group of four is analyzed three or four times. Results shown are the average of these readings in intervals of 2 h. Studies were performed at 25 or 4 C, as indicated. During the period of recording, rats were kept at the specified temperature with free access to food and water and subjected to the same light-dark cycle. Measurements were performed during the daytime hours, usually between 0900-1200 h. Readings of QO2 were corrected by barometric pressure, temperature, humidity, and CO2 and expressed as milliliters of O2 per min~7kg~075; this latter represents a weight-based estimate of the surface area. Since all rats were fed the same diet, and respiratory quotients were similar, results are merely presented as QO2.
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Results are reported as the mean ± SEM. For simplicity, mention of statistical significance is only made when relevant to an argument.
Results Effect of ambient temperature on the kinetics of nuclearplasma exchange and nuclear binding of T3 in hypothyroid rats Since the treatments of the rats (hypothyroidism and cold exposure) and their interaction may shift the Tm, this must be determined under the precise experimental conditions under which estimates of the mass of nuclear T 3 will be made. The results of these studies are shown in Figs. 1 and 2. In the experiment illustrated by Fig. 1, rats were injected with [125I]T3. Ambient temperature (25 C or 48 h at 4 C) did not affect either the clearance of plasma T 3 or the time and magnitude of nuclear binding. Tm occurred between 2 and 3 h after the tracer injections regardless of the ambient temperature, which was not different from the Tm determined for euthyroid rats (16). After the Tm, nuclear tracer T 3 fell, slowly approaching the rate of plasma disappearance. The nuclear serum ratio (N/S) of [125I]T3 at 2.5 h was approximately 0.6 ml/mg DNA for both groups of rats. Figure 2 shows the accumulation of locally produced nuclear [125I]T3 and plasma disappearance of [125I]T4 in hypothyroid rats similarly treated. In rats acclimated at 25 C, maximal nuclear [125I]T3[T4] uptake occurred at 6 h, whereas in the cold-exposed animals, it continued to increase until 8 h (P < 0.001). Both, the higher nuclear
Serum 25°C Serum 4°C
°i
?.
\
.
—m- Nuclei25°C Nuclei 4°C
1 -
8
Statistical analyses Data were analyzed by one (AOV)- and two-way analyses of variance (TWAOV), followed by multiple comparisons using the Neuman-Keuls test. Nuclear T 3 binding data were linearized by plotting nuclear to serum ratio of tracer T 3 us. the calculated mass of T 3 specifically bound to the nuclei and analyzed by linear regression, as previously described (11, 16).
Time after
125
10
I - T3 injection (hr)
FIG. 1. Time course of serum and nuclear concentrations of [125I]T3 in hypothyroid rats injected iv at time zero with 20 /uCi/lOO g BW [125I] T3. Rats were maintained at either 25 C or housed at 4 C for 48 h, as indicated. Serum and nuclear [125I]T3 were measured as described in Materials and Methods. Each point represents the mean ± SEM of four rats. TWAOV showed no significant effect of ambient temperature.
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BROWN FAT, THYROID HORMONE, AND RESPONSE TO COLD
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Endo • 1991 Voll28«No4
/mi;
Serum 25°C 0) V)
o
•o
Serum 4°C
Q O)
E
2-
Nuclei 25°C
|
Nuclei 4°C
B 400
(A
o •o n N
'"3E Q)
CO
ro a> o 3
Z
T3 Nuclear/Serum Ratio (ml/mg DNA) 10
15
20
O Time after 125, l-T 4 injection (hr) 125 FIG. 2. Time course of serum [ I]T4 and nuclear [125I]T3[T4] in hypothyroid rats injected iv at time zero with 100 ^Ci/100 g BW [125I]T4. Rats were maintained either at 25 C or housed at 4 C for 48 h, as indicated. Serum and nuclear radioactive iodothyronines were measured as described in Materials and Methods. Nuclear [125I]T3[T4] corresponds to the corrected, locally produced [125I]T3, as the contribution made by plasma [I25I]T3 has been subtracted, and the resulting value has been multiplied by 2 to correct by the 50% drop in specific activity derived from the outer ring deiodination. Each point represents the mean ± SEM of four rats. TWAOV shows no significant effect of cold on serum [125I]T4, and a marginal effect of cold on nuclear [125I]T3[T4] (P < 0.05) due to the 8 h point, which was significantly higher at 4 than at 25 C (P < 0.001).
[125I]T3[T4] concentrations and the slight delay for [125I] T3[T4] to peak in the nuclei observed in the cold-exposed animals reflect the enhanced fractional T 4 to T 3 conversion induced by cold in BAT, as previously reported (11) and further documented below by a higher 5'D-II activity in the cold-exposed rats. As depicted in Fig. 2, this short cold exposure did not significantly affect serum [125I]T4 levels. As a consequence, the local nuclear [125I]T3[T4] to serum [125I]T4 ratio was about 60% higher in the coldexposed rats ([4.6 ± 0.18 x 10~2] vs. [2.8 ± 0.26] x 10"2; P < 0.005). Maximum binding capacity (MBC) and apparent affinity of BAT NTR in hypothyroid rats maintained at room temperature For forthcoming calculations of NTR occupancy, we estimated the MBC of BAT T 3 receptors by in vivo saturation analysis, as previously described for room temperature-acclimated or cold-exposed euthyroid rats (11, 16). Several groups of three rats each were injected with [125I]T3 and various doses of unlabeled T3, and killed at the Tm (2.5 h after the injection). Results are presented in Fig. 3. Data plotted as indicated fitted a straight line with coefficient of correlation of -0.985 (P < 0.001; F = 417 for the linear correlation, while attempts to fit
FlG. 3. In vivo saturation analysis of BAT nuclear receptor affinity
and MBC in hypothyroid rats. Data have been linearized as shown; the inset represents the nontransformed data. In the linearized plot, each dot represents one rat, and the dose of cold T3, in micrograms per 100 g, has been indicated by different symbols; in the inset, each dot represents the mean nuclear T 3 ± SEM for each dose of cold T3. See Materials and Methods for details regarding conceptual background and techniques. Regression analysis of the linearized data showed a coefficient of correlation of -0.985 (P < 0.0001). The F value was 417.31 for the linear equation; attempts to fit to a curvilinear model resulted in smaller F values. The calculated MBC was 713 pg/mg DNA, with 95% confidence limits of 683 and 743. The slope of the curve represents the serum T 3 level required to occupy 50% of the MBC; the value was 0.95 ng/ml, with 95% confidence limits of 0.85 and 1.05 ng/ml.
the data to curvilinear function gave lower F values). The calculated MBC was 0.71 ng/mg DNA, with 95% confidence limits of 0.683 and 0.743 ng/mg DNA. The MBC was not significantly different from the one observed in euthyroid rats [95% confidence limits, 0.730.69 ng/mg DNA (11)]. The slope of this line represents the plasma concentration of T 3 to occupy 50% of NTR ([T3]50) and was 0.95 ng/ml, with 95% confidence limits of 0.85 and 1.05 ng/ml. This means that at euthyroid plasma T 3 levels (~0.55 ng/ml) nuclear occupancy would not be greater than 40%, and that in order to reach the levels of 95% occupancy or more seen in cold-exposed rats (11), plasma T 3 should be 18 ng/ml or greater, that is about 40-fold the euthyroid level. It is also worth noting here that [T3]50 in hypothyroid rats is significantly lower than that in euthyroid rats at room temperature (1.26 ng/ml) (16) or exposed to cold for 3 weeks (3.12 ng/ml) (11). This progressive increase in [T3]50 as the local BAT T 3 production from T4 increases reflects the dilution of plasma T 3 by the T 3 generated within the brown adipocytes. NTR occupancy and sources of nuclear T3 in rats with varying degrees of hypothyroidism: effects of cold exposure Rats made hypothyroid with MMI were given graded doses of T4 for a week, and the sources and quantity of
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BROWN FAT, THYROID HORMONE, AND RESPONSE TO COLD
exposed rats with regard to the contribution of plasma T 3 to BAT nuclear T 3 (F = 3.86; P = NS), in accord with the lack of effect of cold on the N/S ratio of [131I]T3; in both groups, nuclear T3[T3] was solely a function of T4 dose-dependent plasma T 3 (F = 88.89; P < 0.0001). There was also a T4 dose-dependent increase in nuclear T3[T4] (F = 70.64; P < 0.0001), but in this case cold exposure caused a significant approximately 2-fold increase in nuclear T3[T4] for each T4 dose (F = 104.97; P < 0.0001). Consequently, and because of the significance of the contribution of T3[T4], total BAT nuclear T 3 was also T4 dose-dependent (F = 174.5; P < 0.0001) and significantly greater in the cold-exposed rats (F = 69.24; P < 0.0001). With regard to NTR occupancy, in the rats acclimated at 25 C it plateaued at about 60% with the dose of 2 /ig T4/kg-day, a rate of occupancy that is not different from the value of approximately 70% found in intact rats acclimated to 23 C (16). It should be noted that with this dose of T4, serum T4 was about 30% and serum T 3 about 60% of the corresponding values in fully replaced rats (Table 2) or intact euthyroid rats (2, 3, 16). In coldexposed rats, multiple comparison analysis indicated that nuclear occupancy plateaued at about 85% with the dose of 4 /ug/kg-day, although with this dose of T4, serum T4 and T 3 were significantly lower than in the fully replaced rats (Table 2); most importantly, the contribution of T3[T4] to nuclear T 3 was identical to that of fully replaced rats in spite of a 45% lower serum T4 in the rats given4)iigT4/kg-day. To assess the effect of adrenergic activation of 5'D-II on the maintenance of BAT nuclear T 3 content, groups of animals identically treated and exposed to 4 C for 48
nuclear BAT T 3 were assessed as described in Materials and Methods. Studies were performed in rats acclimated at 25 C and in rats exposed to 4 C for 48 h. Radioisotopic data are presented in Table 1, and the calculated mass of BAT nuclear T 3 in the same rats is presented in Table 2. Table 1 shows the serum concentration and nuclear content of [131I]T3, the serum concentration of [125I]T4, the nuclear content of [125I]T3[T4], and the corresponding N/S ratios. All values were obtained at the respective Tm for which [131I]T3 was injected 2.5 h before killing the rats and [125I]T4 either 6 or 8 h (cold-exposed rats) before killing the rats. TWAOV shows no effect of the dose or ambient temperature on the serum concentrations of [131I]T3 and [125I]T4. The nuclear content and N/S ratio of [131I]T3 decreased significantly with the dose of T4, so that in the rats treated with the replacement dose of T4, they are about 50% the values seen in untreated rats. Likewise, nuclear [125I]T3[T4] and the nuclear [125I]T3[T4] to serum T4 ratio decreased with T4 in a dose-dependent fashion, but in this case the drop with the full replacement dose of T4 was more than 50%. Cold exposure did not affect the T4-induced fall in nuclear [131I]T3 and the corresponding N/S or the T4-induced fall in nuclear [125I]T3[T4] and the ratio of this to serum [125I]T4; however, for each dose of T4, nuclear [125I]T3[T4] and its ratio to serum [125I]T4 were higher in the coldexposed rats by factors of about 1.5 and 2, respectively (P< 0.001). The results in Table 2 show a T4 dose-dependent increase in both serum T4 and T3, but cold did not significantly affect either value by TWAOV. There was no difference between room temperature- and coldTABLE 1. Serum and BAT nuclear concentrations of [l31I]T3) [125I]T4) and with [131I]T3 and [125I]T4 and housed at different ambient temperatures 1
Dose of T4 (^g/kg-day)
Serum (%/ml X 10)
I. Acclimated at 25 C 0 0.5 1 2 4 8 II. After 48 h at 4 C 0 0.5 1 2 4 8
1.2 1.2 1.2 1.1 1.1 1.2
±0.1 ±0.1 ±0.1 ±0.1 ± 0.1 ± 0.1
1.2 ± 1.1 ± 1.1 ± 1.2 ± 1.2 ± 1.2 ±
0.1 0.1 0.1 0.1 0.2 0.1
5
I]T3[T4] at the equilibrium time point in hypothyroid rats injected 5
I]T3
Nuclei (%/mg DNA x 10)
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N/S
Serum T4 (%/ml)
I]T4 or [125I]T3
Nuclear T3 (%/mg DNA x 10)
0.75 ± 0.63 ± 0.59 ± 0.53 ± 0.46 ± 0.45 ±
0.1 0.1 0.1 0.1 0.1 0.1
0.65 ± 0.56 ± 0.50 ± 0.46 ± 0.42 ± 0.38 ±
0.02 0.01 0.01 0.02 0.02 0.01
1.8 ± 1.9 ± 2.1 ± 2.1 ± 2.3 ± 2.6 ±
0.1 0.1 0.1 0.1 0.1 0.2
0.53 ± 0.50 ± 0.50 ± 0.42 ± 0.25 ± 0.19 ±
0.02 0.03 0.01 0.03 0.01 0.01
0.82 ± 0.62 ± 0.53 ± 0.54 ± 0.49 ± 0.44 ±
0.1 0.1 0.1 0.1 0.1 0.1
0.68 ± 0.56 ± 0.48 ± 0.45 ± 0.41 ± 0.37 ±
0.01 0.01 0.01 0.01 0.00 0.01
1.3 ± 0.1 1.6 ± 0.1 1.6 ± 0.1 1.7 ± 0.1 1.9 ± 0.1 2.1 ± 0.1
0.76 ± 0.71 ± 0.68 ± 0.57 ± 0.38 ± 0.31 ±
0.03 0.01 0.04 0.05 0.02 0.01
N-T3/S-T4 x 100 3.0 ± 0.1 2.7 ± 0.1 2.5 ± 0.1 2.0 ± 0.2 1.1 ±0.1 0.74 ± 0.1 6.1 ± 5.1 ± 4.2 ± 3.3 ± 2.0 ± 1.5 ±
0.1 0.2 0.3 0.2 0.1 0.1
All values are the mean ± SEM of four rats. [125I]T4 was injected 6 or 8 h (cold exposed) and [131I]T3 2.5 h before killing the animals. After subtracting from the total observed nuclear [125I]T3[T4] the contribution made by plasma [125I]T3[T4], the corrected values were multiplied by 2 to correct for random deiodination of [125I]T4. TWAOV showed no effect of T4 on serum [131I]T3 and no effect of cold on nuclear [131I]T3) [131I]T3 N/S ratio, and serum [125I]T4. Nuclear [125I]T3[T4] and N-[125I]T3[T4]/S-[125I]T4 were significantly higher in cold-exposed rats (P < 0.001). See text for more details.
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BROWN FAT, THYROID HORMONE, AND RESPONSE TO COLD
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Endo • 1991 Vol 128 • No 4
TABLE 2. Serum concentrations of T4 and T3, and sources and content of BAT nuclear T 3 in hypothyroid rats treated with various doses of T4 and housed at 25 C or exposed to 4 C for 48 h Serum cone (ng/ml)
Dose of T 4
(Mg/kg-day)
Nuclear T 3 (pg/mg DNA)
Nuclear occupancy
T4
T3
T3[T3]
T3[T4]
Total
%
