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How the thyroid controls metabolism in the rat :
different roles for triiodothyronine and diiodothyronines
Maria Moreno * †, Antonia Lanni †, Assunta Lombardi † and Fernando Goglia † ‡
* Dipartimento di Chimica, Universit`a di Salerno, Facolt`a di Scienze, Via S. Allende, 84081 Baronissi, Salerno, Italy and †Dipartimento di Fisiologia Generale ed Ambientale, Universit`a degli Studi di Napoli ‘Federico II’, Via Mezzocannone 8, I 80134 Napoli, Italy
1. Although the first evidence of a relationship between the thyroid and metabolism was
reported in 1895, the mechanism by which thyroid hormones influence resting metabolic
rate in whole animals is still poorly understood. This paper reports an attempt to test
whether diiodothyronines (Tμs) and triiodothyronine (T×) have different roles in the control
of resting metabolism (RM).
2. Changes in resting metabolic rate were measured in hypothyroid rats treated acutely (25 ìg (100 g body weight)−1) either with one of the Tμs or with T×. Injection of T× induced an increase of about 35% in RM that started 25—30 h after the injection and lasted until
5—6 days after the injection, the maximal value being observed at 50—75 h. The injection of Tμs evoked a temporally different pattern of response. The increases in RM started 6—12 h after the injection, had almost disappeared after 48 h, and the maximal stimulation was observed at 28—30 h.
3. When actinomycin D (an inhibitor of protein synthesis) and T× were given together, the
stimulation of RM was almost completely abolished. The simultaneous injection of
actinomycin D and either of the Tμs, on the other hand, did not cause any attenuation of the stimulation seen with the Tμs alone.
4. Following chronic treatment (3 weeks) with either T× or Tμs there was a stimulation of organ growth only after the administration of T×.
5. Chronic administration of either Tμs or T× to hypothyroid rats significantly enhanced the
oxidative capacity of each of the tissues considered. In the case of Tμs the stimulation was almost the same whether it was expressed as an increase in specific activity or total tissue activity. In the case of T× the increases were, in the main, secondary to the hypertrophic or hyperplastic effect.
6. These results indicate that Tμs and T× exert different effects on RM. The effects of Tμs are rapid and possibly mediated by their direct interaction with mitochondria. Those of T× are slower and more prolonged, and at least partly attributable to a modulation of the cellularity of tissues that are metabolically very active.
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Keywords: Thyroid hormone, Metabolism
‡To whom correspondence should be addressed. investigations of the actions of thyroid hormones. Indeed, the use of acute, as opposed to chronic, hormonal treatment and the use of different methods to induce hypothyroidism could have led different authors towards different interpretations (Lanni, Moreno, Lombardi & Goglia, 1996).
In fact, studies on the characteristics of deiodinase enzymes (K.ohrle, 1994) have revealed that very different animal models are produced depending on whether the hypothyroidism
is induced by surgical or chemical thyroidectomy (Lanni et al. 1996).
In the past, the iodothyronines other than TÚ and T× that are present in biological fluids have been regarded as inactive. Consequently, studies of the calorigenic effect of thyroid hormones have focused almost exclusively on TÚ and T×. Indeed, of the thyroid hormones, T× has been
universally considered to be the active form. Recently, however, a growing number of studies have indicated that two diiodothyronines, 3,3'-diiodo-l-thyronine (3,3'_Tμ) and 3,5-diiodo-l-thyronine (3,5-Tμ) (together referred to as Tμs) could be of biological relevance. These iodothyronines are able rapidly to stimulate the oxidative capacity and respiration rate of rat liver mitochondria (Lanni, Moreno, Cioffi & Goglia, 1992, 1993; O’Reilly & Murphy, 1992;
Lanni, Moreno, Lombardi & Goglia, 1994b) and these effects could be mediated by the binding of Tμs to specific mitochondrial sites (Goglia, Lanni, Horst, Moreno & Thoma, 1994; Lanni, Moreno, Horst, Lombardi & Goglia, 1994a).
Recently, we reported that Tμs induce a dose-dependent calorigenic effect when chronically injected into hypothyroid rats (Lanni et al. 1996). In such animals, following the administration of Tμs, we found a good correspondence between the increase in resting metabolism and the increase in the specific oxidative capacity (expressed as cytochrome oxidase (COX) activity in (ng atoms O) min¢ (mg protein)−1) of tissues that are metabolically very active, such as liver, gastrocnemius muscle, heart and brown adipose tissue (BAT). However, a similar close
correspondence could not be shown in hypothyroid rats injected with T×. In fact, while the administration of T× increased their resting metabolism to euthyroid values or above, a relevant stimulatory effect on specific oxidative capacity at the tissue level was observed only in the liver.
This could not account for the observed increase in RM.This apparent discrepancy could be explained if we assume that the effects of Tμs and T× on RM are mediated by different mechanisms at the cellular level. Thus, the effect of T× could be mediated via a nuclear pathway, resulting in a hypertrophic or hyperplastic effect on tissues that are metabolically very active. In contrast, if Tμs have mitochondria as their target, this could result in a direct
and more rapid stimulation of RM, without an effect on organ mass.
We have tested these hypotheses by: (a) treating rats acutely either with Tμs or with T× and measuring the time of onset and following the time course of the calorigenic effect (measured as the effect on resting metabolism in the whole animal); and (b) treating rats chronically with the same substances and following the development of the hypertrophic or hyperplastic effect (in the liver, gastrocnemius muscle, brown adipose tissue and heart) by determining the mass of the tissues as well as their total protein and DNA content.
As a model for both acute and chronic studies, we used hypothyroid animals in which hypothyroidism was induced by combined treatment with propylthiouracil (PTU) and iopanoic acid (IOP). This treatment, which induces a severe hypothyroidism and, at the same time, inhibits all the three known types of deiodinase enzyme, permits us to attribute the observed effects to the iodothyronines injected, ratherthan to any of their deiodinated products. Moreover, in hypothyroid rats the presence of endogenous iodothyronines is strongly reduced and the endogenous occupation of the binding sites should consequently be lowered.
A preliminary version of these results has been published in abstract form
(Moreno, Lanni, Lombardi & Goglia, 1996).
METHODS
Animals and treatments
Male Wistar rats (250—300 g) living in a temperature-controlled room at 28 °C were kept one per cage under an artificial lighting regime of 12 h light:12 h darkness. A commercial mash (Charles- Rivers, Lecco, Italy) was available ad libitum and the animals had free access to water. All experiments were performed in accordancewith local and national guidelines covering animal experiments. At the end of the experiment, the rats were anaesthetized by i.p. administration of chloral hydrate (40 mg (100 g body weight (BW))−1) and killed by decapitation. Hypothyroidism was induced in some rats by the administration of PTU (0·1% wÏv in drinking water for 3 weeks) together with IOP (6 mg (100 g BW)−1) (Lanni et al. 1996). These rats are referred as the ‘P + I’ group.
To enable determination of the time course of the calorigenic effect of iodothyronines, hypothyroid animals were acutely injected i.p. with either (i) a single dose of 25 ìg (100 g BW)−1 of either T× or 3,5-Tμ, or (ii) in the case of 3,3'_Tμ, three doses of 25 ìg (100 g BW)−1 at 12 h intervals. As a control for the possible effects of the injection itself, saline was injected i.p. into hypothyroid control rats. The dose of 25 ìg (100 g BW)−1 was used because it produces a clear-cut effect on RM and indeed restores it
to the level observed in euthyroid control rats (Tata, Ernster, Lindberg, Arrhenius, Pedersen & Hedman, 1963).
This dose given acutely is not a large dose; in fact, it has been established that 200 ìg (100 g BW)−1, given i.v., is the acute dose needed to obtain ü95% nuclear receptor saturation for 24 h (Jump, Narayan, Towle & Oppenheimer, 1984).
It is conceivable that the administration of PTU and IOP and the consequent alteration in iodothyronine metabolism could induce non-physiological rather than physiological metabolic effects. Actually, it is known that the substances we used to induce hypothyroidism have no effect on metabolism in man (Acheson & Burger, 1980). Nevertheless, to be sure that these two substances do not induce any non-physiological effects in rats, we also measured the effect of the acute administration of 25 ìg iodothyronines (100 g BW)−1 on RM in two other control groups of rats in which hypothyroidism was induced either by surgical thyroidectomy
M. Moreno, A. Lanni, A. Lombardi and F. Goglia J. Physiol. 530 505.2
without administration of P + I (referred to as Tx) or by thyroidectomy and concomitant P + I administration (referred to as Tx + P + I). A similar effect on RM in all the hypothyroid groups, regardless of the way hypothyroidism was induced, would indicate that non-physiological effects were not induced in animals treatedwith P + I.
To enable evaluation of the possible involvement of changes in protein synthesis in the action of the iodothyronines tested, actinomycin D (8 ìg (100 g BW)−1) was injected in combination with 25 ìg (100 g BW)−1 of 3,5-Tμ or T×. At the extremely low doses used here, actinomycin D only inhibits the synthesis of messenger RNA (Goldberg & Rabinowitz, 1962).
To enable the hypertrophic or hyperplastic effect of iodothyronines on the tissues to be followed, P + I rats received, by a once-daily i.p. injection over a 3 week period, one of three different doses (2·5, 5 or 10 ìg (100 g BW)−1) of either T×, 3,5-Tμ or 3,3'_Tμ.
This method also allowed us to plot a dose—response relationship. The above doses fall within the range of doses used in a previous study of dose dependency (Oppenheimer, Schwartz, Lane & Thompson, 1991). At the end of the treatment, each rat was killed, the trunk blood collected and the serum separated and stored at −20°C for later measurement of the concentration of T× and TÚ. Interscapular BAT, heart, gastrocnemius muscle and liver were dissected out, cleaned, immediately weighed (wet weight) and processed for the determination of COX activity, protein content and DNA content.
As an index of cellularity, we adopted the proteinÏDNA ratio which, following an increase in organ weight, indicates a hypertrophy if it increases, but a hyperplasy if it decreases or remains
unchanged.
Resting metabolism
The resting metabolism was measured using open-circuit indirect calorimetry. The rat was placed in a respiration chamber (•32 cm ² 20 cm ² 19 cm) with airflow being measured using an Oμ-ECO mass flow controller (Columbus Instruments International Corporation, Columbus, OH, USA). Details of this set-up and of the way that measurements are made have been given by Lanni et al. (1996).
The measurements for the calculation of RM (the lowest metabolic rate of a resting animal when it is not in a postabsorptive or fasting state and is not sleeping) were taken at 28 °C between 11.00 h and 16.00 h, when the energy expenditure was at a lower level than in any other period of the day. Measurements were taken before and at various intervals after the rat was injected with T× or with one of the Tμs (see above).
Analytical procedures
Cytochrome oxidase (COX) activity was determined polarographically at 25 °C, using a Clark oxygen electrode and a modification (Barr`e, Bailly & Rouanet, 1987) of the procedure of Aulie & Grav (1979). This required 1·5 ml of reaction medium containing 30 ìÒ cytochrome c, 4 ìÒ rotenone, 0·5 mÒ dinitrophenol (DNP), 10 mÒ sodium malonate and 75 mÒ Hepes buffer, at pH 7·4. Samples of liver, skeletal muscle, brown adipose tissue and heart were finely minced, diluted 1Ï10 (wÏv) and homogenized in modified Chappel-Perry medium (mÒ): 1 ATP, 50 Hepes buffer adjusted to pH 7·4, 100 KCl, 5 MgClμ, 1 EDTA and 5 EGTA. The homogenate was then diluted 1:2 (vÏv) in the same medium, with lubrol (100 mg (g tissue)−1) being added to unmask the enzyme activity of the tissue. It was then left standing in ice for 30 min.
Cytochrome oxidase activity was measured as the difference between (i) the rate of oxygen consumption observed after the addition of substrate (4 mÒ sodium ascorbate with 0·3 mÒ N,N,-
N',N'-tetramethyl-p-phenylene-diamine (TMPD)) and homogenate and (ii) the rate of oxygen consumption observed after the addition of substrate alone. This method took into account the autooxidation of ascorbate.
For the measurement of the activities of the deiodinases, tissue samples were individually homogenized on ice in 0·25 Ò sucrose and 10 mÒ Hepes (pH 7·0) containing 10 mÒ DTT ( dithiothreitol).
Liver and brain microsomes for type I deiodinase (ID-I) activity and type III deiodinase (ID-III) activity, respectively, were obtained as described by Visser, Kaptein, Terpstra & Krenning, (1988). BAT infranatants, for type II deiodinase (ID-II) activity, were obtained as described by Leonard, Mellen & Larsen (1983).
They were all immediately frozen in a dry-ice—acetone bath and stored at −80°C until assay.
Samples of reverse [3',5'-125I]T× ([3',5'-125I]rT×) and [5'-125I]T× were prepared by radioiodination of 3,3'_Tμ or 3,5-Tμ (Henning, Berlin, Germany), respectively, using the chloramine-T method as described by others (Visser, Docter & Hennemann, 1977; Visser, Krieger-Quist, Docter & Hennemann, 1978). Labelled products were purified by Sephadex LH-20 chromatography (Pharmacia Uppsala, Sweden) and purity was checked by HPLC analysis using unlabelled compounds as references. Free 125I was eliminated from [3',5'-125I]rT× and [5'-125I]T× on Sephadex LH-20 immediately before each experiment. Iodothyronines were obtained from MMDRI, Henning Berlin R&D (Berlin, Germany). [125I]-Na for radioiodination was purchased from Amersham.
The essential step in the assay of type I deiodinase activity was the production of radioiodide from [3',5'-125I]rT× by ID-I deiodinase. A 2 ìg sample of liver microsomal protein was incubated for 30 min
at 37 °C with 0·1 ìÒ rT× and •100 000 c.p.m. [3',5'-125I]rT× in 200 ìl of 0·2 Ò phosphate buffer (pH 7·2) with 4 mÒ EDTA and 5 mÒ DTT. Incubations were performed in triplicate in a shaking waterbath and the reactions were stopped by the addition of 100 ìl 5% bovine serum albumin at 0 °C. Protein-bound iodothyronines were precipitated by the addition of 500 ìl of 10% trichloroacetic acid. After incubation of the mixtures at 0 °C, they were centrifuged and the radioactivity in the supernatant was then
determined. Enzymatic deiodination was corrected for nonenzymatic 125I production (as determined in blank incubations without microsomes) and multiplied by 2 to account for random labelling and the deiodination of the 3' and 5' positions in [3',5'- 125I]rT×.
The activity of the type II deiodinase enzyme was measured in BAT according to the method of Leonard et al. (1983); this involves measuring the release of radioiodide from [3',5'-ÁÂÇI]rT×. A 20 ìg
sample of BAT infranatant proteins (obtained by centrifugation of BAT homogenate at 500 ² g for 10 min) was incubated in triplicate for 60 min at 37 °C in a shaking waterbath with 2 nÒ rT× and
•100 000 c.p.m. of [3',5'-ÁÂÇI]rT× in 200 ìl 0·1 Ò phosphate buffer (pH 7·2) with 2 mÒ EDTA and 20 mÒ DTT. The reactions were stopped by the addition of 100 ìl 5% BSA and the 125I produced
was isolated and analysed as described above. Random labelling and deiodination of the 3' and 5' positions of [3',5'-ÁÂÇI]rT× was taken into account in the calculation of ID-II activity.
Type III deiodinase activity in brain microsomes was determined by measuring the formation of 3[3'-125I]Tμ from [5'-125I]T× by HPLC analysis as reported by Schoenmakers, Pigmans & Visser (1995).
A 100 ìg sample of brain microsomal protein was incubated in triplicate for 60 min at 37 °C in a shaking waterbath with 1 nÒ T× and •100 000 c.p.m. of [5'-ÁÂÇI]T× in 200 ìl of 0·1 Ò phosphate buffer (pH 7·2) with 4 mÒ EDTA and 10 mÒ DTT. The reactions were stopped by the addition of 300 ìl methanol on ice. After centrifugation of precipitated proteins, the supernatants were
Iodothyronines and metabolic J. Physiol. 505.2 rate 531
analysed for 3[3'-ÁÂÇI]Tμ formation by HPLC analysis involvingelution with a 45 : 50 (vÏv) mixture of methanol and 20 mÒ ammonium acetate (pH 4·0) at a flow of 0·8 ml min¢.
Serum total TÚ and T× levels and free T× were determined in samples of serum using reagents and protocol supplied by Becton- Dickinson (Orangeburg, NJ, USA).
The protein concentration was determined by the method of Hartree (1972) using bovine serum albumin as standard. The DNA content was measured by a colorimetric method (Burton, 1956).
Results are expressed as means ± s.e.m. The statistical significance of differences between groups was determined by a one-way analysis of variance followed by a Student—Newman—Keuls test.
Comparison between independent means was performed using a Student’s t test.
RESULTS
Total and free T× and total TÚ concentrations The combined administration of PTU and IOP produces rats with severe hypothyroidism. Indeed, total and free T× and total TÚ levels were significantly lower in such hypothyroid rats than in euthyroid ones (by about 80, 92 and 88%, respectively; Table 1). In Tx and in Tx + P + Irats, the reduction in all three levels was of the same order as that seen in P + I animals (data not shown).
Following the chronic injection of various doses of T× to P + I rats, the total and free T× levels were (i) within the range of values observed in euthyroid animals (at a dose of 2·5 ìg (100 g BW)−1), (ii) slightly higher (at a dose of 5 ìg (100 g BW)−1) or (iii) significantly higher (at a dose of 10 ìg (100 g BW)−1) (see Table 1). Our values for these circulating levels in euthyroid, hypothyroid and hypothyroid + T×treated animals are in agreement with values previously
reported in rats under similar conditions (Francavilla et al. 1991).
Deiodinase activity in hypothyroid rats
The activities of the deiodinase enzymes in P + I animals were either completely blocked or, in the case of the type II deiodinase, greatly reduced (by 66%) (Table 2). Similar results were obtained in Tx + P + I animals, but in TX animals the activities were differently affected. In the latter animals, type I and type III deiodinases were inhibited by about 63% and 67%, respectively, while type II activity was increased nearly 4-fold (Table 2).
Effect of acute injection of iodothyronines on the RM of hypothyroid rats
RM was considerably lower in all the hypothyroid groups than in euthyroid controls (0·94 ± 0·02, 0·90 ± 0·02, 0·89 ± 0·03 and 1·45 ± 0·03 l Oμ (kg0·75)−1 h¢, in P + I, Tx, Tx + P + I and euthyroid control rats, respectively), thus confirming the hypothyroid status of the animals. Figure 1 illustrates the effect of the acute administration of either 3,5-Tμ or T× on the RM of P + I rats. The injection of T× caused an increase of about 35% in resting metabolic rate;
the increase started 25—30 h after the injection, reached maximal values at 50—75 h and lasted until 5—6 days after the injection (Fig. 1, 0). This trend was in accordance with
that previously observed by Tata in his early studies on thyroidectomized rats (Tata et al. 1962; Tata, 1963).
The injection of Tμs, on the other hand, evoked a temporallydifferent pattern of response. The injection of 3,5-Tμ caused an increase of about 40% in RM (Fig. 1, 2). However, in this case the increase started between 6 and 12 h after the injection, peaked at about 30 h and had almost disappearedat 48 h. A similar trend was observed in the response to 3,3'_Tμ (data not shown), but in that case we needed to give at least three injections 12 h apart to produce the effect.
With 3,3'_Tμ, the increase in RM started 6—12 h after thelast injection and the response had almost disappeared by 30 h after the last injection (not shown). As the RM actually decreased during the first few hours of treatment in the case of animals injected with 3,3'_Tμ, the increase was by about 17% if referred to the initial RM value (before the beginning of the treatment), but by about 27% if referred to the RM value measured immediately before the last
injection.
The simultaneous injection of actinomycin D and either of the Tμs did not cause any attenuation of the stimulation
M. Moreno, A. Lanni, A. Lombardi and F. Goglia J. Physiol. 532 505.2
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Table 1. Total and free T× and total TÚ serum levels in euthyroid (N), hypothyroid (P + I) and
hypothyroid rats chronically treatedwith T× (P + I + T×)
––––––––––––––––––––––––––––––––––––––––––––––
Total Free Total
Group serum T× serum T× serum TÚ (nmol l¢) (pmol l¢) (nmol l¢)
––––––––––––––––––––––––––––––––––––––––––––––
N 0·88 ± 0·06 b 4·1 ± 0·4 b 62·0 ± 3·0 b
P + I 0·17 ± 0·02 a 0·5 ± 0·05 a 7·3 ± 0·8 a
P + I + T× (2·5 ìg T× (100 g BW)¢) 1·11 ± 0·11 b 3·6 ± 0·3 b 6·8 ± 0·6 a
P + I + T× (5 ìg T× (100 g BW)¢) 1·28 ± 0·14 b 4·9 ± 0·5 b c 6·5 ± 0·5 a
P + I + T× (10 ìg T× (100 g BW)¢) 1·76 ± 0·16 c 5·8 ± 0·7 c 6·1 ± 0·5 a
––––––––––––––––––––––––––––––––––––––––––––––
Results are the means ± s.e.m. of 5 experiments for each group, each experiment being performed in
triplicate. Values labelled with different letters are significantly different (P < 0·05) from each other.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– ––––––––––––––––––––
seen with the Tμs alone (Fig. 1, 3). In contrast, when actinomycin D and T× were given together, the stimulation of RM by T× was almost completely abolished (Fig. 1, 1).
The effects of the Tμs and T× on RM were almost the same in Tx animals as in Tx + P + I animals and none of these effects was substantially different from the corresponding effect in the P + I group. However, one difference was observed between Tx animals and the others and lay essentially in the steepness of the curves showing changes in RM as a function of time. Anyhow, in Tx rats: (i) the peak increase in RM induced by Tμs preceded that induced by T× by 26—36 h and (ii) the maximal percentage changes following iodothyronines administration were of the same order (+27—35% in the case of Tμ and +36% in the case of
T×).
It should be emphasized that the deiodinase activities were markedly altered in Tx animals as well as in P + I and Tx + P + I animals, but in a different way. In fact, in Tx rats ID-I and ID-III were inhibited, while I-DII was activated (Table 2). Further, in P + I and in Tx + P + I rats these activities (due to the inhibition exerted by PTU and IOP) remain unchanged by administration of iodothyronines.
By contrast, it is known that in Tx rats the deiodinase activities are further altered following T× administration, and in a way that tends to reverse the effect of thyroidectomy (ID-I and ID-III are stimulated (Kaplan & Utiger, 1978; Esfandiari, Courtin, Lennon, Gavaret & Pierre, 1992) while ID-II is inhibited (Leonard, Kaplan,
Visser, Silva & Larsen, 1981)). Consequently, using a Tx model we have a pattern of deiodination that is different before and after T× administration (this alteration may
underlie the aforementioned slight differences in the steepness of the curves observed in Tx animals). This being so, and as stated in the Introduction, it becomes problematic to attribute an observed effect to a given iodothyronine rather than to its possible metabolic product (i.e. T× rather than Tμ). Having considered all the above, we decided that our chronic studies with different doses of iodothyronines should be performed only on P + I rats. Our main reasons
were: (a) the acute effect of iodothyronines were almost the same in the various hypothyroid conditions, (b) in P + I rats the deiodinase activities are always at the same level and
(c) we could thus avoid imposing another form of stress (surgical thyroidectomy).
Iodothyronines and metabolic J. Physiol. 505.2 rate 533
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Table 2. Activity of liver ID-I , BAT ID-II and brain ID-III in hypothyroid ( P + I, Tx + P + I
and Tx) and euthyroid (N) rats
––––––––––––––––––––––––––––––––––––––––––––––
Group ID-I activity ID-II activity ID-III activity
(pmol I (fmol I (fmol 3,3'_Tμ
(min (mg protein))¢) (h (mg protein))¢) (min (mg protein))¢)
––––––––––––––––––––––––––––––––––––––––––––––
P + I Undetectable 14 ± 1 * Undetectable
Tx + P + I Undetectable 13 ± 1 Undetectable
Tx 75±5 * 153 ± 14 * 9 ± 1*
N 202 ± 12 41 ± 3 27±1
––––––––––––––––––––––––––––––––––––––––––––––
Results are the means ± s.e.m. of 5 experiments for each group, each experiment being performed in
triplicate. *Values significantly different (P < 0·05) from N rats.
–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– ––––––––––––––––––––
Figure 1. Changes in the resting metabolic rate of hypothyroid rats following administration
of iodothyronines with and without actimomycin D
Hypothyroidism was induced by combined treatment with PTU and IOP (P + I). The dose was
25 ìg (100 g BW)−1 for both triiodothyronine (T×) and 3,5-diiodo-l-thyronine (3,5-Tμ). 2, 3,5-Tμ
alone; 3, 3,5-Tμ+actinomycin D; 0, T× alone; 1, T× + actinomycin D. Each data point shows the
mean ± s.e.m. from 5 rats. The values are expressed as the percentage change from the value at time 0 (i.e. immediately before the injection).
Effect of chronic treatment with iodothyronines on specific and total tissue COX activity of liver, gastrocnemius muscle, BAT and heart
The values for the specific (SCOX) and total oxidative (TCOX) capacity of the various tissues are reported in Table 3. An analysis of variance revealed that, in the case of 3,3'_Tμ and 3,5-Tμ, hormonal treatment was effective in stimulating both specific and total COX activity (P < 0·05; F > 3·72). The effect of the administration of Tμs was dose dependent, the maximal effect occurring at a dose of 10 ìg (100 g BW)−1. Administration of 3,5-Tμ to hypothyroid rats at a dose of 10 ìg (100 g BW)−1 stimulated the specific oxidative capacity of the tissues by 40—61 %, the effects on heart and muscle (40 and 45%, respectively) being weaker than those on BAT and liver (60 and 61%, respectively). Administration of 3,3'_Tμ to hypothyroid rats at a dose of 10 ìg (100 g BW)−1 stimulated the specific oxidative capacity of the tissues by 32—63 %, the effects on liver and heart (32 and 40%, respectively) being weaker than those on BAT and muscle (51 and 63%, respectively).
When the oxidative capacity was expressed as total activity, a similar pattern was observed. In percentage terms, in fact, the stimulation of COXactivity by Tμs was almost the same whether the activity was expressed as specific or total activity.
In the case of T×, an analysis of variance revealed that hormonal treatment was effective in stimulating specific activity only in the liver (P < 0·02; F = 7·12). Actually, the lack of an effect of T× on specific COX activity in BAT is not surprising since it has been shown that BAT preferentially utilizes plasma TÚ and that it only poorly utilizes T× derived from the plasma and extracellular space (Bianco & Silva, 1987). However, when the oxidative capacity was expressed as total activity, the stimulation was evident and significant in all organs and tissues tested. In percentage terms
(Table 3; values in parentheses), the changes from P + I values were much greater when expressed in terms of total activity than when expressed in terms of specific activity.
At a dose of 10 ìg (100 g BW)−1, in fact, expressing the increases in COX activity in terms of total activity (rather than in terms of specific activity) caused the size of the increase to be more than doubled in the liver (162% vs. 74 %), 3·5 times greater in muscle (91% vs. 26 %), 5·5 times greater in the heart (96% vs. 18 %) and about 5 times greater in BAT (73% vs. 15 %).
Effect of chronic treatment with iodothyronines on body weight, organ mass, tissue protein content and tissue DNA content in hypothyroid rats
Body mass and the mass of individual organs are significantly lower in hypothyroid rats than in euthyroid ones (Table 4).
Following the chronic administration of iodothyronines, all the organs examined were significantly heavier in T×-treated animals than in hypothyroid ones (see Table 4), the greatest
effects being observed in BAT and heart. However, no
M. Moreno, A. Lanni, A. Lombardi and F. Goglia J. Physiol. 534 505.2
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Table 3. Specific (SCOX) and total (TCOX) cytochrome oxidase activity from hypothyroid rats and
from hypothyroid rats treated with various iodothyronines
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Liver Gastrocnemius Heart BAT
––––––––––––– –––––––––––– ––––––––––––– ––––––––––––––
Group SCOX TCOX SCOX TCOX SCOX TCOX SCOX TCOX
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
P+I 353±30 404±32 230±13 35±2·8 1734±168 84±6·7 1442±109 10·0±0·9
P+I+3,5-Tμ
2·5 ìg 402±39(14) 504±40(25) 302±22*(32) 42±4·2(20) 1790±171(3) 83±8·2(0) 1779±121(23) 10·7±0·9(7)
5 ìg 477±6*(24) 696±60*(72) 304±21*(32) 54±4·3*(54) 2419±212(40) 119±12·2(42) 1989±159*(38) 14·8±1·5*(48)
10 ìg 570±53*(61) 651±40*(61) 334±33*(45) 54±5·0*(54) 2425±221(40) 135±15·3*(61) 2307±187*(60) 20·2±2·1*(100)
P+I+3,3'-Tμ
2·5 ìg 416±33(17) 488±20(21) 303±30*(32) 46±4·2(31) 1954±160(13) 88±7·9(5) 1476±122(2) 11·5±1·1(15)
5 ìg 423±19(20) 537±22*(32) 359±10*(56) 63±5·0*(80) 2433±197(40) 126±13·0*(50) 1869±169(30) 17·3±1·4*(70)
10 ìg 469±13*(32) 510±20*(23) 376±35*(63) 51±4·8*(46) 2421±213(40) 116±12·1(38) 2173±132*(51) 14·7±1·2*(47)
P+I+T×
2·5 ìg 473±45(34) 789±80*(95) 265±30(15) 52±4·8*(49) 1800±177(4) 153±13·2*(82) 1668±103(16) 22·8±2·3*(128)
5 ìg 508±48*(44) 749±75*(85) 280±25(22) 62±5·0*(77) 1850±200(7) 134±12·7*(60) 1586±152(10) 18·4±1·5*(84)
10 ìg 614±58*(74) 1060±100*(162) 290±10(26) 67±7·0*(91) 2048±183(18) 165±14·2*(96) 1658±127(15) 17·3±1·4*(73)
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SCOX is expressed as (ng atoms O) min¢ (mg protein)−1; TCOX is expressed as (ìg atoms O) min¢. Results are presented as means±s.e.m. Values in parentheses represent the percentage increase over P + I value.
*Values significantly different (P < 0·05) from P + I rats. n= 9 for P + I group, n = 5 for each hypothyroid iodothyronine-treated group, where n is no of experiments. Groups given different doses of 3,5-Tμ, 3,3'-Tμ and T× are distinguished by the dose which is given per 100 g BWunder the group names in the left column.
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differences were observed between hypothyroid rats and 3,3'_Tμ- or 3,5-Tμ-treated animals, whatever the dose (data not shown).
Calculation of the proteinÏDNA ratio (see Table 5) revealed that chronic T× administration induced a hypertrophic effect in the liver and heart and a hyperplastic effect in muscle and BAT. The evidence for this was as follows. In liver and heart, T× exerted a strong effect on protein content and a weaker effect (heart) or no effect (liver) on DNA content. The opposite was true for gastrocnemius muscle
Iodothyronines and metabolic J. Physiol. 505.2 rate 535
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Table 4. Body and organ masses from euthyroid (N), hypothyroid (P + I) and triiodothyroninetreated
hypothyroid (P + I + T×) rats
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Group n Body mass Liver Gastrocnemius BAT Heart (g) (g) (g) (mg) (mg)
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P + I 9 263 ± 8 † 9·2 ± 0·6 † 1·56 ± 0·08 † 176 ± 11 † 644 ± 33 †
(35·1 ± 1·9) (5·94 ± 0·29) (0·68 ± 0·05) (2·44 ± 0·09)
N 7 312 ± 10 12·5 ± 0·7 1·86 ± 0·08 263 ± 14 870 ± 32
(40·0 ± 2·0) (5·9 ± 0·04) (0·84 ± 0·05) (2·80 ± 0·50)
P + I + T×
2·5 ìg 5 305 ± 15 * 11·4 ± 0·1 1·82 ± 0·12 281 ± 46 * 1125 ± 15 *
(37·7 ± 1·4) (5·96 ± 0·01) (0·91 ± 0·04) * (3·74 ± 0·51) *
5 ìg 5 282 ± 15 11·1 ± 0·6 1·95 ± 0·02 * 359 ± 24 * 973 ± 28 *
(39·5 ± 0·5) (6·93 ± 0·33) (1·29 ± 0·16) * (3·45 ± 0·21) *
10 ìg 5 306 ± 6 * 12·8 ± 1·8 * 2·13 ± 0·13 * 446 ± 63 * 1137 ± 33 *
(41·7 ± 2·0) (6·94 ± 0·53) (1·41 ± 0·21) * (3·7 ± 0·08) *
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Values in parentheses represent tissueÏbody weight ratio ² 103. Results are presented as means ± s.e.m.; n, no. of experiments. *Values significantly different (P < 0·05) from P + I rats; †values significantly different (P < 0·05) from N rats. Because neither 3,3'_Tμ nor 3,5-Tμ treatment had a significant effect on the above parameters (treated vs. hypothyroid rats), the data are not reported. Different P + I + T× groups are distinguished by the doses of T× which are given per 100 g BW under the group name in the left column.
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Table 5. Total protein content (mg), total DNA content (mg) and proteinÏDNA ratio in liver,
gastrocnemius muscle, BAT and heart homogenates from euthyroid (N), hypothyroid (P + I) and
triiodothyronine-treated hypothyroid (P + I + T×) rats
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P + I P + I + T× N
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2·5 ìg 5 ìg 10 ìg
(n = 9) (n = 5) (n = 5) (n = 5) (n = 7)
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Liver Protein 1138 ± 73·1 † 1457 ± 70 * 1553 ± 68 * 1745 ± 153 * 1529 ± 116
DNA 36 ± 3·5 38 ± 0·5 36 ± 2·8 35 ± 2·9 36 ± 1·9
ProteinÏDNA 31 ± 2·7 † 38 ± 1·3 43 ± 3·4 * 50 ± 4·5 * 42 ± 4·00
Gastrocnemius Protein 170 ± 15 192 ± 2 196 ± 16 204 ± 19 195 ± 2 *
DNA 3·6 ± 0·3 4·7 ± 0·1 * 5·2 ± 0·4 * 6·2 ± 0·6 * 4·8 ± 0·3
ProteinÏDNA 47 ± 4·0 41 ± 1·3 38 ± 2·7 33 ± 2·7 * 41 ± 3·9
BAT Protein 6·7 ± 0·6 13·7 ± 0·7 * 11·6 ± 1·2 * 10·3 ± 2·2 * 9·4 ± 0·1 *
DNA 0·38 ± 0·03 0·84 ± 0·01 * 0·98 ± 0·03 * 1·28 ± 0·16 * 0·51 ± 0·05
ProteinÏDNA 18 ± 1·0 16 ± 0·8 12 ± 0·9 * 8 ± 1·5 * 18 ± 1·6
Heart Protein 54 ± 3·8 65 ± 7·5 78 ± 6·8 * 106 ± 10·1 * 56·4 ± 4·3
DNA 2·1 ± 0·2 2·5 ± 0·2 2·7 ± 0·3 3·2 ± 0·4 2·6 ± 0·2
ProteinÏDNA 25 ± 2·2 27 ± 3·4 29 ± 2·7 33 ± 3·4 22 ± 2·0
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Because neither 3,3'_Tμ nor 3,5-Tμ treatment had a significant effect on the above parameters
(treated vs. hypothyroid rats), the data are not reported. Results are presented as means ± s.e.m.; n, no. of experiments. *Values significantly different (P < 0·05) from P + I rats; †values significantly different (P < 0·05) from N rats. Different P + I + T× groups are distinguished by the doses of T× which are given per 100 g BWunder the group name.
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