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J Appl Physiol 95: 1584-1590, 2003. First published June 27, 2003; doi:10.1152/japplphysiol.00363.2003
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Mitochondrial respiration in muscle and liver from cold-acclimated hypothyroid rats

Angel A. Zaninovich,1 Inés Rebagliati,1 Marcela Raíces,2 Conrado Ricci,1 and Karl Hagmüller3

1Thyroid Research Laboratory, Nuclear Medicine Center, University Hospital, and 2Institute of Genetic Engineering and Molecular Biology, University of Buenos Aires, 1120 Buenos Aires, Argentina; and 3Institute of Zoology, University of Graz, 8010 Graz, Austria

Submitted 10 April 2003 ; accepted in final form 24 June 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The effects of long-term cold exposure on muscle and liver mitochondrial oxygen consumption in hypothyroid and normal rats were examined. Thyroid ablation was performed after 8-wk acclimation to 4°C. Hypothyroid and normal controls remained in the cold for an additional 8 wk. At the end of 16-wk cold exposure, all hypothyroid rats were alive and normothermic and had normal body weight. At ambient temperature (24°C), thyroid ablation induced a 65% fall in muscle mitochondrial oxygen consumption, which was reversed by thyroxine but not by norepinephrine administration. After cold acclimation was reached, suppression of thyroid function reduced muscle mitochondrial respiration by 30%, but the hypothyroid values remained about threefold higher than those in hypothyroid muscle in the warm. Blockade of {beta}- and {alpha}1-adrenergic receptors in both hypothyroid and normal rats produced hypothermia in vivo and a fall in muscle, liver, and brown adipose tissue mitochondria respiration in vitro. In normal rats, cold acclimation enhanced muscle respiration by 35%, in liver 18%, and in brown adipose tissue 450% over values in the warm. The results demonstrate that thyroid hormones, in the presence of norepinephrine, are major determinants of thermogenic activity in muscle and liver of cold-acclimated rats. After thyroid ablation, cold-induced nonshivering thermogenesis replaced 3,5,3'-triiodothyronine-induced thermogenesis, and normal body temperature was maintained.

cold acclimation; oxygen consumption; norepinephrine; adrenergic-receptor blockers

ENDOTHERMIC ANIMALS LIVING in a cold environment maintain normothermia and energy balance through an activation of mechanisms that increase heat production and heat conservation. Heat production is commonly classified as either obligatory or adaptive (for reviews, see Refs. 15, 23, 29, 38). The obligatory thermogenesis results from the widespread metabolic activity in many tissues and serves to maintain normal body temperature, which in homeotherms is usually higher than ambient temperature. The thyroid hormones stimulate obligatory thermogenesis by a number of mechanisms that are still incompletely understood (8, 32, 38). The additional heat needed to preserve body temperature in a cold environment is obtained through adaptive or facultative thermogenesis, a process regulated by the hypothalamus through the sympathetic nervous system. Brown adipose tissue (BAT) is the main site for adaptive thermogenesis primarily because of the mitochondrial uncoupling protein-1 (UCP-1), which uncouples electron transport from the phosphorylation of ADP and appears to be the only protein able to respond to thermal challenge (31, 30).

UCP-1 expression is stimulated by norepinephrine (NE) but requires the presence of thyroid hormones (3, 4, 37). If hypothyroid rats are transferred from thermoneutrality to a cold environment, BAT cannot raise UCP-1 synthesis sufficiently and rapidly, and the animal falls into a severe hypothermia and succumbs within hours of cold exposure (3, 35). This effect can be prevented by the administration of thyroid hormone but not by the administration of NE (3, 33). In fact, hypothyroid BAT is overstimulated by catecholamines, but hypothyroid animals have reduced responsiveness to adrenergic stimulation, owing to alterations at several levels of adrenergic signal transduction (34).

Although the specific role of the thyroid hormones during the acute phase of cold acclimation was clarified by numerous studies for the past 20 years, the influence of thyroid on thermogenesis during long-term cold exposure has drawn little attention. In fact, some studies suggested that thyroid hormones may not be necessary for the maintenance of normothermia in a cold-acclimated state (see review in Refs. 17, 26).

Because NE has metabolic actions in numerous organs and activation of BAT accounts for ~40% of the total oxygen consumption after NE stimulation (13), it may be inferred that cold-induced, NE-mediated nonshivering thermogenesis (NST) occurs in other tissues. We have addressed this question by studying 1) whether cold-induced, NE-mediated NST occurs in skeletal muscle, a tissue in which the existence of NST is contradictory (see review in Ref. 23), and 2) whether thyroid-induced thermogenesis is a major factor in muscle and liver adaptation to cold. To determine these parameters, we measured mitochondrial oxygen consumption in muscle and liver of both normal and hypothyroid rats acclimated to 4°C. Furthermore, under these conditions, we determined the actions of thyroid hormone replacement, NE administration, or NE-receptor blockade.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals and Materials

The experiments were conducted in accordance with the National Institutes of Health Guide for the Care and the Use of Laboratory Animals and were approved by the animal research committees of the institutions involved. Male and female Wistar rats with an initial body weight of 110-130 g were housed individually in wire mesh cages on a 12:12-h light-dark schedule (0800-2000) and a room temperature of ~24°C. They had free access to tap water and food pellets. The diet contained 26% proteins, 6% fat, and 44% carbohydrates calculated from the nitrogen-free extract. Hypothyroidism was induced at room temperature or after 8 wk of cold acclimation by a single injection of 300 µCi 131I/100 g body wt ip. To eliminate any possible residual thyroid hormone production, a group of rats was given, in addition, 2-mercapto-1-methyl-imidazole (methimazole, kindly provided by Laboratorios Gador, Buenos Aires, Argentina) at 0.05% in the drinking water, for 30 days. Other drugs and reagents, unless otherwise specified, were purchased from Sigma Chemical (St. Louis, MO). Radioactive 131I was obtained from the Atomic Energy Commission of Argentina.

Procedures

Experiments at room temperature. Rats rendered hypothyroid at room temperature were kept in individual cages. A group of hypothyroid rats was treated with 1.5 µg/100 g body wt sc thyroxine (T4) in divided doses daily for 3 days. Other group of hypothyroid rats was injected with 100 µg/100 g body wt sc NE, and animals were killed by cervical dislocation at 1, 4, or 6 h after the injection of the catecholamine. The values given in the text correspond to 4-h measurements.

Experiments after long-term cold exposure. Groups of normal rats were placed in individual wire mesh cages either in a cold room at 4°C or were at ambient temperature. After 8 wk of cold exposure, baseline values of muscle and liver mitochondrial oxygen consumption were determined in six rats. On the same day, a group of normal rats was made hypothyroid by an intraperitoneal injection of 131I, and the remaining rats were left intact and served as euthyroid controls. Hypothyroid and normal rats were then maintained for an additional 8-wk period in the cold room. Colonic temperature and body weight were determined, and blood was collected for the measurement of serum thyroid hormones concentration and creatine kinase and {alpha}-glycerophosphate dehydrogenase ({alpha}-GPD) activities. Animals were killed by cervical dislocation, and samples from gastrocnemius and biceps femoral muscles, liver, and BAT were obtained. Even though BAT thermogenesis in cold-acclimated hypothyroid rats had been studied before (43), it was considered of interest to determine the state of BAT function in the present animals for comparison with the findings in skeletal muscle and liver from the same rat.

Effects of thyroid or NE treatment in cold-acclimated rats. At the end of 16 wk of cold exposure, a group of hypothyroid rats was injected with 2.5 µg/100 g body wt sc T4, in divided doses daily for 3 days, and thereafter animals were killed. Another group of hypothyroid rats was injected with a single dose of 100 µg/100 g body wt sc NE, and rats were killed at 1, 4, or 6 h after the injection of the catecholamine.

Blockade of sympathetic activity in cold-acclimated rats. Groups of cold-acclimated normal or hypothyroid rats were injected with 0.5 mg/100 g body wt ip DL-propranolol in combination with 0.4 mg/100 g body wt prazosin at 4 and 2 h before animals were killed. Other groups of normal or hypothyroid rats were injected with 0.1 mg/100 g body wt ip reserpine at 18 and 2 h before animals were killed. Reserpine depletes catecholamine stores by mechanisms not involving the adrenergic receptors. Untreated controls were injected with saline. Colonic temperatures and rats' behavior were monitored before the first injection of NE blockers, at 2 h, and at the end of the 4-h experiment. Animals were killed by cervical dislocation, and muscle, liver, and interscapular BAT were obtained.

Isolation and purification of mitochondria. Muscle (1 g/4 ml buffer), liver (1 g/9 ml buffer), and whole BAT pad were ground and homogenized, and their mitochondria were isolated as described in our laboratory's previous reports (7, 43). The final pellet was resuspended in 1-ml volume in sucrose buffer (0.24 M). Protein content was measured in aliquots of mitochondrial pellets by the Lowry assay using bovine serum albumin as standard.

Oxygen consumption in muscle and liver mitochondria was measured in an oxygraph (Gilson Medical Electronic) using a Clark electrode placed in a 1-ml respiratory chamber at 30°C, as detailed before (7, 43). An aliquot of mitochondrial suspension was added to the respiration medium to reach a final concentration of 1.5 mg protein/ml of respiratory medium. Measurement of oxygen uptake was initiated in state 4 (without ADP).

After the velocity of oxygen uptake was determined, a phosphate acceptor (0.42-0.82 mM ADP) was added to the medium, and respiration was allowed to proceed. L-Malate (6 mM), L-glutamate (6 mM), and malonate (3 mM) were used as substrates for muscle and liver respiration. {alpha}-Glycerophosphate was used as substrate for BAT respiration.

Unlike the equal values of state 3 (with added ADP) and state 4 respiration (without added ADP) in cold-exposed BAT mitochondria because of total uncoupling (43), the addition of ADP to muscle and liver mitochondria from cold-acclimated rats sharply accelerated oxygen uptake, indicating that mitochondria were in a coupled state. The values of muscle and liver oxygen consumption given in the text correspond to state 3 respiration. Results in state 4 respiration are shown in Table 2. Results of mitochondrial oxygen consumption are expressed in nanograms oxygen per minute per milligram of mitochondrial protein. The rates of oxygen consumption in mitochondria from gastrocnemius and biceps femoral muscles were similar.


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  		Table 2. State 4 mitochondrial oxygen consumption and respiratory control ratio in muscle and liver from rats shown in Figs. 1 and 2

 



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  		Fig. 1. State 3 muscle mitochondrial oxygen consumption in normal (n = 15), hypothyroid (Hypo; n = 18), and thyroxine (T4)-treated hypothyroid (n = 8) rats exposed to 4°C for 16 wk. Norepinephrine (NE) and NE+T4 effects were studied in 5 rats per group. Thyroid ablation was performed after 8 wk of cold exposure. Experiments at room temperature used 6 rats per group. One rat per experiment was used. All rats in the warm and in the cold had normal body temperature (values above bars). Normal in the warm vs. normal in the cold, P < 0.001; hypothyroid in the warm vs. hypothyroid in the cold, P < 0.001; normal vs. hypothyroid in the warm, P < 0.001; normal vs. hypothyroid in the cold, P < 0.001 (ANOVA and Duncan's test).

 


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  		Fig. 2. Liver mitochondrial oxygen consumption in rats used for experiments depicted in Fig. 1. Normal vs. hypothyroid in the warm, P < 0.001; normal vs. hypothyroid in the cold, P < 0.02; normal in the warm vs. normal in the cold, P < 0.05; hypothyroid in the warm vs. hypothyroid in the cold, P < 0.005 (ANOVA and Duncan's test).

 
Other procedures. The hepatic mitochondrial {alpha}-GPD activity, a reliable index of thyroid status (28, 18), was measured spectrophotometrically at 500 nm and 37°C as described before (43). Plasma creatine kinase activity was measured by spectrometry at 37°C and 340 nm (CK-NAC, Wiener Laboratories, Buenos Aires, Argentina). Plasma T4 and 3,5,3'-triiodothyronine (T3) concentrations were measured by a double-antibody technique (5). The limits of sensitivity were 5 nmol/l for T4 and 0.12 nmol/l for T3. Body temperature was measured with a digital thermometer (Digi-thermo, Soar, Buenos Aires, Argentina) introduced 4 cm into the rectum and kept in place for 3 min. Baseline temperature was not altered during this procedure. Comparisons were made by ANOVA and Duncan's test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Body Weight

After an initial weight loss, all animals in the cold room had increased energy intake and weight gain throughout cold acclimation (Table 1). Daily growth of cold-acclimated rats was 2.4 ± 0.3 g/day. The rate of growth was not altered by thyroid ablation. In rats maintained at room temperature, the average daily growth rate was 1.9 ± 0.2 g in normal rats and 1.6 ± 0.2 g in hypothyroid rats. Because hypothyroid rats at room temperature displayed normal activity, the lower body weight was likely the result of decreased energy intake (average 4.5 g food · 100 g body wt-1 · day-1 in hypothyroid vs. 6 g · 100 g body wt-1 · day-1 in normal rats). Body weight in normal or hypothyroid males in the cold exceeded that of females by 27.4%. As in the case of BAT (43), muscle and liver mitochondrial respiration showed no sex differences, and data were pooled.


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  		Table 1. Thermogenic parameters in normal or hypothyroid rats after exposure to 4°C for 16 wk or left in the warm

 



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  		Fig. 3. Brown adipose tissue (BAT) mitochondrial respiration in rats shown in Fig. 1. Room-temperature experiments were only performed in normal rats (n = 6) for comparison with the effects produced by cold acclimation. Animals injected with propranololprazosin (prop-praz) or reserpine were those used for experiments shown in Fig. 4. Normal BAT in the warm vs. that in the cold, P < 0.001; normal BAT in the cold vs. adrenergic blocker-treated rats, P < 0.001; reserpine-treated vs. propranolol-prazosin-treated rats, P < 0.001 (ANOVA and Duncan's test).

 


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  		Fig. 4. Effects of sympathetic blockade on muscle and liver mitochondrial oxygen consumption in cold-acclimated rats. Normal rats (n = 15) were those used in experiments depicted in Fig. 1. Propranolol-prazosin-treated rats (n = 12) were injected with propranolol (0.5 mg/100 g body wt ip) plus prazosin (0.4 mg/100 g body wt) at 4 and 2 h before the animals were killed. Reserpine (8 rats) was administered in a dose of 0.1 mg/100 g body wt ip at 18 and 2 h before animals were killed. Muscle, liver, and BAT were obtained from the same animal. One rat per experiment was employed. All rats treated with propranolol-prazosin developed signs of severe hypothermia. Untreated vs. propranolol-prazosin-treated results in all groups, P < 0.001; untreated vs. reserpine-treated results in muscle, P < 0.001. Reserpine had no significant effects on liver oxygen consumption (ANOVA and Duncan's test).

 
Mitochondrial Respiration at Room Temperature

Muscle. Impairment of muscle thermogenesis after the suppression of thyroid function was shown by the 65% decrease in mitochondrial oxygen consumption compared with values in normal rats (Fig. 1). This effect was observed in earlier studies (12) and did not alter normal body temperature (see Table 1). Mitochondrial respiration was normalized by the low T4 replacement dose (1.5 µg) but not by NE treatment, confirming that thyroid hormones are major determinants of muscle thermogenesis.

Liver. Mitochondrial oxygen consumption in normal liver was about one-half that of muscle mitochondria (Fig. 2). Thyroid ablation reduced consumption by 32%, a deficit reverted by T4 treatment, although not by NE treatment.

BAT. Only normal rats were studied. Mitochondrial oxygen consumption was 75 ± 8 ng oxygen·min-1·mg mitochondrial protein-1. This figure represents a mere 18% of the oxygen consumption rate reached after cold acclimation, as shown in Fig. 3.

Mitochondrial Respiration in Cold-acclimated Rats

Muscle. Thyroid ablation of cold-acclimated rats reduced oxygen consumption by 30% from the levels of normal rats (Fig. 1). Nevertheless, the hypothyroid values remained almost threefold higher than those in the warm, indicating an active thermogenic process in the absence of thyroid hormone. Serum creatine kinase in hypothyroid rats was below normal levels, an indication that there was no muscle shivering to compensate for the reduced heat production in muscle and liver. Shivering has been shown to markedly increase creatine kinase activity (9). The deficit in mitochondrial respiration was corrected by T4 replacement, whereas exogenous NE failed to stimulate a thermal response above the levels achieved by endogenous NE. In normal rats, cold acclimation increased muscle mitochondrial oxygen consumption by 35% from values in the warm. This change was associated with a slight but significant rise in mitochondrial proteins (Table 1) and with normal body temperature.

Liver. Thyroid ablation produced a 20% drop in mitochondrial oxygen consumption, but the values remained ~40% higher than those in the warm (Fig. 2). T4 treatment normalized mitochondrial respiration, whereas NE treatment was without effect. In normal rats, cold exposure enhanced oxygen consumption by 18% from values at room temperature, but no changes in mitochondrial proteins were observed.

Figure 3 shows the typical BAT response to cold exposure in normal rats, with an increase in consumption of 450% from values at room temperature. The enhanced respiratory activity was not altered by thyroid ablation, T4 replacement, or NE administration, suggesting that BAT response to sympathetic stimulation was maximal.

Effects of Adrenergic-receptor Blockers in Cold-acclimated Rats

The injection of propranolol-prazosin caused a marked fall in body temperature and mitochondrial oxygen consumption in muscle, liver, and BAT (Fig. 4). The effects were more pronounced in hypothyroid rats, but normal rats were also affected. Muscle and BAT mitochondria from hypothyroid rats had an 80% fall in oxygen consumption. The fall in BAT respiration resembled the effects of BAT surgical sympathectomy (6). Liver mitochondrial respiration decreased by 50% after treatment with adrenergic blockers. All animals injected with propranolol-prazosin had physical signs of severe hypothermia, such as shivering, nesting behavior, piloerection, and progressive unresponsiveness to external manipulation. The in vivo effects may have been produced by the combination of lower heat production and impaired heat conservation, as analyzed in the DISCUSSION.

Reserpine treatment reduced muscle and BAT oxygen consumption in all cold-acclimated rats by one-half (Figs. 3 and 4). The effects on body temperature (36.6 ± 0.3°C in normal rats and 35.4 ± 0.4°C in hypothyroid rats, Table 1) were nevertheless moderate and were not accompanied by signs of severe hypothermia. The effects of reserpine on liver mitochondria were small and their significance is not clear. The depletion of catecholamines stores by reserpine varies among tissues, and it is possible that a full depletion in the liver was not reached after 18 h of the first injection.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The present data demonstrate that thyroid hormones and NE are required for long-term, increased thermogenic activity in muscle and liver of normal rats living in a cold environment, and when thyroid hormones are suppressed, normal body temperature is maintained mainly by NST. A novel aspect of this study was to conduct the thyroid ablation after cold acclimation had been achieved, resulting in a hypothyroid state associated with normal body temperature and survival. This approach allowed us to assess the effects of cold-induced NST in tissues free of thyroid-induced thermogenesis. In contrast, room temperature-adapted hypothyroid rats are unsuitable for these studies, because the animals rapidly develop hypothermia and die within hours of cold exposure.

Despite the fact that sympathetically induced heat production results mainly from thermogenesis in BAT, NE has metabolic actions at different organ systems, and activation of BAT may account for ~40% of total oxygen consumed after NE stimulation (13). Skeletal muscle and liver also are major oxygen consumers, and their combined activities at thermoneutrality take up near 40% of cardiac oxygen delivery (42). In the cold, however, muscle and liver heat production may not raise substantially above the levels in the warm, a phenomenon that has generated diverse interpretations (for review, see Ref. 23). Thus previous studies have shown that blood flow in muscle of cold-acclimated rats or in rats administered NE was not increased (14), which was taken to indicate that NST did not play a significant role in muscle adaptation to cold. On the other hand, studies performed in perfused hind-limb of cold-acclimated rats have shown an increase in oxygen extraction and oxygen consumption after NE injection (27, 36). Other works reported an enhancement (10, 25) or no changes (16, 22) of resting metabolism after cold acclimation of normal rats.

In the present study, cold-acclimated normal rats had an active T3-induced thermogenesis, as indicated by the high levels of {alpha}-GPD activity, and increases in muscle and liver mitochondrial respiration of 35 and 18%, respectively, above the values at room temperature. These increments were modest compared with the 450% rise in BAT oxygen consumption, reflecting that NST in muscle and liver may not be a major factor for cold adaptation of normal rats, a view expressed in previous works (13, 14). However, blockade of NE activity significantly reduced mitochondrial respiration and elicited signs of severe hypothermia, reflecting that NE action is needed for the promotion of muscle and liver thermogenesis by thyroid hormones. The NE effects are in agreement with previous studies showing augmented NE turnover (11, 40), increased vascularity (21), and sympathetic innervation of muscle fibers (1) in muscle of cold-acclimated normal rats, but the pathways mediating NE effects on thyroid hormone-induced thermogenesis are not clear.

Our study shows a central role for NST in muscle and liver heat production in rats deprived of T3-induced thermogenesis. The experiments depicted in Fig. 1 demonstrate that mitochondrial respiration in hypothyroid muscle, even though lower than in normal rats, was almost threefold higher than that of hypothyroid rats at room temperature, revealing an active thermogenic process in the absence of thyroid hormones. The fall in mitochondrial respiration produced by treatment with propranolol-prazosin agrees with studies of intact animals and perfused muscle of cold-acclimated rats indicating the participation of {beta}- and {alpha}1-adrenergic receptors in muscle NST (27, 36). Even though the {alpha}1-receptor does not stimulate thermogenesis by itself, it mediates NE action on vasoconstriction (20) and greatly potentiates the action of {beta}-adrenergic receptor by increasing the ability of cAMP to stimulate thermogenesis (44). These actions by the adrenergic receptors suggest that the fall of body temperature induced by propranolol-prazosin was likely the combined result of reduced heat production in mitochondria and augmented heat loss produced by vasodilation derived from blocking the {alpha}1-receptors (20).

Because the hypothyroid rats were normothermic, it is germane to address the following points: 1) the mitochondrial respiration rate in muscle and liver, but not in BAT, failed to reach normal levels; and 2) reduced thermogenesis in muscle and liver did not affect the maintenance of body temperature. Concerning the first issue, one assumes that the thermogenic process seen in hypothyroid muscle and liver reflects the maximal biochemical capacity for NST in these tissues, which are primary sites of thyroid-induced obligatory thermogenesis. This view is concordant with the failure of exogenously administered NE to raise mitochondrial thermogenesis above the values achieved by endogenous NE. This phenomenon resembles that seen in BAT in Fig. 3, in which maximally stimulated mitochondria failed to respond to additional NE supply. On the other hand, the rapid normalization of oxygen consumption induced in muscle and liver by T4 replacement suggests that the capacity of the hypothyroid mitochondria to produce heat through thyroid-mediated pathways remained intact. It is unclear whether the different responses of muscle and liver, on one side, and BAT, on the other, stem from the fact that muscle and liver fulfill specific functions that generate heat as an obligatory metabolic by-product in a mitochondrial coupled state (see comments on UCP-3 below). Contrariwise, mitochondria-rich BAT in cold-acclimated rats maintains maximal responsiveness to NE and maximal uncoupling through UCP-1 action, in the presence or absence of thyroid (43), all of which renders BAT the central organ for cold-induced NST.

Another issue of importance refers to the normothermia in rats with reduced muscle and liver mitochondrial thermogenesis. Shivering thermogenesis is the primary mechanism used by the body to increase metabolic activity when a warm-acclimated rat is placed in the cold, or during experimental conditions of acute cold exposure of rats. However, after 4 mo of cold exposure and maximal BAT thermogenic activity, shivering does not appear to make up the shortfall in mitochondrial respiration in the two tissues, a view supported by the smaller creatine kinase activity in hypothyroid animals. Moreover, BAT weight and oxygen consumption in cold-exposed hypothyroid rats was similar to that of normal rats, whereas UCP-1 synthesis, measured in an earlier report (43), reached the levels of normal rats. Because thermal homeostasis is regularly achieved through coordinated adjustments in heat production or heat loss from peripheral vasodilation, it seems reasonable to think of an increased sympathetically induced insulation in compensation for the moderate decrease in heat production in muscle and liver, thus maintaining normothermia.

It is germane to mention here that the synthesis of UCP-3 in hypothyroid rats was most likely altered by the lack of T3. Recent studies by Gong et al. (19) and Vidal-Puig et al. (41) performed in UCP-3 knockout mice have provided clear biochemical evidence that even though UCP-3 has uncoupling activity, its absence did not alter cold-induced thermogenesis, whole body oxygen consumption or body temperature and have suggested the possibility of some other function of UCP-3, but the exact nature of this role has not been delineated. Other workers have postulated a physiological role for UCP-3 as exporter of fatty acid anions from muscle and BAT mitochondria when fatty acids are the predominant substrates, a function that does not involve the uncoupling properties of this protein (24). It is instructive that even though UCP-1 expression is unique to BAT, the uncoupling of muscle mitochondria in transgenic mice expressing UCP-1 in skeletal muscle did not alter the ability of muscle mitochondria to adjust oxygen consumption to changes in ambient temperature, an adaptation requiring sympathetic activity (2).

Taken together, the results demonstrate that thyroid hormones, in the presence of NE, are major determinants of long-term thermogenic activity in muscle and liver of cold-acclimated normal rats. After thyroid ablation, NST replaced thyroid-induced thermogenesis, and normal body temperature was maintained. These lifesaving effects of the sympathetic system, previously unknown in tissues of cold-acclimated hypothyroid rats except BAT (43), appear to result from an integrated operation from multiple organ systems aimed at preserving normal body temperature when the supply of thyroid hormones to tissues has ceased and body temperature falls.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
The purchase of animals, drugs, and reagents used in this study was financed by private funds. The Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina, the University of Buenos Aires, and the University of Graz supported the personnel, equipment, and laboratories in which the studies were carried out.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. A. Zaninovich, Hospital de Clínicas, Nuclear Medicine Center, Av. Córdoba 2351, 1120 Buenos Aires, Argentina (E-mail: azaninovich{at}sinectis.com.ar).

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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