Endocrinology, doi:10.1210/en.2004-1544
Endocrinology Vol. 146, No. 7 2872-2884
Copyright © 2005 by The Endocrine Society
Temperature Homeostasis in Transgenic Mice Lacking Thyroid Hormone Receptor-
Gene Products
Husnia Marrif,
Aria Schifman,
Zaruhi Stepanyan,
Marc-Antoine Gillis,
Angelino Calderone,
Roy E. Weiss,
Jacques Samarut and
J. Enrique Silva
Division of Endocrinology (H.M., A.S., Z.S., J.E.S.), Jewish General Hospital, McGill University, Montréal, Québec, Canada H3T 1E2; Montréal Heart Institute (M.-A.G., A.C.), Montréal, Québec, Canada H1T 1C8; Department of Medicine (R.E.W.), University of Chicago, Chicago, Illinois 60637; and Ecole Normal Superior (J.S.), 69364 Lyon Cadex 07, France
Address all correspondence and requests for reprints to: J. Enrique Silva, M.D., Jewish General Hospital, Division of Endocrinology, Room E-104, 3755 Cote-Ste-Catherine, Montréal, Québec, Canada H3T 1E2. E-mail: enrique.silva{at}staff.mcgill.ca.
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Abstract
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We studied temperature homeostasis in male mice lacking all thyroid hormone receptor-
gene products (TR-0/0). As other TR-deficient mice, TR-0/0 mice have lower core body temperature (TC) than cognate wild-type controls. We found that obligatory thermogenesis is normal in TR-0/0 and that the lower TC at room temperature (RT, 20–22 C) is caused by a down setting of the hypothalamic thermostat. However, TR-0/0 mice are cold intolerant due to impaired facultative thermogenesis. Norepinephrine-induced brown adipose tissue (BAT) thermogenesis is blunted, even though BAT-relevant genes and T4 deiodinase respond normally to cold stimulation, as do serum T3, serum glycerol (marker of lipolysis), and heart rate. BAT normally contributes to maintain TC at RT, 9 C below thermoneutrality, yet TR-0/0 mice do not show signs of being cold stressed at 20–22 C. Instead, oxygen consumption is greater in TR-0/0 than in wild-type mice at RT, suggesting the recruitment of an alternate, cold-activated form of thermogenesis to compensate for the lack of BAT thermogenesis. These results indicate that TR is necessary for T3 to modulate the central control of TC and for an essential step in norepinephrine activation of BAT thermogenesis but not to sustain obligatory thermogenesis. In addition, the results provide evidence for an alternate form of facultative thermogenesis, which probably originates in skeletal muscle and that is less effective and more energy demanding than BAT thermogenesis.
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Introduction
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THE PHYSIOLOGICAL EFFECTS of thyroid hormone (TH, T3) are mediated by receptors belonging to the super family of nuclear receptors. There are two genes encoding the known TH receptors (TR), TR and TRß, each of which generates several products by alternate splicing and use of internal promoters. Four of these, namely TR1 and TRß1, -2, and -3 bind T3 and can mediate T3 actions and are, thus, bona fide receptors, whereas some products such as TR2, or 
TR1, TR2, and TRß3 do not bind the hormone and their function remains to be elucidated (1). Even though TRs do not show marked differences in affinity for T3 or the capacity to mediate gene transactivation by T3 in vitro, differences in the phenotypes of several models of TR genes disruption indicate that some receptors may mediate some effects of TH better than others in vivo. For example, TRß appears more important for TH control of cochlear development, the feedback by TH at the hypothalamic and pituitary level, and certain aspects of intermediary metabolism, such as lipogenesis and cholesterol metabolism, whereas lower heart rates and lower body temperatures have been consistent findings in all models of TR1-deficient mice (1, 2). Such phenotypic differences may result from differences in the abundance of receptor isoforms in relevant tissues as well as intrinsic properties of a given isoform that make it better suited for the regulation of some genes. On the other hand, even isoform-specific phenotypes are more accentuated in all-TR-deficient models (TRß-KO) than in either TR- or TRß-deficient ones. Thus, although TRß2 appears as best suited for the feedback mechanism at the hypothalamic-pituitary level, serum TSH concentrations are higher in TRß-KO mice than in the various models of selective TRß deficiency (1, 2). As discussed later, TRß-KO mice have lower body temperature than TR-deficient mice, even though mice lacking only TRß have normal body temperature. Such findings suggest that TRs may be exchangeable to some extent or that they mediate different actions of T3 that converge to produce a given effect.
With the advent of homeothermy, TH acquired a key role in supporting thermogenesis and temperature homeostasis, but the underlying mechanisms are unknown or poorly understood (3). Reduced body temperature has been consistently found in TR1-deficient mice, a finding never reported in the various forms of TRß deletion or in the human thyroid hormone resistance syndrome, which is due to mutations of the TRß (Ref. 1 and references therein), implicating TR in the mediation of TH thermoregulatory function(s). Understanding how the lack of TR leads to this phenotype can provide valuable insight on the thermogenic role of TH and is, hence, an endeavor worthwhile to undertake. In addition, understanding why a given TR isoform may better mediate an action of TH may pave the way to design T3 analogs that selectively elicit medically desirable effects of TH. We aimed therefore to define the aspects of temperature homeostasis affected by the lack of TR.
The reduced core body temperature (TC) of TR-deficient mice has been so far documented at the so-called room temperature (20–22 C), which represents a moderate but significant cold stress. Because at such ambient temperature both obligatory and facultative thermogenesis contribute to maintain TC (4), the lower TC could hence be a sign of a deficiency in obligatory thermogenesis, in facultative thermogenesis or in both, that is, TC could be forced by a thermogenic deficiency; but it is also possible that TC is simply regulated at a lower level, being the expression of a down setting of the hypothalamic thermostat. Interestingly, these possibilities are not mutually exclusive. The information relating TR isoforms to temperature homeostasis is scarce and difficult to integrate. The administration of GC1, a TRß-selective agonist, to hypothyroid mice did not restore cold resistance, and this was associated with the failure of GC1 to restore the responsiveness of brown adipose tissue (BAT) to norepinephrine (NE), suggesting that TR is somehow necessary for T3 to sustain NE signaling, at least in BAT (5), and furthermore, that the cause of the lower body temperature of TR knockout mice may be a defect in facultative thermogenesis (4). In another recent study, a spontaneously occurring inactivating mutation in the human TRß was introduced in the TR. Transgenic mice expressing such a mutated receptor developed obesity, cold intolerance, and a defect in lipolytic response to adrenergic stimulation of adipose tissues (6). The problem with this model is that the mutant TR may be acting as a dominant negative, interfering with the function of both TR and TRß, a view that is in agreement with the absence of such a rich phenotype in TR-, TRß-, or even double, TRß-deficient mice (TRß-KO). These TRß-KO mice have been reported to have lower body temperature than pure TR-deficient mice (7, 8), although the pure TRß KO mice have normal body temperature (1). Moreover, mice lacking both TR genes also have lower oxygen consumption and show signs of BAT chronic stimulation and desensitization to NE (8), all suggesting that these animals are chronically cold stressed. Such observations strongly suggest that both receptors participate in mediating TH thermogenesis, but how they interact is not readily evident.
We have undertaken the systematic analysis of temperature homeostasis in mice with deletion of all known TR gene products (from now on TR-0/0 mice) (7), which has the advantage of avoiding dominant-negative effects of c-erb2 and truncated products of the TR gene. The studies presented here provide meaningful insight into the role of TR on the temperature homeostatic function of TH and on those hard to reconcile observations mentioned above. The results show that TR-0/0 mice have normal obligatory thermogenesis and that the lower temperature of TR-deficient mice at room temperature is the reflection of a down resetting of the hypothalamic thermostat, and not forced by a thermogenic deficiency. However, TR-0/0 mice do have a severely limited facultative thermogenesis, which is revealed as intolerance to colder environments and is caused by an impaired responsiveness of BAT to sympathetic stimulation. Finally, the results provide evidence for the recruitment of an alternative form of facultative thermogenesis, possibly from skeletal muscle, which prevents TR-0/0 mice from being cold stressed at room temperature, with little or no BAT thermogenesis.
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Materials and Methods
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Animals
All experimental procedures, as well as housing, handling, and breeding protocols, were approved by the McGill University’s Animal Care and Use Committee (protocol 2990). TR-0/0 mice, lacking all known products of the TR gene, were created in the laboratory of Dr. Samarut, as described elsewhere (7, 9). These mice were at the University of Chicago for several generations, being backcrossed more than 10 times into the C57BL/6 background, before TR-0/0 male and female mice were transferred to our lab, in which we crossed them with wild-type (WT) C57BL/6 mice. We generated our colony by intercrossing the resulting heterozygous so that both WT and TR-0/0 mice used in the present experiments are at least the fourth generation from common ancestors in the same C57BL/6 genetic background. Results presented here are from comparisons between WT mice, homozygous for the intact alleles of both TR and TRß genes and TR-0/0 mice. Experiments were performed in 3- to 5-month-old males, ranging 25–31 g, but matched within 1-month age and 5-g body weight differences in individual experiments. Unless indicated otherwise, experiments were performed with at least four mice per genotype. Animals were kept at 20–22 C (usually 21 C), with a 12-h light, 12-h dark cycle starting at 0600 h in standard plastic cages, with wood shaving beddings, at five mice of the same sex per cage when not breeding or under experimentation. As indicated where appropriate, animals were either acutely exposed or acclimated for at least 2 wk to thermoneutrality (as defined below) or other ambient temperatures, for which they placed in individual cages. During acute cold exposure, bedding was kept to a minimum, and animals had food and water ad libitum. Mice were fed a standard mouse chow, catalog no. 5001 (Agribrands Purina Canada Inc., St-Hubert, Québec, Canada) containing 3.3 kcal/g, with 13, 60, and 27% of the calories from fat, carbohydrate, and proteins, respectively.
In vivo measurements
Energy expenditure.
Energy expenditure was measured by indirect calorimetry in an open-circuit system (Oxymax System, Columbus Instruments Inc., Columbus, OH), as described previously (10). Mice were housed in individual, airtight plastic cages through which air was pumped at a constant rate, and where oxygen consumption (QO2) and carbon dioxide production were calculated from the corresponding concentrations in the air going in and out of the cage. Unless otherwise indicated, measurements were done for 24 h, four mice at a time. Within an experiment, measurements were made on contiguous days. For each mouse, readings were taken every 17.5 min because this is what it takes to complete the gas readings on four cages, so that each mouse QO2 was measured approximately 84 times. Twenty-four-hour profiles shown were generated by the means obtained from four mice in successive 17.5-min cycles. QO2 is expressed in [milliliters x hour–1 x 100 g–0.75] based on accepted principles (4). Measurements were performed either at thermoneutrality temperature (30 C) or at room temperature (21 C), as indicated, for which the Oxymax System was installed in a walk-in environmental chamber that maintains ambient temperature within 0.1 C of the set temperature (SureTemp, Raleigh, NC). Thermoneutrality temperature, approximately 30 C in the mouse (4), is defined as the ambient temperature at which body temperature is maintained solely by obligatory thermogenesis, without the participation of heat-saving or dissipating mechanisms and without activation of facultative thermogenesis. Thus, QO2 measured at thermoneutrality is the closest approximation to obligatory thermogenesis or the energy spent to sustain vital functions. However, because mice are not restrained or sedated, measurements as described here include nonexercise physical activity such as moving in the cage, grooming, etc. (4).
Food intake
Food intake was measured in individual metabolic cages placed at the desired ambient temperature. Food consumed was recorded daily for at least 3 d, averaged, and expressed on a per-day and 100 g0.75 body weight basis.
TC
TC was measured with a flexible rectal probe YSI 423 (Yellow Springs Instrument Co., Dayton, OH) connected to a high-precision thermometer (Yellow Springs Instrument Precision 4000A thermometer). Animals were not anesthetized or sedated and were restrained for not longer than 30 sec for the measurement. A small rubber ring, placed at 2.5 cm of the tip of the probe, ensured uniformity in the depth the probe was introduced in the rectum. TC was measured between 0900 and 1100 h unless indicated otherwise. In the acute cold exposure experiments, TC was measured in the morning before the mice were moved to the cold room (1700–1800 h) and then the next morning when animals were removed from the cold. TC was simultaneously measured in control groups maintained at room temperature.
BAT temperature in response to norepinephrine infusion
The whole procedure was done under anesthesia consisting of ketamine-xylazine-acepromazine, 50:5:1 µg/g body weight, given im in a volume of 1 µl/g. A temperature (YSI 427; Yellow Springs Instrument) probe was anchored under the interscapular BAT pad through a small skin incision, basically as described (11). Another probe (YSI 423; Yellow Springs Instrument) was inserted into the rectum as described above to measure TC. Through another small incision, a 27G catheter was inserted in one of the jugulars and anchored to the pectoral muscle. Throughout the procedures and observation period, mice were kept over an electrically heated pad set at 25 C to avoid profound hypothermia. Under anesthesia, during baseline observation, rectal temperature was usually 33–34 C. Pilot experiments showed no effect of saline infused iv at 1–2 µl/min on either BAT or rectal temperature. This latter was consistently 1–2 C less than that of BAT. After a baseline-recording period on the saline infusion, NE was given at a rate of 0.4 nmol/min, a submaximal dose (11) that in our hands produced a consistent and clear response in pilot experiments with WT mice.
Heart rate response to isoproterenol
These studies were performed at the Montréal Heart Institute. Mice were anesthetized with 2.5% isoflurane in oxygen, given on a loose mask at 0.5 l/min, and immobilized over a heating pad, as above. Electrocardiogram was recorded from lead I, for which needle electrodes were inserted in the forelimbs and right leg (ground). Signals were amplified with a single-lead electrocardiogram amplifier connected to the A/D recorder using IOX 1.8 software (EMKA technologies, Falls Church, VA). A 27G catheter was inserted in one of the jugulars exposed through a small skin incision. After a period of 5–8 min of observation during which heart rate remained stable, a bolus of 0.1 µg/g of isoproterenol was injected and recording was continued for additional 8–10 min. Maximal heart rates were reached within 1 min ± 10 sec and remained elevated for the period of observation (see Fig. 8
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Blood and tissue sampling
Blood was obtained either from the tails tip or inferior vena cava, under light isoflurane anesthesia, when mice were killed by exsanguination. Baseline fasting samples were obtained approximately 6 h after removing the food, which was done around 0800 h. Blood was allowed to coagulate at room temperature, and cleanly collected serum was stored at –20 C until analyzed. Tissues were snap frozen in liquid nitrogen and kept at –80 C until analyzed.
Biochemical assays
Enzymatic assay kits were used for measurement of serum triglycerides (Infinity triglyceride reagent, Sigma Diagnostics, Inc., St. Louis, MO), serum-free glycerol (glycerol colorimetric method, Randox, Mississauga, Ontario, Canada), and serum-free fatty acids (NEFA C, Wako Chemicals USA, Richmond, VA). Because triglycerides are measured by the release of glycerol, free glycerol was subtracted from the total glycerol measured in the triglyceride assay. Blood glucose was measured with a clinical glucometer in a few microliters of fresh blood, as recommended by the manufacturer (ADVANTAGE, Roche Diagnostics Corp., Indianapolis, IN). Total T4 and T3 levels were measured by RIA using commercial kits (Diagnostic Products Corp., Los Angeles, CA) with the modifications described elsewhere to adapt it for use with mouse serum (12). Type II iodothyronine 5'deiodinase (D2) activity was measured in the 1000 x g supernatant of BAT homogenates, using 2 nM 125I-rT3 in the presence of 1 mM propylthiouracil and 20 mM DTT, basically as described (13).
Uncoupling protein (UCP)1 measurement
UCP1 was measured by immunoblotting, essentially as described (14), using a highly specific rabbit antibody generated in our laboratory (15). This antibody reveals a single 32,000-Da band in Western blots of BAT mitochondria of either rats (15) or mice (10) but not in mitochondria from other tissues. Mitochondria from individual mice were blotted onto nitrocellulose paper using a dot blot apparatus with a 96-well manifold (Schleicher & Schuell, Keene, NH) at 6 and 3 µg of total mitochondrial protein. Same amounts of liver mitochondria were run in parallel as negative controls. First antibody was used in excess at a dilution of 1:1000, and the amount of UCP1 was quantified by the amount of first antibody bound to peroxidase-conjugated antirabbit IgG (Sigma-Aldrich, St. Louis, MO) as revealed by Western blot ECL (enhanced chemiluminescence) detection reagents (Amersham Biosciences, Piscataway, NJ). Blots were exposed to Kodak Biomax MS film (Amersham Biosciences), the OD of the dots quantified with Scion Image software (Scion Corp., Frederick, MD), and the results expressed in arbitrary densitometric units normalized to 6 µg of mitochondrial protein. Blots were stripped and then blotted with an anticytochrome C monoclonal antibody (Zymed Laboratories, Inc., South San Francisco, CA) to document the mitochondrial protein loading and fixation to the paper. Under the assay conditions described, UCP1 antibody produced no signal with liver mitochondria, whereas the CytC antibody gave signals proportional to the amount of mitochondrial protein loaded from both BAT and liver.
RNA isolation and PCR analysis
Tissue RNA was extracted with acid guanidinium-phenol-chloroform (16), quantified by absorbance at 260 nm, and stored in DEPC-treated water at –80 C until further analysis. Integrity of RNA was routinely verified by agarose gel electrophoresis. The mRNA for UCP1 was quantified by competitive RT-PCR, using primers, competitors, and conditions described elsewhere (10). mRNA peroxisomal proliferator-activated receptor-
coactivator-1 (PGc1) (17) was also measured by RT-PCR, using the same approach and the following primers: sense, 5'-ATGTGTCGCCTTCTTGCTCT and antisense, 5'-GCGGTATTCATCCCTCTTGA, obtained from the published sequence (17) (accession no. AF049330). Primers were designed using the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). They generated a 350-bp product from cDNA and a 452-bp product from the competitor DNA. Levels of mRNAs are expressed in attomoles per microgram of total RNA, except for UCP1, which is expressed in femtomoles.
Other tissue measurements
DNA and tissue protein content were measured by standard methods (18, 19).
Statistical analysis
Results are expressed as mean ± SEM. Experiments were repeated at least once, and experimental groups included a minimum of four mice, unless indicated otherwise. Experiments involving several treatment or time groups were analyzed by ANOVA followed by post hoc tests for multiple comparisons (Newman-Keuls). Two-way ANOVA was used to compare the effects of treatments on two experimental groups, e.g. ambient temperature acclimation on two genotypes. Individual means were then compared by the Bonferroni’s test if across experimental groups or the Newman-Keuls test if within one experimental group.
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Results
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The basic phenotype of TR-0/0 mice has been previously described (7). These animals grow somewhat less than WT controls, which accounts for their slightly lower body weight, their body composition being the same as cognate controls (7). Their fasting levels of blood glucose and serum triglycerides, free fatty acids, and glycerol were not significantly different from the WT controls and are not shown. Serum concentrations of T4 have been reported to be modestly but significantly reduced in TR-0/0 mice, whereas TSH and T3 levels do not differ from those of WT controls (20, 21). Note that the small differences both in serum T4 concentrations and body size need a large number of animals to reach statistical significance; hence, such differences may not be apparent in experiments involving fewer animals, which does not necessarily contradict published results (20, 21). Accordingly, it was always possible to find animals of similar weight in litters within a 1-month age difference.
Are TR-0/0 mice kept at room temperature under cold stress?
The so-called room temperature, 20–22 C, is substantially lower than the thermoneutrality temperature, which is approximately 30 C for the mouse (4). In all the studies where reported, the approximately 1 C lower TC of TR-deficient mice has been detected in animals housed at room temperature and therefore may reflect the inability of these mice of maintaining TC in a cool environment. Alternatively, this difference may reflect a down setting of the hypothalamic thermostat. To discern between these two possibilities, we carefully measured TC in WT and TR-0/0 mice acclimated at various temperatures, namely at 20 C (room temperature), 30 C (thermoneutrality), and 34 C. As shown in Fig. 1, TC was about 1 C lower in TR-0/0 than WT at room temperature, as reported previously for these mice (9), as well as for other TR-deficient mouse models (2), but the difference was not narrowed as the ambient temperature was progressively increased, up to 34 C. Had the lower TC of TR-0/0 reported at 20–22 C been the result of forced reduction in temperature, i.e. due to limiting thermogenesis, the difference should have progressively narrowed, and eventually abrogated, as the animals were acclimated at progressively warmer environments surpassing thermoneutrality.
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FIG. 1. TC in TR-0/0 mice and WT controls. The abscissa indicates the temperature at which the animals were acclimated for at least 2 wk, except for those exposed to 34 C, for which the exposure to the temperature was only 5 d for safety. *, P <0.05; **, P <0.01; ***, P <0.001. See Materials and Methods for details on procedures.
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BAT is the major site of thermogenesis in small rodents (22). This tissue shows signs of increased stimulation if animals are under cold stress, which has been documented in several experimental models, notably in hypothyroidism (23), or in the complete absence of TR (8), indicating that thyroid hormone is not necessary for the tissue to exhibit such signs. Signs of increased BAT stimulation have also been seen in more subtle reductions of obligatory thermogenesis, as occurs in mice lacking mitochondrial glycerol phosphate dehydrogenase (10). As shown in Fig. 2 and Table 1, BAT of TR-0/0 mice did not show signs of being more stimulated than that of WT controls. The histology of the tissue does not show typical signs of increased stimulation, namely reduced fat content, present in smaller droplets; a more acidophilic cytoplasm, reflecting increased mitochondrial mass; or more fluffy chromatin, reflecting the augmented transcriptional activity (23, 24). Biochemical signs of increased stimulation are increased DNA, RNA, and protein per milligram of tissue, which we did not find in TR-0/0 mice because we did not find either an increase in the expression of genes relevant to the BAT response to cold, such as UCP1, mRNA and protein, or PGC1 mRNA or in D2 activity (Table 1). As noted farther below (also see Fig. 6), these genes and D2 respond normally to acute cold stress in TR-0/0 mice, so their normalcy at 21 C indicates that stimulation of BAT in these mice is not substantially greater than in WT controls. Consistent with these findings, TR-0/0 do not show behavior signs of cold stress, such us reduced activity, curling of the body, or huddling (4). Altogether, these findings argue against TR-0/0 mice being under more cold stress than the WT when living at room temperature and favor a central down-regulation of body temperature, i.e. a down setting of the hypothalamic thermostat.
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FIG. 2. Representative histology of BAT obtained from WT controls and TR-0/0 mice reared at 20–22 C. Upper sections were stained with hematoxylin-eosin and the lower with osmium tetroxide to stain the fat. Magnification, x400.
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Obligatory thermogenesis in TR-0/0 mice acclimated at thermoneutrality
Obligatory or basal thermogenesis is the heat that results from the vital processes, the energy that is dissipated as heat in the energy transformations inherent to life (3). As mentioned in Materials and Methods, when the environmental temperature is in the so-called thermoneutral zone, this amount of heat is sufficient to maintain body temperature, without the participation of facultative thermogenesis or of heat dissipating or saving mechanisms (3, 4). Accordingly, BAT is quiescent in mice and rats maintained at thermoneutrality, and within minutes of exposing rats to this temperature, the transcription of the UCP1 gene is suppressed, as documented by others and us (10, 25, 26). Thus, measuring oxygen consumption at 30 C is a good estimate of obligatory thermogenesis. Results from mice acclimated at this temperature are summarized in Fig. 3. Figure 3A shows the 24-h profile of QO2, with its circadian variation due to increased nocturnal activity. Each curve represents the average of four mice measured simultaneously. The curves are virtually superimposed and the means obtained from the 24-h profiles are not significantly different (Fig. 3B). In agreement with QO2 data, food intake measured at thermoneutrality was not affected by the genotype either (Fig. 3C); nor did the genotype differentially affect the respiratory exchange ratio (or respiratory quotient), the mean of 80 measurements during 24 h being 0.83 ± 0.004 for WT and 0.82 ± 0.003 and indicating no change in fuel preference.
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FIG. 3. QO2 of WT and TR-0/0 mice acclimated at 30 C measured over 24 h. A, Each line was generated by the successive means obtained every 17.5 min over 24 h from four mice per genotype, housed in separate cages but measured simultaneously. The abscissa indicates the correlative number of the measurement, and the bar over the abscissa depicts the time lights were on (open) or off (black). Measurements were commenced at 1030 h. B, Mean 24-h QO2 obtained for each genotype over 80 measurements on four mice per group. C, Food intake measured in mice acclimated at 30 C. None of the differences is statistically significant.
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Facultative thermogenesis in TR-0/0 mice
TH is also necessary for facultative thermogenesis, and hypothyroidism is associated with a remarkable cold intolerance (Ref. 3 and references therein). The lack of signs of cold stress at room temperature and the normality of obligatory thermogenesis do not exclude a limitation in the capacity of TR-0/0 mice to respond to an acute cold challenge. Besides, the demonstration that hypothyroid mice treated with the TRß-selective analog GC1 did not recover the capacity to maintain body temperature when acutely exposed to cold (5) suggests TR may be somehow necessary for TH to support facultative thermogenesis. Figure 4 shows the results of a representative experiment wherein WT and TR-0/0 mice acclimated at 22 C were challenged with an acute overnight exposure (approximately 16 h) to 6 C. Figure 4A shows the TC of cold-exposed mice and controls kept at 21 C, measured at the end of the cold exposure. At 21 C, TC was approximately 1 C lower in the TR-0/0, as expected, whereas the difference widened after the cold challenge, with TR-0/0 becoming frankly hypothermic (31.9 ± 0.1 C) and torpid. Figure 4B shows the mean drop in TC with the cold challenge in WT and TR-0/0 mice in a paired manner, i.e. relative to the individual TC measured the morning preceding the cold exposure. TC fell 1.5 ± 0.3 C in the WT and three times more, 4.4 ± 0.4 C, in the TR-0/0 mice (P < 0.002). Figure 4C shows the amount of food consumed and weight change with the cold exposure in a parallel experiment. Initial weights were the same, 27.6 ± 1.1 and 27.3 ± 1.9. Even though food intake was the same in both genotypes, TR-0/0 mice lost significantly more weight than the WT controls. Thus, TR-deficient mice have markedly reduced cold tolerance and lose more weight than the controls during an acute cold challenge. Note, however, that in hypothyroid WT mice, TC descends even faster [<30 C within a few hours of exposure to 6 C, with > 50% mortality overnight (data not shown)].
Responses of circulating TH levels to cold exposure
An integral part of the response to cold is the stimulation of the hypothalamic-pituitary-thyroid axis and increased T4-to-T3 conversion. A blunted response in TR-0/0 mice could explain their subnormal response to cold, but as shown in Fig. 5, this was not the case. Whereas serum T4 levels did not change significantly with the overnight cold exposure, both serum T3 levels and the T3 to T4 ratio, an indicator of T4-to-T3 conversion, increased significantly with cold in both genotypes (P < 0.001). Moreover, serum T3 increased significantly more in TR-0/0 than in WT mice (P < 0.001), whereas serum T4 was not less, representing a more vigorous response both at the central and peripheral levels, the central response providing more T4 to sustain a greater peripheral conversion to T3. Therefore, a reduced thyroid axis response to cold cannot be invoked on the inability of TR-0/0 mice to maintain their temperature when acutely exposed to cold.
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FIG. 5. Serum T4 and T3 concentrations and T3 to T4 ratios in WT and TR-0/0 mice acclimated at 21 C and exposed to 6 C overnight for 16 h. Differences with the corresponding genotypes at 21 C are indicated (***, P < 0.001), whereas significance level between genotypes at a given temperature are indicated over the bars connecting the groups compared. N.S., Not significant.
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BAT responses to cold
BAT is essential for the response to sustained cold exposure (22). TH, actually locally generated T3 by the adrenergic activation of D2, is essential for the response to such a challenge in rats (15). The T3 surge derived from such activation rapidly increases the transcription of the UCP1 gene and the level of relevant enzymes in BAT (15, 27, 28). Preventing the activation of D2 with iopanoic acid (15) or the lack of this enzyme in transgenic D2-deficient mouse (29) is associated with blunted responses to sympathetic stimulation. Also PGC1, a cofactor necessary for transactivation by peroxisomal proliferator-activated receptor- (PPAR), retinoic acid receptor, and TR has been found essential for a full thermogenic response (30). Therefore, we investigated whether the lack of TR-0/0 was associated with reduced D2 activity or UCP1 and PGC1 gene responses to T3 causing the cold intolerance. As depicted in Fig. 6
, overnight cold exposure was associated with a 40–50-fold stimulation of D2 activity, and this response was not different in WT and TR-0/0 mice. Likewise, the UCP1 and PGC1 gene responses to such a cold challenge did not differ in magnitude in WT and TR-0/0 mice. The cold challenge as done here (16 h at 6 C) in mice acclimated at 22 C is not enough to see a robust response of UCP1 (protein) in lean C57BL/6J mice (31, 32, 33), which we confirmed because levels of were 5–15% higher after 16 h of cold exposure, not significantly different from controls at room temperature or between genotypes (data not shown).
Although essential for the BAT thermogenic response, UCP1 is not sufficient, and factors such as fatty acids may be necessary for its activation (reviewed in Ref. 22). Hypothyroidism is associated with failure of BAT to elevate its temperature in response to NE, and this defect is not corrected by the TRß-selective agonist GC1 (5), suggesting that TR is ultimately necessary for actual BAT heat generation, even if not required for the stimulation of thermogenic genes such as UCP1 and PGC1. Accordingly, we directly measured BAT temperature in response to a short-term intravenous NE infusion in anesthetized WT and TR-0/0 mice as described in Materials and Methods, basically with the same approach used to test the effect of GC1 (5, 11). Within the short time of the infusion, heat generated is the expression of the activation of preexisting UCP1 and the ensuing uncoupled mitochondrial respiration. Because the levels of UCP1 in the basal state were not affected by the genotype (Table 1), a failure to normally increase heat production during a short NE infusion would be indicative of a deficiency of the activation process. In addition, such an experiment with exogenous NE would exclude the potential variable of reduced BAT sympathetic (endogenous) stimulation. Results are shown in Fig. 7. BAT temperature increased linearly with time during the NE infusion in WT mice, whereas TC increased in parallel, remaining 1–2 C below, in agreement with the idea that BAT is the source of the increase in body heat. The response was so vigorous that infusion was cut short in WT mice to avoid dangerous hyperthermia. In contrast, in TR-0/0 mice, NE virtually failed to increase both BAT and TC, suggesting a failure of NE to activate heat production in BAT and the ensuing rise in TC.
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FIG. 7. Effect of iv NE infusion on BAT and TC in WT and TR-0/0 mice acclimated at 21 C. Because of the anesthesia, TC at the start of the NE infusions was 33–34 C, with no differences between the two genotypes (see Materials and Methods for details and rationale).
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Other responses to adrenergic stimulation
The response of D2 to cold exposure shown above (Fig. 6A) indicates that the central sympathetic response to cold is normally robust and that the responsiveness BAT to NE is not globally reduced in TR-0/0 mice. In vivo, the adrenergic D2 activation depends on 1-adrenergic receptors (ARs), but it occurs even in the presence of propranolol (34, 35), i.e. the vigorous response of D2 to cold is not evidence in favor of normal responsiveness to ß-AR-mediated NE stimulation. An adrenergic response mediated by ß-AR and relevant to cold adaptation is lipolysis. Inhibition of cold-associated lipolysis reduces the thermogenic response to cold, whereas enhancing lipolysis with adenosine deaminase increases it (36). The lipolytic response to cold is most evident in the first hours after the start of the cold stress (37). To assess the global lipolytic response to cold, we chose to measure serum glycerol levels 2 h after placing the mice at 4 C (as opposed to free fatty acids, which may be more affected by differences in oxidation rates). As shown in Fig. 8A, glycerol clearly increased in response to cold but both the baseline levels and the stimulated values were not affected by the genotype. On the other hand, baseline heart rate has consistently been reported lower in TR-deficient mice (1, 2, 38), which could be possibly due to reduced responsiveness to ß-AR stimulation. As shown in Fig. 8B, heart rate was, as expected, lower in TR-0/0 than WT mice but responded equally to isoproterenol in both genotypes. The statistical analysis is shown in Table 2. Both basal and isoproterenol-stimulated mean heart rates were significantly lower in the TR-0/0 mice, but the increment induced by isoproterenol was the same (110 min–1) as that of WT mice. Thus, the reduced response to adrenergic stimulation in TR-0/0 is not global, sparing at least the heart chronotropic response to exogenous catecholamines and the overall lipolytic response to cold.
Temperature homeostasis of TR-0/0 mice at 20–22 C
As already mentioned, for small rodents the so-called room temperature (20–22 C) represents a significant cold stress that requires the contribution of BAT thermogenesis to maintain TC (22, 39). Reflecting the recruitment and activation of BAT thermogenesis, QO2 progressively augments when normal rodents are moved from thermoneutrality to increasingly cooler environments, in inverse proportion to the ambient temperature (4), and this is paralleled by an increase in BAT mitochondria GDP binding and UCP1 concentration (39). However, we have shown here that despite the lack of responsiveness of BAT to NE, TR-0/0 reared at room temperature do not seem under cold stress, BAT does not show signs of being more stimulated than in WT controls (Fig. 2, Table 1), and indeed the lower TC of these mice is not due to a thermogenic deficiency. These findings indicate that overall thermogenesis at room temperature is preserved in TR-0/0 mice. Indeed, we found that QO2 was not only normal in TR-0/0 kept at room temperature, but much to our surprise, it was higher than in WT controls. Figure 9A depicts the 24-h profile of WT and TR-0/0 mice. Each line is generated by the means of four mice per genotype measured simultaneously, every 17.5 min, as described in Materials and Methods. The differences between the two genotypes are highly significant, which is more tangibly depicted in Fig. 9B by the relative frequency of QO2 over the 24-h period. Mean 24-h QO2 of 81 measurements per mouse times four mice was 209 ± 6 ml x hour–1 x 100 g–0.75 in WT and 275 ± 7 in TR-0/0 mice (P < 0.0001). For purposes of comparison, we show in Fig. 9C the mean QO2 of WT and TR-0/0 mice measured at 30 or 21 C (in separate groups of mice). In WT mice, QO2 was about 60% higher at 21 C than at 30 C, reflecting the recruitment of BAT thermogenesis, whereas in TR-0/0 mice kept at 21 C QO2 was more than twice those kept at 30 C. This experiment has been repeated, with very similar results (Table 3). Note in Table 3 that food intake was higher at room temperature than that at 30 C (Fig. 3) and that it was higher in TR-0/0 in proportion to the increase in QO2, showing internal consistency of both measures of 24 h energy expenditure.
Because BAT of TR-0/0 mice kept at room temperature does not show signs of being more stimulated and responds poorly to NE, such findings suggests the participation of a mechanism different from BAT. The 24-hour profile of QO2 in mice at 21 C shows that the difference between the two genotypes is not the same through the day (Fig. 9A), being greater during darkness, when mice are more active, and smaller during the light hours, when the animals are more quiet in their cages. Thus, the TR-0/0 QO2 computed at dark was 1.42 ± 0.05-fold greater than that of WT, whereas during daytime the corresponding fold difference was only 1.24 ± 0.04 (P < 0.007 for the relative increase at night). This contrasts with findings at thermoneutrality, during which the circadian variability is the same in both genotypes, with the curves generated by both genotypes being superimposed (Fig. 3). Altogether, the data show that at thermoneutrality energy expenditure is the same in both genotypes, i.e. they have same obligatory thermogenesis, whereas in cooler environments, needing the activation of facultative thermogenesis, energy expenditure increases in both genotypes but clearly and significantly more in TR-0/0 than WT controls, with the difference being wider during periods of activity.
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Discussion
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We report here that TR-0/0 mice, as other models lacking TR (2), have lower TC at room temperature, but we show that this is not forced by a thermogenic deficiency but due to a down setting of the hypothalamic thermostat. We also show that TR is not necessary for TH to sustain obligatory thermogenesis because this is normal in TR-0/0 mice, but we demonstrate that TR is necessary for BAT thermogenic response because TR-0/0 mice become severely hypothermic after an overnight exposure to cold (4–6 C) and BAT fails to generate heat in response to adrenergic stimulation (exogenous NE). Interestingly, even though BAT normally contributes significantly to the maintenance of TC at room temperature, TR-0/0 seem perfectly comfortable at this temperature, which is associated with increased energy expenditure, suggesting that BAT thermogenesis is substituted by another form of thermogenesis, possibly related to muscle activity. Each of these points will be discussed below.
That the lower TC observed in different forms of TR-deficient mice at the so-called room temperature is not the reflection of an uncompensated thermogenic deficiency is supported by the observation that the difference with WT is not narrowed but remains constant when ambient temperature is raised to as high as 34 C, well over the thermoneutrality temperature. In addition, TR-0/0 mice do not exhibit any behavioral differences with WT at room temperature such as being less active in a curled position or huddling, as seen in cold stressed animals (4). Most importantly, TR-0/0 mice show no evidence of increased BAT sympathetic stimulation relative to appropriate WT controls at 20–22 C. Regardless of the responsiveness of BAT, this shows signs of increased stimulation when there is cold stress, as is observed in hypothyroidism (23), in the complete lack of TR, i.e. TRß-KO mice (8) (discussed later) or in mice with deletion of the mitochondrial glycerol 3-phosphate dehydrogenase gene (10), contrasting with the normal findings in TR-0/0 mice (Table 1 and Fig. 2). Thus, the BAT of TR-0/0 mice, neither morphologically nor biochemically, seems to be more stimulated than that of WT controls, including the expression of UCP1 and PGC1 as well as the activity of D2. Because we also showed that D2, UCP1, and PGC1 mRNA respond normally to cold stress (Fig. 6), the normal level at 21 C indicates lack of increased stimulation rather than lack of response.
Another important finding in the present studies is the down setting of the hypothalamic thermostat by about 1 C in TR-0/0. It has been reported that in addition to the thermogenic deficiency derived from the lack of TH, the thermostat is set lower in hypothyroid rats (40, 41), much as it occurs here in TR-0/0 mice. Such a lowering of the set point in hypothyroidism, or for that matter in TR-deficient mice, has adaptive value because it lessens the need for thermogenesis. This latter is directly proportional to the difference between the thermostat set point and the ambient temperature and the thermal conductance of the animal (4). Thus, assuming that thermal conductance is not affected by the lack of TR, the 1 C lower set point represents a reduction of approximately 6% in the thermogenic demands when the animals live at 21 C: 100 x [1 – (36-21)/(37-21)]. It is evident that the saving is greater as the temperature is warmer, i.e. at 28 C is 11%. Interestingly, hypothyroid rats freely moving in experimental ambient temperature gradients stay at a temperature 2–3 C higher than euthyroid controls (40), altogether suggesting that energy conservation in heating the body is a physiological priority in the absence of TH action.
To examine obligatory thermogenesis, we measured QO2 at thermoneutrality, the ambient temperature in which TC is sustained solely by obligatory thermogenesis, without the need of facultative thermogenesis or heat-dissipating or -saving mechanisms (4). Under such conditions, QO2 was the same in WT and TR-0/0 mice, in contrast with the 30–40% reduction seen in hypothyroid mice (not shown, but see Ref. 10); not only was the 24-h mean QO2 the same, but also the circadian profile was the same as well (Fig. 3). In agreement with this result, food intake at thermoneutrality was also the same in both genotypes. Lastly, respiratory exchange ratio was not affected by the genotype under these experimental conditions. These are important findings because they indicate that none of the genes or mechanisms used by TH to stimulate obligatory thermogenesis strictly requires TR. By inference, the low metabolic rate reported in all-TR-deficient mice (8) is the result of the lack of TRß-mediated effects, which is further discussed later.
Even though TR-0/0 mice do not show signs of cold stress in cool environments, e.g. room temperature, these mice have a limited facultative thermogenesis, becoming significantly more hypothermic than WT mice within hours of being exposed to 6 C. The problem seems to be the inability of BAT to generate heat in response to NE (Fig. 7). Note that we infused NE at comparatively high rate, which caused BAT from WT animals to respond vigorously, bringing body temperature to dangerously high levels, whereas TR-0/0 mice barely increased BAT and TC during the infusion. Our observations are in agreement with those by Ribeiro et al. (5), who found that whereas T3 could correct the unresponsiveness to NE in WT mice, a TRß-selective agonist, GC1, could not. They further found that the response of BAT cells to forskolin was also impaired and not corrected by GC1, suggesting that the impairment is at a post-ßAR level, probably at or beyond the generation of cAMP. Intriguingly, other responses of BAT to sympathetic stimulation, such as D2, and UCP1 and PGC1 mRNAs were not impaired, nor were global lipolysis or heart rate responses impaired. NE signaling in BAT is complex. Whereas D2 activation by cold or exogenous NE can be fully suppressed by AR antagonists, and specifically prazosin, an 1-AR antagonist and not affected by ßAR blockers (34), there is a complex interaction between AR and ßAR-initiated pathways that is TH dependent (42) as well as a cAMP response element in the enzyme gene (43), so that some cAMP is necessary for 1-AR agonists to stimulate the enzyme. Likewise, UCP1 gene stimulation requires cAMP, which interacts synergistically with T3 to stimulate gene transcription (44), but the response of UCP1 to T3 in hypothyroidism can be maximal with comparatively small amounts of cAMP because it is fully restored well before T3 normalizes the ßAR receptor and cAMP levels (45). One would infer then that the failure of NE to increase heat production derives from the need of maximal levels of cAMP or the need of a yet to-be-identified, TR-dependent factor needed to activate mitochondrial heat production. Further studies are doubtlessly important to define the defect leading the failure of NE to ultimately induce heat production in BAT in mice lacking TR.
The results presented add new insights to previous observations, furthering our understanding of TH thermogenesis and TH-adrenergic interactions. As mentioned, TRß-KO mice have lower body temperature than TR-deficient mice. At room temperature, body temperature can be as low as 32–34 C in these mice (7), and they show clear signs of increased BAT stimulation (8), even though pure TRß-deficient mice do not have lower body temperature (1, 2). Golozoubova et al. (8) found that even though BAT showed signs of hyperstimulation in double-TR knockout mice, the tissue did not respond normally to adrenergic stimulation, which they interpreted as sign of desensitization due to chronic exposure of BAT to increased sympathetic stimulation. Interestingly, we have found that pure TRß–/– mice have signs of chronic BAT stimulation (data not shown), but these mice, as others have reported (1, 2), have normal body temperature at 21 C, suggesting that the compensation is effective. On the other hand, as shown here, BAT responsiveness is markedly reduced in TR-deficient mice, suggesting that the elimination of TR in mice lacking TRß negates the BAT compensation by causing a defect in the NE signaling pathway. Rather than desensitization, the defect of BAT responsiveness to NE in TRß-KO mice primarily results from the lack of TR-mediated T3 stimulation of a critical step in the signaling cascade. Desensitization may compound the problem but is not the primary event.
Despite the impaired BAT response to NE, TR-0/0 mice tolerate well the moderate cold stress of being at room temperature, 8–10 C below thermoneutrality. As indicated previously, there is sufficient evidence to support the concept that BAT normally contributes significantly to temperature homeostasis at such ambient temperature. The increase in QO2 in the transition from thermoneutrality to 21 C, shown here and elsewhere (10), reflects in normal rodents the increase in BAT thermogenesis needed to adapt to the lower temperature (4, 46). Notwithstanding the lack of response of BAT to adrenergic stimulation, we show here that QO2 not only is not reduced, but also it is greater in TR-0/0 mice living at room temperature than in WT controls (Fig. 9). Besides, BAT does not show signs of being more stimulated than in WT controls as if there were no need for BAT thermogenesis in TR-0/0 mice living at 20–22 C. These observations strongly suggest the substitution of BAT thermogenesis by another form of heat generation. It is interesting that this putative form of alternate thermogenesis is more energy demanding than that present in WT because both QO2 and food intake are increased in TR-0/0 mice reared at room temperature. Furthermore, in acute cold exposure, TR-0/0 lost more weight than WT, yet they ate the same amount (Fig. 4C). In this regard, our results are similar to observations made in mice with deletion of the UCP1 gene (47), another form of BAT function abrogation. Like TR-0/0 mice, UCP1-ablated mice did not show evidence of being cold stressed at room temperature but were cold intolerant (47), yet to everybody’s surprise, they were not obese. On subsequent investigation, these UCP1-deficient mice were resistant to a high-fat diet when housed at room temperature, but when studied near thermoneutrality, they gained weight more rapidly than the WT (48), which was interpreted as the abrogation by thermoneutrality of an alternative form of thermogenesis, more energy demanding, and that hence prevented obesity when activated, i.e. at room temperature. It would appear then that when BAT is congenitally or chronically disabled, its function may be replaced by another form of facultative thermogenesis that is both more energy demanding and less thermogenic. What could this alternate form of thermogenesis be and where does it occur?
The concept that BAT is the main, if not the only, site of facultative thermogenesis in rodents (22) has been reinforced by additional observations made in UCP1 knockout mice. As mentioned, these mice have cold intolerance (47), but their gradual exposure to cold improves their resistance to cold without the emergency of a form of NE-activated thermogenesis (49). These authors demonstrated persistent muscle electrical activity during cold adaptation in these UCP1-deficient mice, whereas in WT controls shivering was rapidly replaced by BAT thermogenic activity, and they consequently attributed the cold tolerance to a form of chronic shivering (49) and concluded that UCP1 remains the only protein the activity of which can be recruited for the purpose of facultative thermogenesis (50). On the other hand, there is good evidence that skeletal muscle is a site of nonshivering facultative thermogenesis in birds (51, 52), but there is no agreement whether this tissue, or for that matter other tissues, may be a site of nonshivering facultative thermogenesis in mammals. Certainly BAT is not prominent in large mammals, at least during adulthood, and it has been proposed on solid grounds that muscle can take that function (see Ref. 53 for a recent review and additional references).
The fraction of energy dissipated as heat in muscle may vary and may be subject to regulation, possibly by two mechanisms that are not mutually exclusive, namely Ca2+ movement between cytoplasm and sarcoplasmic reticulum (54, 55) and the flux of reduced nicotinamide adenine dinucleotide (NADH) via the glycerol-3-phosphate shuttle, as suggested by observations made in mice lacking the mitochondrial glycerol-3-phosphate dehydrogenase (10, 56). On the other hand, whereas the circadian variation of TC was the same in WT and TR-0/0 mice at thermoneutrality (Fig. 3), the difference between TR-0/0 and WT mice at room temperature was wider at night (42%), when animals are physically more active, than in daytime (24%), lending support to the idea that the alternate source of heat could be the skeletal muscle. Thus, all evidence taken into consideration, long-term BAT deficiency results in the recruitment of an alternative form of thermogenesis that may be akin to chronic shivering, but this does not exclude the possibility that muscle increases heat production associated with its activity, be this shivering or normal muscle contraction. In this regard, there is substantial evidence that TH makes muscle activity more thermogenic (57), probably by stimulating the expression of sarcoendoplasmic reticulum Ca2+-ATPase-1 (SERCA-1), predominantly in slow twitch muscle (53). Slow-twitched muscle is under more sustained nervous stimulation than fast-twitch muscle, and the increased expression of sarcoendoplasmic reticulum Ca2+-ATPase-1 in it is associated with increased Ca2+ flow through the sarcoplasmic membrane and lower coupling of Ca2+ transport to ATP use (53). In TR-0/0 mice, this would be mediated by TRß, the response to which may be enhanced either because of the absence of c-erbA2 and other negative products of the TR gene (21) or the facilitated interaction with transcription coactivators (58).
In summary, we have shown here that the lack of TR receptor is associated with a reduction in the set point of temperature regulation, preservation of obligatory thermogenesis, and a severe limitation in BAT facultative thermogenesis. Such results suggest that this receptor is important for TH to modulate central regulation of temperature and to stimulate some step in the NE-signaling pathway that is critical for a normal thermogenic response of BAT. The limitation in BAT facultative thermogenesis, the data further suggest, results in the activation of alternative form of thermogenesis, which is less efficient and more energy demanding than BAT thermogenesis, which may be a combination of chronic shivering associated with enhanced heat production. In addition to defining some of the mechanisms of temperature homeostasis that require TR, our results raise a series of interesting questions, the pursuit of which may improve our understanding of temperature homeostasis and energy balance.
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Acknowledgments
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The authors thank Nadia Ait Chalal for her technical support.
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Footnotes
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This work was supported by a grant from the Thyroid Research Advisory Council of the American Thyroid Association with funds provided by Abbott Laboratories, Inc. H.M. was supported in part by a Diana Metzer Abramsky Research Fellowship of Thyroid Foundation of Canada.
First Published Online April 21, 2005
Abbreviations: AR, Adrenergic receptor; BAT, brown adipose tissue; D2, type II iodothyronine 5'deiodinase; NE, norepinephrine; QO2, oxygen consumption; TC, core body temperature; TH, thyroid hormone, T3; TR, TH receptor; TR-0/0, mice with deletion of all known TR gene products; TRß-KO, TR-deficient model; UCP, uncoupling protein; WT, wild type.
Received November 29, 2004.
Accepted for publication April 12, 2005.
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References
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Abstract
Introduction
21 C)