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Baystate Medical Education and Research Foundation, Department of Medicine, Division of Endocrinology, Baystate Medical Center, Tufts University Medical School, Springfield, Massachusetts
ABSTRACT I. THERMOGENESIS AND BODY TEMPERATURE HOMEOSTASIS A. Evolutionary Aspects: Homeothermy and the Need for Additional Heat Production B. Regulation of Body Temperature: Obligatory and Facultative Thermogenesis C. Thermogenic Mechanisms 1. Thermogenic mechanisms that increaseATP utilization 2. Thermogenic mechanisms that reduce the efficiency of mitochondrial ATP synthesis II. HORMONAL REGULATION OF THERMOGENESIS A. Thyroid Hormone Acquires a New Role With the Advent of Homeothermy B. Thyroid Hormone and Obligatory Thermogenesis 1. How does TH affect the thermodynamic efficiency of the homeothermic machine? 2. TH stimulation of ATP consumption as a thermogenic mechanism 3. TH reduction of ATP synthesis efficiency as a thermogenic mechanism C. Thyroid Hormone and Facultative Thermogenesis 1. TH-adrenergic interactions 2. TH and BAT thermogenesis 3. Is there another site of nonshivering facultative thermogenesis in mammals? III. OTHER HORMONE ROLES IN THERMOGENESIS A. Insulin, Glucagon, and Epinephrine 1. Insulin and thermogenesis 2. Glucagon and epinephrine B. Glucocorticoids C. Leptin IV. THERMOGENESIS AS A SOURCE OF VARIABILITY IN ENERGY EXPENDITURE V. SUMMARY AND CONCLUSIONS GRANTS ACKNOWLEDGMENTS REFERENCES
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I. THERMOGENESIS AND BODY TEMPERATURE HOMEOSTASIS |
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A. Evolutionary Aspects: Homeothermy and the Need for Additional Heat Production
Studies in poikilothermic species show that biological processes have in general an optimal temperature, particularly regarding locomotive activity and the utilization of energy (5). The advent of homeothermy, that is, of mechanisms to keep body temperature constant, independently from a broad range of environmental temperatures represented a leap forward in evolution as it allowed species to expand their niche. While mechanisms to save or dissipate heat are important in adjusting to sudden changes in temperature, if the body could not produce significant amounts of heat, the range of ambient temperatures for effective regulation would be severely limited. Biological machines are not any different from any other in that they require energy, in the form of fuels, to perform some form of work, which in the case of the biological machines is the functions inherent to life. In biological machines, the energy contained in substrate fuels is captured in an utilizable form, as purine nucleotides, largely ATP, which can be thus considered the energy currency of the cells, an immediate source of energy to sustain biological functions (Fig. 1A). Now, whenever there is energy transformation or transfer, some energy is lost as heat, and the energy transformations in the biological machine are no exception. Life, therefore, naturally produces heat, but this heat is insufficient to maintain TC if the environment is significantly colder, as evident in poikilothermic species, the body temperature of which parallels that of the environment. To maintain TC in cold habitats with just the minimal amount of heat derived from vital functions would require both vast thermal insulation and minimizing the body surface area for heat loss by irradiation and evaporation. Insulation would have to be massive, particularly in smaller species, and would limit substantially activity and displacements, as it would the adoption of sphere-shaped postures to minimize the heat-losing surface. Instead, mechanisms to increase heat production have evolved, as it is readily evident when comparing poikilothermic and homeothermic species metabolic rate. Thus, whether one looks at the whole animal or separate organs, mice have much faster metabolism than lizards of similar size and studied at the same ambient temperature (78, 119) (Fig. 1B). The fact that smaller animals, with a higher surface area-to-volume ratio, hence more prone to lose heat, have higher metabolic rates than larger species (102) is, in and of itself, evidence that species "learned" how to upregulate metabolism to produce sufficient heat to keep up with the demands.
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The heat normally resulting from sustaining vital functions is called obligatory thermogenesis, and as mentioned above, homeothermic species have evolved an obligatory thermogenesis (ObT) higher than that in poikilothermic species (78).
Operationally, one can define a thermoneutral zone, a range of ambient temperatures where ObT is sufficient, without the participation of any other temperature homeostasis mechanism, to maintain TC. In this range of ambient temperatures, the body is in thermal equilibrium with the environment (102). The thermoneutral zone is narrow, yet it is possible to define a lower and a higher limit to the range, but for the purpose of this discussion, I prefer to use the term thermoneutrality temperature (TN), defined as the lowest ambient temperature at which ObT suffices to maintain body temperature without the participation of other thermoregulatory mechanisms. Thus TN is identical to the lower limit of the thermoneutral zone. TN depends on the magnitude of ObT relative to the thermal conductance of the animal. As mentioned, this latter is strongly influenced by the surface area-to-volume ratio. Smaller species not only have higher metabolic rates relative to their mass than larger animals, but by virtue of their so much larger surface area-to-volume ratio, their thermal conductance is proportionally higher, and their TN cannot be as low as that of larger species. Thus TN is 30 and 28°C in mice and rats, respectively, compared with 23°C in humans (102).
When ambient temperature goes below TN, the immediate response is to activate heat-saving mechanisms, which include vasoconstriction, piloerection, and the adoption of a curled posture to reduce the surface of heat irradiation. Animals also reduce their mobility. Such mechanisms, as mentioned, are very limited, and additional thermogenesis is promptly activated. This additional heat, produced on demand, is called facultative or adaptive thermogenesis. Shivering is the earliest and most primitive response to increase heat production and is called shivering facultative thermogenesis. This form of thermogenesis consumes large amounts of energy, is not effective during severe cold (e.g., 4–6°C for rodents) or prolonged cold exposure, and interferes with normal activity. Understandably, homeothermic species have evolved a more efficient and long-lasting form of nonshivering facultative thermogenesis that uses pure metabolic mechanisms to generate heat. For simplicity, we will call this latter facultative thermogenesis (FcT) and the former just shivering. The site and mechanisms of FcT differ in the two classes of homeothermic animals, birds and mammals. In birds, the major site of FcT seems to be the skeletal muscle (72), whereas in mammals the major site of FcT is brown adipose tissue (BAT) (51). As discussed later, BAT can heat the body effectively and with a lower energy cost probably by virtue of its particular anatomical locations, rather than by the nature of the biochemical mechanism used to generate heat.
As mentioned above, as machines, cells are not different from any other in that there is an obligatory loss of energy as heat during energy capture and utilization to support work or endothermic reactions. Therefore, one form of generating more heat is to increase energy utilization, in biology, basically to produce more ATP. Another form of increasing heat generation is by reducing the thermodynamic efficiency of the energy utilization, that is, by sacrificing the work output for the sake of generating more heat. The comparison of the homeothermic with the poikilothermic machine suggests that both types of mechanism are used by homeotherms to increase heat production. As already mentioned, mammals have significantly faster metabolic rates than reptiles, all variables such as shape and ambient temperature being controlled for (78, 119), but also the thermodynamic efficiency of the homeothermic machine is lower. This is illustrated in Figure 1C, with data from Woledge (254), that shows that the ratio of mechanical work per change of enthalpy (W/
H) is significantly lower in mouse and rat muscle than in muscle from frog or tortoise.
1. Thermogenic mechanisms that increaseATP utilization
Being an endothermic reaction, ATP synthesis can only be increased by accelerating its utilization. ATP utilization is certainly a function of metabolic rate, that in turn is a function of ambient temperature, the so-called Q10 effect, but this explains only a minor fraction of the high metabolic rate of homeothermic species because the difference remains wide when metabolic rates are compared at the same ambient temperature (78). This leads to the conclusion that warm-blooded species spend ATP in a futile manner. Metabolic cycles resulting from coupling lipolysis and lipogenesis, glycolysis, and gluconeogenesis, etc., account for a significant fraction of ATP consumption. Such cycles are quantitatively important in liver and in the cycling substrates between this latter and muscle or adipose tissue. However, such cycles are activated as a consequence of increased metabolism rather than as a means to increase metabolic rate, and they do not seem to be used to a significant extent as thermogenic mechanisms. Even if activated by TH, metabolic cycles account for a comparatively low fraction of the thermogenic effect of this hormone (211). There seems to be significant differences between poikilothermic and homeothermic species in the reutilization of substrate, e.g., muscle lactate used as gluconeogenesis substrate in liver (43, 159, 178), but to the best of my knowledge no comparative quantitative studies are available in the literature.
On the other hand, cells spend an important fraction of their energy in maintaining certain ion gradients, and there is evidence that such mechanisms may be used for the purpose of increasing ATP consumption in a futile manner. Thus Hulbert and Else (119) find that the metabolic rate is more sensitive to ouabain, an inhibitor of Na+-K+-ATPase, in tissues of mammals than in reptile counterparts. Not only the energy spent maintaining this gradient is higher in homeothermic than in poikilothermic species when expressed in absolute terms, but also when expressed as a fraction of total energy expenditure. On the other hand, when these ion gradients across the membrane are reduced either by leakage or by utilization of them to support transmembrane transport, the cell is forced to spend more ATP to keep the gradient. It has been postulated that this is one mechanism whereby TH increases thermogenesis, namely, by increasing the permeability of the cell membrane to Na+ and K+, allowing the passive entry of the former and exit of the latter, thus forcing the Na+-K+-ATPase to spend more energy in maintaining the gradients (108–110).
Another such mechanism to increase thermogenesis is the Ca2+ exchange between cytosol and sarcoplasmic reticulum. Cytosolic Ca2+ is an important intracellular second messenger and a signal to activate a number of processes, muscle contraction among others, but the cessation of the signal and the maintenance of the responsiveness to the signal requires the rapid reuptake of the cytosolic Ca2+ back into the endoplasmic reticulum, the energy for which is provided by the coupling of ATP hydrolysis by a Ca2+-dependent ATPase located in the membrane of the organelle. Ca2+ cycling between cytosol and sarcoplasmic reticulum in a modified muscle segment around the eye of deepwater fish provides heat to maintain the function of the eye and adjacent brain at temperatures as high as 14°C over the water temperature (24, 26). This is a good example of a mechanism used by nature to produce heat that antecedes homeothermy. Interestingly, the ryanodine receptor (RyR) channel expressed in this organ is similar to that in slow-twitch muscle (161), which may be more important for thermogenesis than fast-twitch muscle, as discussed below. The "leakage" of Ca2+ from the reticulum will force the activity of the enzyme, with ensuing energy expenditure. A dramatic illustration of the potential of this mechanism to produce heat in mammals is malignant hyperthermia, wherein in genetically predisposed individuals or animals, certain environmental factors such as some anesthetics can make the sarcoplasmic reticulum frankly leaky (see Refs. 158, 166 for reviews), with an ensuing life-threatening hyperthermia. Furthermore, it is possible that some leakage occurs in the normal resting muscle contributing to ObT. We estimated based on observations made in isolated sarcoplasmic vesicles that the Ca2+ recycling across the sarcoplasmic membrane could account for 30–70% (depending on the calcium pool size in the muscle) of the resting muscle energy expenditure (153). As mentioned, muscle is a major site of FcT in birds (72), and it has been found that SERCA1 and RyR channels increase in muscle of ducklings during cold adaptation (74). Such observations altogether add support to the hypothesis that sarcoplasmic reticulum Ca2+ leak, coupled to rapid recapture by the sarco/endoplasmic reticulum Ca2+-dependent ATPase (SERCA), could subserve a thermogenic role (reviewed in Refs. 25, 59).
It would appear then, largely from comparisons of Na+-K+-ATPase activity between poikilothermic and homeothermic species, as well as from comparative observations regarding muscle calcium cycling, that nature has increased these ionic exchanges, to a significant extent in a futile manner, for the sake of producing heat in homeothermic species. In general, these mechanisms generate heat largely by forcing cells to produce more ATP to support the maintenance of the gradients. In contrast to other thermogenic mechanisms discussed below, the thermodynamic efficiency of ADP phosphorylation seems to be preserved. In agreement with this idea, ADP phosphorylation in the heater organ of deepwater fish, even under stimulation by Ca2+, seems to remain well coupled (172). However, it is possible that the net efficiency of ATP utilization in pumping Ca2+ into the sarcoplasmic reticulum be also reduced to some extent to producing heat, which has been called "uncoupling" of the Ca2+ pump (67, 219), as will be discussed below.
2. Thermogenic mechanisms that reduce the efficiency of mitochondrial ATP synthesis
The energy resulting from the electron transfer through the respiratory chain in the mitochondria is transiently trapped in a proton gradient across the inner membrane of this organelle, the so-called proton-motive force, which is then utilized by ATP synthase to produce ATP by phosphorylation of ADP (144) (Fig. 2A). In the presence of an oxidizable substrate, but in absence of ADP, the gradient builds up and respiration is kept at a minimum (state 4 respiration) (Fig. 2B), illustrating the low permeability of the membrane to protons and the "braking effect" of the gradient on respiration. If ADP is added, the ATP synthase will phosphorylate it to ATP utilizing the energy trapped in the gradient (Fig. 2A). This will reduce the gradient to some extent and allow respiration to proceed as a function of the gradient's reduction, i.e., the rate of ATP synthesis (state 3 respiration) (Fig. 2B). However, the coupling of oxidations to ATP synthesis is not perfect, and a significant part of the energy liberated from the substrate will normally be dissipated as heat. If a low-impedance route is provided for protons to move back into the mitochondrial matrix, which can be done with substances appropriately called uncouplers [p-trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP) in Fig. 2B], protons will more readily move back into the matrix, bypassing the ATP synthase and collapsing the gradient, causing respiration to be accelerated but uncoupled from ADP phosphorylation. Since the passage of protons across the membrane, in the direction of the gradient, is an exergonic reaction, the energy will be now dissipated as heat instead of being captured in ATP (Fig. 2B) (144).
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Attempts to demonstrate such leak through artificial membranes with the exact composition of mitochondria membrane failed, leaving it unexplained how it could occur in native mitochondria (44). The cloning of two new UCPs, called UCP2 and UCP3 (BAT UCP became then UCP1), of more ubiquitous tissue distribution, brought the expectation that they could explain the proton leak detected in mitochondrial of tissues other than BAT (88, 98, 243), and indeed, when expressed in yeast mitochondria, these novel proteins could mimic the action of UCP1. The cloning of new UCPs in mammals stimulated the search in other species. Observations in potato mitochondria had already revealed the presence of a 32,000 kDa protein that conferred this mitochondria properties reminiscent of BAT mitochondria (241), and thus a UCP1 homolog was cloned from potato (138, 240) and subsequently demonstrated in all higher plants investigated (126). Homologs have been also found in birds (83, 183, 242). The phylogenic analysis of these proteins shows they belong to a large family of mitochondrial anion carriers and that UCP1, UCP2, UCP3, and the bird homologs are clustered close to plant UCPs, whereas the other two mammalian homologs UCP4 and BMPC1 are more distant (30). UCP1 and bird and plant UCPs share many properties, such as being inactivated by nucleotides, activated by fatty acids, and induced by cold. Such observations clearly demonstrate that uncoupling of mitochondria respiration from ATP synthesis is not a late but an ancient strategy used by nature to produce heat. Notwithstanding these observations, a major role for all the UCPs in mammalian FcT is still to be convincingly demonstrated. Transgenic mice lacking either UCP2 or UCP3 do not exhibit a readily evident thermogenic deficiency (7, 99, 244). While mice overexpressing UCP3 at very high levels are hyperphagic, yet leaner than controls (58), the expression of UCP2 in yeast mitochondria in amounts similar to those normally found in tissues failed to produce a detectable proton leak, suggesting the uncoupling originally observed in forced expression experiments in yeast was an artifact derived from the high levels of expression (227). On the other hand, it is possible that some of these proteins have different roles, one of which could be to contribute to ObT, as suggested by some genetic studies in humans (32, 203, 247), and by some data from our laboratory that will be discussed below. These proteins may also, or instead, accomplish other functions. For example, the deletion of the UCP2 gene in mice is associated with resistance to Toxoplasma gondii, as a consequence of uncontrolled production of reactive oxygen species in macrophages (7). In addition to limiting the production of reactive oxygen species, a role that UCP3 and plant UCPs (224) may share with UCP2, UCP2 may modulate insulin secretion (55, 258) and, along with UCP3, may be important for fatty acid metabolism (reviewed in Ref. 196). In the author's opinion, the lack of striking phenotypes in transgenic models of gene deletion must be taken cautiously, because it has been repeatedly found that phenotypes could be obscured by compensations or redundant mechanisms, as illustrated later with specific examples. More research is obviously needed.
Looking for additional thermogenic mechanisms, we have recently initiated the investigation of the NADH glycerol-3-phosphate (G3P) shuttle. Reducing equivalents (H+, e–) generated from substrates oxidized in the cytoplasm ultimately reduce NAD to NADH, H+. This cannot, however, enter the mitochondria to complete the reduction of O2 to H2O, and thus cannot directly transfer the energy to ATP. This is accomplished by shuttle molecules than can enter the mitochondria and are reduced by NADH (144). There are two major shuttle systems: the so-called malate-aspartate shuttle and the G3P shuttle (Fig. 3). In the former, the molecule reduced by NADH is oxaloacetate, which is reduced to malate by the cytoplasmic malate dehydrogenase. The malate enters the mitochondria and in the matrix is reoxidized to oxaloacetate by the mitochondrial malate dehydrogenase that then transfers the reducing equivalents to the complex I of the respiratory chain, generating three ATP molecules per atom of oxygen reduced. The resulting oxaloacetate is returned to the cytosol in the form of aspartate, generated by transamination from glutamate, and in the cytosol, aspartate regenerates oxaloacetate by the transamination of 
-ketoglutarate to glutamate.
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By catalyzing the interconversion between DHAP and G3P, the G3P shuttle constitutes a crossroad between lipid and carbohydrate metabolism as well as the main entry of glycerol into gluconeogenesis. Such relationships are shown in Figure 4. Thus G3P can be converted to DHAP by mGPD and thus enter the gluconeogenesis pathway mainly in liver and kidney. Another fate of G3P, quantitatively important in lipogenic tissues (e.g., adipose tissues, liver), is to be acylated to form triglycerides and other lipids. On the other hand, G3P can also be generated by phosphorylation of glycerol resulting from lipolysis, i.e., triglyceride hydrolysis, in a reaction catalyzed by glycerol kinase (GK). Liver has a large content of GK, and because of its size, this organ is the main site of production of G3P from circulating, lipolysis-derived glycerol (120). It should be noted that other tissues also have significant amounts of GK that plays the role of locally recycling glycerol derived from lipolysis. Notable among these are muscle, brain, and BAT (121, 248), where the enzyme is stimulated by the sympathetic nervous system (86), but interestingly GK is not present in white adipose tissue (WAT).
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-cells. In rodents, mGPD is highly expressed in BAT (173). In the mouse, skeletal muscle ranks second, next to BAT in mGPD abundance, followed by brain, kidney, and liver (136), which we have just confirmed (3). This hierarchy is similar in the rat (142). The preferential use of G3P shuttle in muscle allows the rapid aerobic generation of ATP from NADH produced by glycolysis, reducing the accumulation of lactate. In the liver, and probably other gluconeogenic tissues, G3P is more of a glucose precursor, whereas in lipogenic tissues, such as adipose tissues and liver itself, G3P is the backbone for TG synthesis. Although a rapid way to generate ATP aerobically, ATP synthesis via this pathway is less efficient because only 2ATPs are generated per atom of oxygen fully reduced to H2O. This results from the reducing equivalents entering the respiratory chain in complex III, with the energy of the missing third ATP being dissipated as heat (64). Thus the predominant use of the G3P shuttle could subserve a thermogenic function. The function of the G3P shuttle in different tissues depends not only on the abundance of its enzymes, particularly the rate-limiting one, mGPD, but also on the makeup of other enzymes in the tissue, which in turn reflects the role the tissue plays in whole body metabolism. This will obviously depend both on how active this shuttle is and on how active the alternative malate-aspartate shuttle is. As mentioned, mGPD in rodents is most active in BAT and muscle (100, 136, 142, 173), whereas liver for comparison has lower levels of activity and a very active malate-aspartate shuttle. Thus respiration in liver, but not in muscle, of normal mice is inhibited by aminooxyacetate, an inhibitor of the malate-aspartate shuttle (Fig. 5A). One would also predict from this information that G3P would be mainly a fuel for muscle and BAT, whereas in liver G3P would be less of, or not at all, an oxidizable substrate. This is exactly what we have observed, as shown in Figure 5, A–C. The addition of G3P in sequence after succinate did not change or even decreased respiration in liver but increased it by a factor of four in muscle (tibialis anterior) (Fig. 5, A and B). Note that the absence of mGPD did not have an effect in liver respiration, whereas it completely prevented the G3P-stimulated respiration in muscle. When substrates were added on tissues respiring on Ringer glucose, G3P increased QO2 by sixfold in muscle and by threefold in BAT slices, to a level not different from that induced by succinate, whereas it did not significantly increase respiration of liver slices (Fig. 5D).
To investigate a role for mGPD in thermogenesis and intermediary metabolism, we have been studying a transgenic C57Bl/6J mouse with targeted disruption of the mGPD gene (81) (mGPD –/–). Results have been recently published (3, 71). The most salient characteristics of the phenotype are summarized in Table 1. Briefly, we have found that these animals do have a mild reduction in energy turnover, as judged by both food intake and indirect calorimetry (oxygen consumption, QO2) (71). They have lower blood glucose and higher triglycerides as well as G3P plasma levels. Consistent with the G3P shuttle being more active in muscle, G3P levels and the lactate-to-pyruvate ratio were elevated in muscle, but not in liver. Actually, in liver the lactate-to-pyruvate ratio was reduced probably due to large influx of lactate and the large capacity of the liver to oxidize it to pyruvate, dumping the reducing equivalents into the mitochondria via the malate-aspartate shuttle (3). Most interestingly, mGPD –/– mice had 15–20% higher levels of both T4 and T3, which was due to increased thyroidal secretion, as plasma binding or clearance was not affected by the deletion of the mGPD gene. Because these animals have reduced energy turnover (QO2 and food intake), this suggests that the elevated levels of thyroid hormones are part of a response to cold stress. Consistent with this idea, BAT had signs of being chronically stimulated when animals were reared at 22°C, but raising the ambient temperature to 32°C, slightly over thermoneutrality, resulted in regression of such signs, atrophy of the tissue, and reduction of the circulating levels of T4 and T3 to those of the wild-type genotype (WT) controls. We also found increased UCP3 expression in skeletal muscle in mGPD –/– males but not in the female counterpart. Altogether, these results suggested an attempt to compensate a thermogenic deficiency (71).
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Intriguingly, even though male and female mGPD –/– mice did not perceptibly differ in basal levels of glucose, TG, FFA, G3P levels, etc., markers of qualitative changes in intermediary metabolism, the thriftiness revealed by the high-fat diet or by energy restriction was evident only in females (3). The reasons for such gender differences have not been elucidated, but the data suggest that the increase in TH levels and UCP3 probably play a role. As mentioned, plasma T3 was 15–20% higher in mGPD –/– males than in the WT males, whereas in mGPD –/– females plasma T3 concentration was not higher than in their WT controls. Likewise, UCP3 was not as elevated in mGPD –/– females as it was in the male counterpart (3). In view of the responsiveness of UCP3 mRNA to T3 (71), it is possible that the lack UCP3 elevation in mGPD –/– females was due to their failure to elevate T3. On the other hand, while UCP3-deficient mice are not obese (99), in the context of absence of mGPD, UCP3 may play a role in the resistance to diet-induced obesity of mGPD –/– males because double-knockout males, i.e., UCP3 –/– mGPD –/–, gain weight as mGPD –/– females when challenged with a high-fat diet (Stepanyan, Schifman, and Silva, unpublished observations).
While the work described above was in course, Brown et al. (45) published their observations on another mGPD-deficient model. They reported their mGPD-deficient mice have reduced viability and grew less than WT controls, as we find too. However, they did not detect a reduction in QO2 or in food intake. Most notably, their female mGPD-deficient mice did not gain more weight than the WT controls when fed a high-fat diet since weaning, whereas mGPD-deficient males identically treated gained less weight than WT. In addition, they did not demonstrate a thermogenic deficiency in mGPD-deficient mice of either gender, but this was investigated less thoroughly than we did. While there are many differences in the experimental approach in both studies, it is likely that the major reason for the different findings derives from some unidentified difference(s) in the genetic background. While the nominal genetic background of both genotypes was the C57Bl, the original mGPD transgene carrying the disruption was of different origin, and the C57Bl background of our mice is one that has been for innumerable generations in Japan. The differences between the two models illustrate the caveats of using transgenic models of gene deletion, as the consequences of it may be obscured by compensatory mechanisms and genetic background. Notwithstanding, rather than finding these differences disturbing, their investigation could be revealing of unsuspected factors contributing to the phenotype.
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II. HORMONAL REGULATION OF THERMOGENESIS |
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The thyroid gland is present in all vertebrates, where it is essential for a coordinated development and to control specific functions. The effects of TH on amphibian metamorphosis have been known for nearly a century (105; for a review, see Ref. 92), and the devastating effects of congenital hypothyroidism on mammalian brain development are well known as well (see Refs. 14, 75, 143 for most recent reviews and references). However, only in homeothermic species TH increases metabolic rate and thermogenesis (107, 249; reviewed in Ref. 207). The important role TH plays in temperature homeostasis is backed by the well-known cold or heat intolerance of animals and humans with hypothyroidism or hyperthyroidism, as well as by the hypothermia and hyperthermia associated, respectively, with extreme forms of hypo- and hyperthyroidism (see Ref. 207 for review). TH stimulates ObT, and it is essential for FcT.
As already mentioned, homeothermic species generate more heat than poikilothermic species, and this is due to both the more active metabolism and the lower thermodynamic efficiency of the homeothermic machine (78, 254). These two differences are largely dependent on TH. Indeed, the absence of TH makes homeothermic animals (humans included) regress into a quasi-poikilothermic state. Not only TH increases the number of energy transformations, as evidenced by 30–50% reduced basal metabolic rate, but it also reduces the thermodynamic efficiency of the homeothermic machine for the sake of producing more heat. For example, for any amount of mechanical work, euthyroid rat muscles generate more heat than the hypothyroid counterpart (145) (Fig. 6, further discussed below). Likewise, the energy cost of producing a given amount of glycogen from lactate is higher in euthyroid than hypothyroid hepatocytes (15). Moreover, studies in hepatocytes from rats with manipulated thyroid status show that the difference in oxygen consumption between the hypo- and the euthyroid state is much greater than the difference in ATP turnover (an estimate of the amount of effective work) between the two states (114).
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In addition, TH is essential for FcT, which is inherent to homeothermy. In mammals, particularly in small ones including the human newborn, BAT is the main site of FcT. Interacting with sympathetic nervous system, TH is essential for the full thermogenic response of BAT (reviewed in Refs. 207, 212). Hypothyroid rodents develop profound hypothermia if placed in the cold, and this is promptly corrected, within 24–48 h of giving small doses of T4 that normalize the thyroid status of BAT but not other tissues (21). In both hypothyroid rats and mice, norepinephrine fails to increase BAT temperature, and this defect is again rapidly corrected by the administration of small doses of T4 or T3 (187, 188). TH is essential to sustain the norepinephrine signaling cascade, acting at the receptor as well as the postreceptor level (see Refs. 52, 197, 198, 230 and references therein), but T3 and norepinephrine also interact synergistically at a more distal level, regulating the expression of several genes (20), most notably the expression of the UCP1 gene (reviewed in Ref. 218). Moreover, both UCP1 expression and BAT temperature elevation in response to norepinephrine correlate nicely with TH receptor occupancy by T3 (36).
B. Thyroid Hormone and Obligatory Thermogenesis
1. How does TH affect the thermodynamic efficiency of the homeothermic machine?
It has been known for more than a century that TH increases basal metabolic rate (150), the closest expression of resting ObT, yet still we do not know the precise underlying mechanisms. As reviewed above (Fig. 1), there are basically two major ways to generate more metabolic heat, namely, to increase ATP turnover, by stimulating its utilization, and to reduce the thermodynamic efficiency of ATP synthesis, that is, capturing less energy of the substrate in the form of ATP and dissipating more of it as heat (207). There is ample evidence that TH may stimulate both types of thermogenic mechanisms in homeothermic species.
2. TH stimulation of ATP consumption as a thermogenic mechanism
TH can increase ATP utilization, to a significant extent in a futile manner, for the sake of producing heat. Thus TH increases lipolysis and lipogenesis, proteolysis and protein synthesis, glucose oxidation, and gluconeogenesis. However, the energy cost of stimulating these metabolic cycles is small, probably explaining together not more than 10% of the TH-dependent stimulation of energy expenditure (see, for example, Ref. 91). Perhaps more important is the increase in ATP consumption to maintain ion gradients, namely, the Na+ and K+ gradient across the cell membrane and the Ca2+ gradient between the sarcoplasmic reticulum and cytosol. Directly or indirectly, TH can stimulate the entrance of Na+ in the cells or the extrusion of K+, forcing Na+-K+-ATPase activity to restitute the gradients of these two ions across the cell membrane, with the attendant ATP consumption (110, 125), whereas TH also stimulates the expression of Na+-K+-ATPase (124). There was initially great enthusiasm over this mechanism (76), but today it is felt that it only explains a comparatively minor fraction of TH thermogenesis (59, 90, 205). Even though estimates derived from measurements of ouabain-sensitive QO2 suggest that 
20% of basal metabolic rate (BMR) could be explained by the Na+-K+-ATPase in humans (59), about two-thirds are accounted for by brain and kidney, where the effect of TH on ouabain-sensitive QO2 is small or nil. Revisiting early studies examining the effect of ouabain on QO2 in rats suggests that the contribution of Na+-K+-ATPase to TH-dependent energy expenditure is very small in the euthyroid state, i.e., in the transition from hypothyroidism to euthyroidism, whereas it could be more important in thyrotoxicosis (77).
Another postulated mechanism of increased ATP consumption is the stimulation of Ca2+ transfer from cytosol to sarcoplasmic reticulum (59). This is thought to be largely due to increased activity of the sarcoplasmic/endoplasmic reticulum Ca2+-dependent ATPase (SERCA). TH indeed stimulates the expression of SERCA1 in skeletal muscle (reviewed in Ref. 219). This sole effect could explain the increase in the sarcoplasmic calcium pool, the increased release of calcium during contraction, and ultimately the additional energy expenditure to return the calcium back into the sarcoplasmic reticulum. TH administration to hypothyroid rats increases the SERCA activity and the amount of sarcoplasmic reticulum, which is associated with a substantial increase in the amount of energy spent in Ca2+ pumping (221). Slow-twitch muscle is more responsive to T3 than fast muscle, largely owing to the predominant stimulatory effect of TH on SERCA1 gene expression, the baseline expression of which is low in this type of muscle (219, 220, 239). While TH can stimulate the expression of SERCA2 in individual muscle fibers, the effect is modest and diluted by a reduction in the number of SERCA2-expressing fibers so that the global effect on SERCA2 can even be negative (163, 239).
It has been debated whether there is flux of calcium across the sarcoplasmic membrane in the resting muscle, i.e., a leak of Ca2+. The most recent evidence suggests that the resting cytosolic calcium is in a dynamic equilibrium with the sarcoplasmic calcium (153 and references therein). Independent calculations of the ATP's need to maintain the resting cytosolic Ca2+ concentration in muscle indicate that it may amount to 30–70% of resting muscle ATP demands in the rat (153). This is interesting, since TH also increases the number and activity of ryanodine receptors, involved in the release of Ca2+ from sarcoplasmic reticulum into the cytosol, both in heart and skeletal muscle (60, 127), suggesting that TH could also stimulate sarcoplasmic Ca2+ efflux into the cytosol, both in the resting state and during contraction, enhancing the demands of ATP to return it into the sarcoplasmic reticulum.
Overall, skeletal muscle QO2 increases 30% in the transition of hypothyroidism to euthyroidism (11), and SERCA activity in such transition increases 2.5- to 3-fold and 1.5-fold in slow- and fast-twitch muscle, respectively (219, 220). The stimulation of calcium pumping by TH thus accounts for a large fraction of the physiological TH-dependent stimulation of resting muscle energy expenditure. Because skeletal muscle normally contributes 20–30% of REE or BMR (260 and references therein), Ca2+ transport in muscle could explain as much as 10% of the effect of TH on resting thermogenesis. Because the flux of Ca2+ is increased during muscle activity, the associated heat production is also increased. This has been examined by direct measurement of heat production as a function of tension developed in either twitch or tetanus in both a fast-twitch muscle (extensor digitorum longus, EDL) and a slow-twitch muscle (soleus) (145). Results relevant to this discussion are depicted in Figure 6. In both muscles, either twitch or tetanus activity produced heat linearly as a function of the developed tension, but for any level of tension, there was significantly more heat produced in the euthyroid muscle than in the hypothyroid counterpart, that is, the energy cost of mechanical work is greater in the euthyroid than in the hypothyroid state, the difference being accounted for by the extra heat produced in the euthyroid state. The slopes of the curves, i.e., the increase in heat production with tension, was not affected by the thyroid status in EDL, but it was significantly greater in the euthyroid soleus than in the corresponding hypothyroid muscle, suggesting that in a slow-twitch muscle TH stimulates heat production by an additional mechanism. This may be the predominant stimulatory effect on SERCA1 expression, which is more accentuated in slow- than in fast-twitch glycolytic muscle (219) and the greater capacity for reducing the Ca2+/ATP ratio of this SERCA isoform (68). Interestingly, when the lines are extrapolated to tension zero, the intercepts, which the authors call tension-independent heat production (145), are significantly greater, by >50%, in the euthyroid than in the hypothyroid muscles, providing independent support to the idea that resting muscle heat production is also greater in euthyroidism.
Finally, it is important to recall that slow-twitch muscles are relatively more abundant in larger mammals, such as humans, who in addition have less brown fat, so that these mechanisms are quantitatively more important in larger species. The presence of type II iodothyronine 5'-deiodinase in human but not in rodent skeletal muscle (201) may also be an indication that muscle TH-dependent thermogenesis is more important in humans. As mentioned earlier, resting energy expenditure is very sensitive in athyreotic humans to changes in the dose of thyroxine. The small dose changes in these studies did not significantly affect serum concentrations of the active thyroid hormone T3, suggesting that local T4-to-T3 conversion in the site of thermogenesis could be more important than circulating levels of T3 (2).
3. TH reduction of ATP synthesis efficiency as a thermogenic mechanism
The concept that TH could uncouple oxidative phosphorylation in the mitochondria is not new, but it was ignored for many years (118) until Brand and colleagues (111) postulated, based on observations made in isolated mitochondria, that TH increased the leak of protons through the mitochondrial inner membrane, thus reducing the proton-motive force and stimulating oxidations to maintain the rate of ATP synthesis. These authors showed later in liver cells that in the transition from hypo- to euthyroidism there was an increase in respiration, without a significant increase in ATP turnover, indicating that the TH-induced energy expenditure in this transition was probably dissipated as heat via the stimulated proton leak. Indeed, about half the TH-induced increment in QO2 could be accounted for by the stimulation of the proton leak, whereas the other half was thought to be nonmitochondrial. In the transition from the euthyroid to the hyperthyroid state, there was a more marked increase in QO2, and now half was accounted for by ATP synthesis whereas the other half was explained by the proton leak, with no significant change in the nonmitochondrial component (114) (Fig. 7). Such findings suggest that physiological levels of TH primarily stimulate heat production, rather than ATP consumption. Such interpretation is in agreement with the early observation that Na+-K+-ATPase activity in liver membranes is clearly increased in the thyrotoxic state but is not significantly reduced in the hypothyroid state relative to the euthyroid level (77).
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As mentioned earlier, the NADH-G3P shuttle constitutes another mechanism of producing ATP with lower efficiency, particularly in skeletal muscle, where there is abundant extramitochondrial generation of NADH and where the alternate NADH-malate-aspartate shuttle is of little or no significance (144). It has been known for a long time that TH stimulates the activity of several mitochondrial enzymes, one of which is the mGPD, the rate-limiting enzyme of the shuttle (142 and references therein). Most interestingly, there is a good correlation between the stimulation of QO2 by TH in rat tissues (11) and the stimulation of mGPD activity (142) or its mRNA (97). Moreover, TH does not stimulate mGPD in those tissues where it does not stimulate QO2, nor does it stimulate the enzyme in species where it does not increase QO2 (249). For all these reasons, we undertook the study of transgenic mice lacking mGPD, as described earlier. Results have been published (3, 71), are partially summarized on Table 1, and have been discussed above. Relevant to this part of the discussion, male mGPD –/– mice living at room temperature (21°C), which represents a modest but significant cold stress for mice, have elevated levels of T4 and T3 and increased UCP3 mRNA and protein, and their BAT shows signs of being more stimulated than in controls, yet they have modestly reduced QO2, altogether suggesting a reduction in ObT despite the elevated levels of TH (71). These phenotypic differences were reduced or abrogated when animals were acclimated at 32°C, slightly over the nominal TN for mice (30°C), and the reduction of QO2 of mGPD –/– mice relative to WT was doubled, altogether indicating such phenotype represented a compensatory response to an ObT deficiency.
The lower QO2 in the presence of elevated TH levels is an indication that mGPD is somehow necessary for the stimulation of ObT by T3, but is obviously not essential, nor is it the only mechanism because hypothyroid mice have a QO2 30–40% lower than euthyroid controls (71). In agreement with this view, when hypothyroid mGPD –/– mice were treated with a moderately high dose of T3 (5 times the estimated production rate, 20 ng · g–1 · day–1) for 3 wk, they increased QO2 with the same temporal profile and to the same level as the WT controls (71), so higher levels of T3 can totally overcome the lack of mGPD. Interestingly, this was associated with a greater increase of UCP3 mRNA in mGPD –/– than in WT mice (71). As mentioned earlier, the elevation of UCP3 mRNA in mGPD –/– mice is both ambient temperature dependent and TH dependent, since this mRNA was reduced by acclimating both mGPD –/– and WT mice to 32°C, but was still higher in mGPD –/– mice, whereas levels decrease further, to an equally low level, when mice acclimated at 32°C were rendered hypothyroid (71). Furthermore, as indicated earlier, mGPD –/– males, but not females, have an elevation of plasma T3, and only the males show increased muscle expression of UCP3 (3), whereas mGPD –/– females but not males were sensitive to diet-induced obesity and exhibited a thrifty phenotype. Finally, double-knockout male mice, UCP3 –/– mGPD –/– become sensitive to diet-induced obesity, as mGPD –/– females. Thus the elevation of UCP3 in muscle of mGPD –/– males appears to be a relevant compensatory response to the lack of mGPD and is TH dependent. The participation of several mechanism may well explain why the injection of high doses of T3 to transgenic UCP3 –/– mice induces comparable response to those of WT controls (99). In these studies there was no measurement of circulating TH levels nor was attention paid to alternative mechanisms, e.g., mGPD, that could compensate for the lack of UCP3.
An important corollary from the results summarized in the preceding paragraphs is that more care should be exerted in interpreting the phenotypes of transgenic animals with targeted gene deletions, particularly when the phenotype does not show an expected deficiency. As we showed, when made moderately thyrotoxic, mGPD –/– mice increase QO2 as the WT controls (71). Had we not done the other experiments described above, the conclusion would have been that mGPD is not necessary for the thermogenic effect of TH, in the same way that such experiments led the investigators to conclude that UCP3 was not necessary for TH thermogenesis (99). Neither mGPD nor UCP3 is per se essential for thyrotoxicosis-induced increase in metabolic rate, but they may both contribute to the physiological stimulation of ObT by TH.
In summary, TH has been known to stimulate basal metabolic rate, the closest expression of ObT at rest, for over a century. Everything staying equal, increased basal metabolic rate alone is coupled to proportionally more heat production. A good fraction of the acceleration of metabolism by TH may be futile, that is, TH increases ATP consumption for the sake of heat production. Such mechanisms work basically by increasing ATP utilization, forcing cells to increase oxidations to maintain ATP levels for other cell functions. Although imprecise, the estimation is that these mechanisms increasing the utilization of ATP, whether futile or not, do not account for more than 50% of the thermogenic effect of TH. A large fraction of TH thermogenesis may result from the hormone reducing the efficiency of ATP synthesis, i.e., increasing the fraction of energy lost as heat in its synthesis, forcing the cells to burn more fuel to maintain the required production of ATP to sustain vital functions. Thus studies in mitochondria isolated from animals with manipulated thyroid status, notably from liver and skeletal muscle, indicate that TH reduces the proton-motive force by facilitating the leak of protons through the inner membrane. The recently cloned UCP homologs raised the expectation that these proteins could mediate the TH-mediated increase in proton leak, but the evidence has not so far supported this expectation, although such evidence does not exclude that these proteins, particularly UCP3, be necessary to support the thermogenic effect of TH. Finally, we have explored the possibility that mGPD could participate or be necessary for the stimulation of ObT by TH and obtained evidence that this enzyme is at least necessary because ObT is 17% lower in mGPD –/– mice when the contribution of BAT and compensatory mechanisms are minimized. However, as it occurred with UCP3, the induction of mild thyrotoxicosis caused the same stimulation of QO2 as in WT genotype controls. We must accept the concept that TH uses several mechanisms to stimulate ObT.
C. Thyroid Hormone and Facultative Thermogenesis
The discovery of type II iodothyronine 5'-deiodinase (D2) in BAT and its stimulation by the SNS (146, 216) allowed the demonstration of how important BAT FcT is in cold adaptation and how important TH is for its activation. Indeed, as discussed further below, numerous studies demonstrated previously that TH was somehow necessary for normal BAT responses to cold challenges (see Ref. 117 for a review of the time), but the activation of D2 by the SNS allowed the demonstration of a more complex and synergistic interaction between this latter and TH. Prompted by the potential of BAT to generate T3 both locally and for circulation (215), and the estimated important contribution of D2 to the levels of T3 in BAT even at room temperature (19), we performed experiments in hypothyroid rats treated acutely with T4 or T3. Two days of T4 treatment in doses equal to the daily production rate sufficed to restore cold tolerance and were associated with normalization of the UCP levels in BAT (21), while during this limited regimen of T4 there was no change of the thyroid status of the liver and plasma T3 levels remained subnormal, that is, these animals could tolerate severe cold (4°C) for 48 h without normalizing ObT, only normalizing BAT, as judged by the levels of UCP1. The inhibition of D2 with iopanoic acid prevented the normalization of both cold tolerance and BAT UCP1 levels by T4, even though we gave T3 to avoid the reduction of plasma T3 that could result from the inhibition of T4-to-T3 conversion elsewhere (20, 21). In contrast, to accomplish the same result with T3 injections required longer treatment and doses that increased plasma T3 and liver enzymes, used as markers of general thyroid status, to thyrotoxic levels (21). These observations have recently received decisive support from transgenic mice with disruption of the D2 gene (dio 2), which show cold intolerance, in spite of normal plasma T3 concentration (65).
The SNS and the adrenal medulla are considered part of a system, centrally controlled at the level of the hypothalamus and brain stem, called sympathoadrenal system. Norepinephrine, the main SNS neurotransmitter, is synthesized and stored in peripheral sympathetic nerve endings and released in response to coordinated nerve impulses targeted to specific tissues or organs. The adrenal medulla is an endocrine gland that produces predominantly epinephrine, which is secreted in response to impulses carried in the splanchnic nerves and which globally influences processes throughout the body, including many also stimulated by norepinephrine. The activity of the SNS and adrenal medulla is regulated centrally, in a coordinated manner, suited to changes in the environment. Thus both SNS and adrenal medulla are activated together in response to cold and strenuous exertion, whereas in hypoglycemia the adrenal medulla is stimulated but the activity of the SNS is suppressed.
TH plays an important role in the responses of the sympathoadrenal system to the environment (reviewed in Refs. 209, 210). The sympathoadrenal system triggers rapid adjustments, whereas TH enhances the capacity of the cells to respond to most actions of catecholamines and maintains a metabolic rate appropriate for the availability and mobilization of substrates essential to ensure vigorous adrenergic responses. On the other hand, catecholamines increase T4-to-T3 conversion in selected tissues, notably BAT, and may increase the retention of the TH receptor in the nucleus or the recognition of DNA sequences through PKA-mediated phosphorylation of the T3 receptor (170, 231, 237), the significance of which is yet to be defined. In general, a synergistic interaction is needed in circumstances when the delivery of substrate or release of energy is required, such as during cold adaptation or overeating. In opposite situations, for example, starvation, both systems are turned down independently, at separate levels: sympathetic outflow decreases (257), and thyroidal secretion as well as T4-to-T3 conversion are reduced. In general, sympathetic output to the tissues is reduced in hyperthyroidism and increased in hypothyroidism (139, 156, 236). In patients with thyrotoxicosis, plasma and urinary levels of norepinephrine have been reported as either normal or diminished (12, 62), whereas in hypothyroid patients urinary norepinephrine excretion and plasma norepinephrine levels are elevated (57, 62), reflecting an augmented production rate (61, 179).
The coordinated responses between the sympathoadrenal system and TH are particularly important in temperature homeostasis. As outlined above, BAT D2 plays a key role in the response to cold. Cold exposure triggers vigorous SNS stimulation of BAT, which, in addition to activating the thermogenic response of the tissue, activates D2, with the resulting increase in BAT T3 amplifying SNS effects such as lipolysis and stimulation of the UCP1 gene. In the hyperthyroid states, ObT is increased and there is less need for BAT FcT. Appropriately, SNS activity is reduced and as a result D2 is less stimulated. In addition, D2 is rapidly inactivated by T4 (213, 217), which makes the enzyme more susceptible to ubiquitination and degradation by proteasomes (226; reviewed in Ref. 17). Also, as it will be discussed later, TH reduces the expression of 3-AR in BAT. In the opposite situation, the hypothyroid state, there is an attempt to compensate the reduction of ObT with increased BAT thermogenesis, and this tissue shows indeed signs of increased stimulation (162), and there is an increase in norepinephrine turnover, a sign of increased SNS activity (256). The resulting increase in D2 activity, further enhanced by the reduced levels of T4, is critical to maintain the levels of T3 in BAT with plasma T4 concentrations as low as 25–30% of the euthyroid ones (53, 213). However, with the deepening of the hypothyroidism, the D2 response will be insufficient, there will be lack of T3 in BAT, and the tissue responses to norepinephrine will be ultimately reduced. These relationships are schematically illustrated in Figure 8A.
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