• Brian W. Kim, M.D.
  Instructor
  bwkim@partners.org
  
• Scott Ribich, Ph.D.
  Post-doctoral fellow
  sribich@partners.org
  
• Renata Grozovsky, Ph.D.
  Post-doctoral fellow
  regroz@biof.ufrj.br
  
• Beatriz Freitas
  Graduate student
  beatrizcgf@yahoo.com
  
• Jessica Hall
  Graduate student
  jessica_hall@hms.harvard.edu
  
• Matthew Rosene
  Research Assistant
  matt.rosene@gmail.com
  
• John W. Harney
  Research Assistant
  jharney@rics.bwh.harvard.edu
  
• Anita Nichols
  Administrator
  anichols@partners.org

In adults, the main biological effect of thyroid hormone is to accelerate energy expenditure, a paradigm that is based on observations made during experimental and clinical hypo and hyperthyroidism (16). In patients with severe hypothyroidism, total body energy expenditure can fall as much as ~50% and in thyrotoxic patients it can be increased by ~50%, totaling a ~3 fold induction over hypothyroid baseline. However, given that under normal circumstances serum thyroid hormone is remarkably stable, it is not immediately apparent how thyroid hormone modifies energy expenditure under physiological conditions.

Thyroxine (T4) is the main product of thyroid secretion. However, T4 is only a pro-hormone that must be activated by deiodination to T3 in order to initiate thyroid hormone action. This reaction is catalyzed by the type 1 and 2 iodothyronine deiodinases, D1 and D2 (1). Because D2-generated T3 has a longer residence time within the cell (17), an advantage of this pathway is the possibility of controlling intracellular T3 concentration and thus T3-receptor (TR) saturation independently of serum T3. This allows for D2-mediated rapid changes in intracellular thyroid status in a tissue-specific manner, without affecting systemic thyroid hormone

homeostasis. Perhaps, this is best illustrated by the essential role played by cochlear D2 in the early postnatal development of hearing. A transient isolated surge in D2 activity and local T3 production is critical for not only the onset of auditory function but also the subsequent maturation of auditory sensitivity, without which severe hearing impairment develops (1). In infants and other small mammals energy expenditure is controlled by a surge in D2 that fully saturates TR in the brown adipose tissue (BAT), their main thermogenic site, thus creating a state of tissue-specific thyrotoxicosis. In the absence of the D2 induction mice develop hypothermia when exposed to cold, confirming the critical role of D2-dependent adaptive T3 production (1). In addition, adaptive T3 production also plays a role in controlling energy expenditure in a number of mouse models of resistance to diet-induced obesity (18; 19). However, adult humans lack substantial amounts of BAT and the major site for adaptive energy expenditure is skeletal muscle. It is thus quite striking that, unlike rodents, adult humans express D2 significantly in skeletal muscle (20; 21), and this has been implicated as a determinant of energy expenditure (22-24), widening the interest in understanding endogenous, pharmacological and environmental stimuli that potentially influence D2 expression.

To understand the overall spectrum of thyroid hormone-related effects on energy homeostasis it is important to define the major categories of energy expenditure. The metabolic rate (or rate of metabolism) is an essential feature of all earthly functioning things, and is the sum total of all energy consumption or expenditure occurring in a system (machine, vegetal or animal) at any given time. The minimum energy expenditure that allows normal function is called basal metabolic rate (BMR). In animals it is the energy expenditure necessary to sustain minimal homeostatic functions as measured at rest, in a 12 h-fasted, fully relaxed subject kept at room temperature. It includes various forms of biological work, of which the major categories are (i) ionic and substrate cycles, particularly in the excitable tissues and kidney, (ii) metabolic cycles, particularly in the liver, muscle and adipose tissue, (iii) muscle function, e.g. heart beats, respiratory movements, vasomotor tonus, peristalsis, (iv) basal secretion of exocrine glands and glands annex to the intestinal tract.

The maintenance of the ATP pool that fuels BMR results in substantial heat production, which is known as obligatory thermogenesis. This is explained by the intrinsic thermodynamic inefficiency of energy transformation. In animals, this heat increases body temperature to one at which enzymatic reactions and biological functions operate optimally. In general, the higher the metabolic rate the higher the body temperature. In turn, the higher the temperature, the higher the rates of chemical reactions, especially those catalyzed by enzymes.

Two major biochemical processes explain heat production in living organisms, i.e. (i) ATP synthesis and (ii) ATP hydrolysis. The thermodynamic efficiency of the ATP synthesis is approximately 65%, that is, ~35% of the energy released during the oxidation of energy substrates is lost as heat. Thus, most of the energy is transiently stored in the ATP molecule (ATP synthesis). Later, once the ATP molecule is hydrolyzed in order to support biological work, more heat is lost. The thermodynamic efficiency of this second step is even lower, approximately 45%. Combined the overall efficiency of mammals is

Given the generalized metabolic sensitivity to thyroid hormone documented during hypo- and hyperthyroidism one would anticipate a major physiological role of this hormone in energy homeostasis. However, as mentioned before, serum T3 concentration is remarkably constant, thus precluding a major role in the BMR variations observed after a meal or during sleep. In the last 20 years new light was shed into this problem by studies demonstrating that in some tissues the cellular actions of thyroid hormone are not only determined by serum T3.

Thyroid hormone action is initiated through binding to nuclear receptors (TR), which are high affinity nuclear T3 binding proteins that regulate transcription of T3-dependent genes. TR occupancy is determined by their affinity and the T3 concentration in the nucleus. These values are such that, at normal serum T3 concentration, the contribution from serum T3 alone results in approximately 50% TR saturation in most tissues. However, in tissues expressing D2 there is an additional source of T3 contributed by intracellular T4 to T3 conversion (25; 26). As a result, TR saturation can reach as much as 100%, of which more than half of this T3 is locally produced (27-29).

While we still do not understand all the intricacies of this system, it is known that T3 generation by D2 occurs in the perinuclear region, a cellular compartment with preferential access to the nucleus. This is in contrast to D1, which is localized to the plasma membrane, from which the T3 produced more readily enters the plasma (5). Thus, for cells lacking D2, intracellular thyroid status is determined predominantly by the serum T3 concentration. In contrast, cells expressing D2 have the ability to generate intracellular T3 from T4. This D2-generated T3, referred to as T3(T4), can also bind to nuclear receptors. Thus, cells expressing D2 have two potential sources of nuclear T3: plasma T3, or T3(T3), and T3(T4). On the other hand, it has been recently recognized that D3 is localized to the plasma membrane and undergoes recycling in the endosomes. Remarkably, D3 expression causes cell hypothyroidism as a result of its

inactivating effect on thyroid hormone (30), thus creating a virtual barrier that prevents access of thyroid hormone to the cell nucleus.

Thus, despite steady serum T3 levels, intracellular thyroid status varies along a wide range according to the type and level of deiodinase expression. TR saturation is expected to be minimum in cells expressing D3 and maximum in cells expressing D2. In addition, because of the plasticity of deiodinase expression, particularly D2, TR saturation of a single cell type might change rapidly and dramatically without affecting serum T3. Thus, deiodinase expression in metabolically active tissues is a potent mechanism by which energy dissipation can be controlled. Top

Most of the time endothermic animals function at higher rates than the BMR. This is because any activity that disrupts the resting state promotes ATP breakdown. In mammals, the metabolic rate can be increased by

many factors, e.g. voluntary or involuntary physical activity, transference to a cold environment, hypercaloric feeding, and during fever. The heat derived from an increase in the metabolic rate is known as adaptive thermogenesis. As opposed to obligatory thermogenesis, adaptive thermogenesis may fluctuate rapidly in response to triggering signals, including thyroid hormone. This provides a tremendous evolutionary advantage that has allowed endothermic animals to live in and dominate virtually all planet environments ((31) for review).

Cold exposure prompts the hypothalamus to initiate shivering, the most important involuntary mechanism of cold-induced adaptive thermogenesis in adult humans and in large mammals. However, shivering inevitably causes convective heat loss due to body oscillations and is therefore a less economical form of heat production, particularly in smaller organisms with a high surface to mass ratio. Thus, nonshivering adaptive thermogenesis is the most important heat source in human newborns and other small mammals. This is also initiated at the hypothalamus through the activation of the sympathetic nervous system (SNS), increasing the release of catecholamines throughout the body, particularly in the BAT, the key organ in adaptive thermogenesis. BAT is intensely innervated by the SNS and its thermogenic capacity is largely due to uncoupling protein-1 (UCP1), a mitochondrial protein that short-circuits the proton gradient across the inner mitochondrial membrane, bypassing the less abundant ATP synthase and thereby uncoupling fuel oxidation from the phosphorylation of ADP (32-34).

The mediators of the SNS in the BAT involve  and 1-3 receptors, which act in a synergistic fashion (35-38). The resulting NE-induced increase in cAMP in brown adipocytes rapidly activates lipolysis, initiating mitochondrial heat production, and increases up to ~50 fold intracellular T4 to T3 conversion via D2 (35) - the human, rat and mouse Dio2 genes contain a highly functional canonical CREB-binding site in the promoter (39). This transcriptional mechanism is potentiated by the prolongation of D2 half-life. In addition, the adrenergic induction of a D2-binding protein, the pVHL-interacting deubiquitinating enzyme-1 (VDU1), catalyzes D2 deubiquitination and rescues it from proteasome degradation (9). This increases BAT T3 concentration 4-5-fold within a few hours after cold exposure is initiated (40), creating an isolated tissue thyrotoxicosis (7). As a result, the nucleus of brown adipocytes and other D2-containing cells has a higher T3 concentration and a higher saturation of the TRs, one that would cause thyrotoxicosis in most other tissues (41). Using the dual-labeling technique it was found that in brown adipocytes the level of TR saturation fluctuates primarily according to D2 activity and not plasma T3, the former being a major determinant of adaptive thermogenesis and survival in the cold (7; 29; 42). As a result, the physiological changes that take place during cold and/or NE stimulation of BAT reflect a composite interaction between NE- and T3-generated signals that are linked in a feed-forward mechanism that leads to sustained heat liberation. Blockade of D2-catalyzed T4 to T3 conversion by iopanoic acid blocks the thermogenic response in T4-treated hypothyroid rats, indicating the essential role of this enzyme (11).

Brown adipocytes constitute a unique example of an intricate interaction between the thyroid and the SNS. Interscapular BAT of hypothyroid rats does not respond thermogenically to NE infusion whereas in intact rats, BAT temperature rapidly increases up to ~3 C (43). This is in part explained by mechanisms operating at the UCP1 gene, which is under tight control by NE and thyroid hormones. This has been extensively studied in vivo (11; 42-47), in freshly dispersed (48) or cultured brown adipocytes (49). Cold exposure induces a rapid increase in UCP1 gene expression by transcriptional and post-transcriptional mechanisms (45; 46). As a result, UCP1 mRNA levels are up 3-4 fold after just 4 h and mitochondrial UCP1 content increase 2-3 fold within 4-5 days of cold exposure. Both in vivo and in vitro studies indicate a strong synergism between T3- and NE-generated signals to stimulate UCP1 gene transcription, culminating in approximately 8 fold induction in just a few minutes (48). The molecular basis of this synergism relies on two functional TREs and a CRE in the UCP1 gene promoter (50). However, after a few hours of cold exposure, UCP1 gene transcription returns to baseline values and the high UCP1 mRNA levels during prolonged cold exposure are sustained by a 4 fold increase in its half-life, a phenomenon that is also thyroid hormone dependent (46; 48). T3 plays an important role in sustaining a higher UCP1 concentration during this post-acute phase of cold exposure and this T3 effect can be detected even under conditions of minimal sympathetic activity (47).

The normal response of UCP1 to cold exposure is blunted in hypothyroid rats (11; 44; 51) and requires complete saturation of BAT TR (44). This is evident from plots of BAT mitochondrial UCP1 levels against TR occupancy during acute thyroid hormone treatment of cold-exposed hypothyroid rats. From the hypothyroid levels of TR occupancy up to ~70% TR occupancy the response of UCP1 to cold exposure reaches only 1/5 of that observed in euthyroid rats. As TR saturation increases further, however, the UCP1 response is augmented up to the levels seen in cold-exposed euthyroid rats (44). As with total body O2 consumption, normalizing the UCP1 response with exogenous T3 requires doses that cause systemic hyperthyroidism (42). In contrast, the same result occurs with only replacement doses of T4. This implies an important role for T4 per se in the response to cold, which is explained by the D2 expressed in BAT. D2-catalyzed T4 5’ deiodination generates the additional T3 required for adaptive thermogenesis in BAT (35). Since it is impossible to generate an acute increase in circulating thyroid hormones, the brown adipocytes provide the extra T3 required locally, a process that can be termed adaptive T3 production.

An additional role played by D2 and thyroid hormones in BAT is to mediate the 3-4 fold increase in the activity of lipogenic enzymes, i.e. malic enzyme (ME) and glucose 6-phosphate dehydrogenase (G-6PD), observed in this tissue during cold exposure, a response that is also blunted in hypothyroid rats (44; 52). T3, in turn, stimulates these enzymes, including the expression of Spot-14, a lipogenesis related protein, both in vivo (44; 52) and in differentiating brown adipocytes (53; 54). During cold exposure, BAT lipogenesis is a very active pathway, accounting for >50% of the de novo fatty acid synthesis in the rat (55). BAT lipogenesis is particularly important because it generates the necessary fuel to sustain the high oxidation rate of BAT mitochondria. In freshly isolated brown adipocytes, NE stimulates lipogenesis (incorporation of tritiated water into lipids) and the activity of key lipogenic enzymes, e.g. malic enzyme and acetyl-CoA carboxylase only in the presence of T4 or TR saturating concentrations of T3. In their absence, NE markedly inhibits BAT lipogenesis. D2 blockade with iopanoic acid prevents the NE-mediated surge in lipogenesis in the presence of T4, indicating its essential role in this process (56). Top

The lack of BAT thermal response to NE infusion in hypothyroid animals was first attributed to the decreased UCP1 levels in these animals (11; 44-46; 48). However, in subsequent studies it was found that BAT thermogenesis is restored in T3-treated hypothyroid rats well before UCP1 levels are normalized, indicating that the UCP1 gene is not the limiting locus where adrenergic and thyroid signals interact (12; 43; 57).

Alternatively, it has been proposed that defects in the NE-signaling pathway itself could be involved in limiting BAT adaptive thermogenesis as hypothyroid rat brown adipocytes have decreased NE responsiveness and generate 5-6-fold less cAMP in response to adrenergic agents (58-60). Treatment with T3 in vivo restores the adrenergic transduction and BAT thermogenesis in 72h but the identity of the T3-dependent gene(s) involved remains unknown (57). Interestingly, treatment of hypothyroid mice with the GC-1 compound, a TR-selective agonist, normalizes mitochondrial UCP1 but fails to normalize BAT thermogenesis and cAMP production in response to adrenergic stimulation in vitro, confirming both the poor correlation between UCP1 levels and BAT thermogenesis and the critical role played by the adrenergic transduction mechanisms. A byproduct of these studies was the first demonstration that the two TR isoforms TR and TR are necessary for the full normalization of BAT thermogenesis. Stimulation of UCP1 is mediated by TR, whereas TR potentiates the adrenergic responsiveness (57).

A closer look into the hypothyroid animal model, however, reveals a significant limitation of this model arising from its inherent increased activity of the SNS. NE turnover in most tissues is accelerated by hypothyroidism presumably to compensate for the generalized decrease in NE-responsiveness (61). The increase in NE release causes adrenergic desensitization, ultimately decreasing the responsiveness to NE (62; 63). This phenomenon mitigates the suitability of the hypothyroid animal as model to investigate thyroid-adrenergic synergism. It is not possible to differentiate clearly between the primary effects of hypothyroidism and desensitization-related events. Even if the studies in hypothyroid animals were performed at thermoneutrality, in order to minimize differences in NE turnover, the results would not be relevant because thermoneutrality is not the physiological temperature at which animals are normally acclimated; the BAT is technically turned off so that the results would reflect thyroid-adrenergic synergism when most of the adrenergic component is absent. Top

The mouse with targeted disruption of the D2 gene (Dio2-/-) constitutes an improved system to study thyroid-adrenergic interactions (12; 64). These animals are systemically euthyroid, as their serum T3 is normal and serum T4 is only slightly elevated. Thus, they do not develop homeostatic adaptations at room temperature. However, the absence of D2 impairs BAT thermogenesis by precluding the adaptive increase in T4 to T3 conversion (12).

Non-shivering thermogenesis in BAT is impaired in the Dio2-/- animals, as documented in vivo and in in vitro. When exposed to cold, the Dio2-/- mouse develops hypothermia and survives by increased shivering thermogenesis. Remarkably, the administration of a single TR-saturating T3 dose 24 h before cold exposure completely restores brown adipocyte function to normal, demonstrating that the critical role of D2 is to catalyze the production of TR-saturating amounts of T3 in these cells (12). Near-infrared fluorescence imaging confirmed that Dio2-/- BAT presents a significant deficiency during cold-induced activation (65). Thus, contrary to the hypothyroid animal model, the Dio2-/- mouse lends itself to the mechanistic understanding of why D2 is critical for BAT thermogenesis.

The analysis of gene expression using microarray technology indicates that the expression of many genes involved in the control of energy expenditure, but not UCP1, is altered in the Dio2-/- brown adipocytes. This is critically relevant because the thermogenic mechanisms promoted by D2-generated T3 are thus largely UCP1-independent and likely to operate in other tissues that have a high capacity to transform chemical energy into heat and express D2 such as the skeletal muscle, the main site of adaptive thermogenesis in humans.

The non-stimulated Dio2-/- BAT has normal amounts of mitochondria and normal uncoupling protein-1 (UCP1) concentration (12). Contrary to the hypothyroid animal model, the NE turnover in the Dio2-/- BAT is not increased at room temperature and thus there is no adrenergic desensitization, making the Dio2-/- mouse an ideal system to study thyroid-adrenergic synergism. Nevertheless, isolated Dio2-/- brown adipocytes displayed impaired cAMP generation, lipolysis and induction of UCP1 mRNA during incubation with a wide range of NE, CL and forskolin concentrations, indicating that the absence of D2 makes the Dio2-/- brown adipocytes relatively hypothyroid and insensitive to catecholamine stimulation.

In order to bypass the relative adrenergic insensitivity, the Dio2-/- mouse develops a sustained compensatory ~9-fold increase in BAT SNS stimulation during cold exposure that normalizes the expression of cAMP-dependent genes and metabolic pathways (66). However, this compensatory mechanism comes with a price tag attached: suppression of the otherwise normal lipogenic surge observed during cold exposure. Thus, this creates a state of metabolic imbalance, rapidly depleting the brown adipocytes of their source of fatty acids and impairing adaptive thermogenesis (66). Top

The potential role of D2 in human energy homeostasis has been ignored because human newborns grow less dependent on BAT thermogenesis and, unlike small mammals, adult humans do not have substantial amounts of BAT (67). However, with the cloning of the human D2 cDNA and the finding of cAMP-inducible D2 mRNA and activity in human skeletal muscle (20; 21), its role in controlling human adaptive thermogenesis has been revisited. Thyroid hormone per se is known to increase energy expenditure in skeletal muscle (for review (68)) and could also regulate local energy homeostasis through its interaction with the SNS. Accordingly, human skeletal muscle is under the influence of the thyroid-adrenergic synergism and an increase in local cAMP production is known to activate glycolytic enzymes, sarcolemmal Na+/K+ pumps, phospholamban and voltage-sensitive and sarcolemmal Ca++ channels (69; 70), resulting in increased glucose uptake and utilization (71; 72). The expression of GLUT4, the insulin responsive-glucose transporter that mediates the rate-limiting step of glucose metabolism in skeletal muscle, is also up-regulated by thyroid hormone (73).

Various studies support a previously unrecognized role of D2 in determining the thyroid status and metabolic rate of the skeletal muscle, analogous to its role in BAT. Earlier experimental studies on humans (74) have consistently found diet-induced changes in serum thyroid hormones that could be explained by changes in D2 activity. As an example, the increase in BMR observed in subjects fed a high carbohydrate diet is typically associated with an increase in serum T3/T4 ratio (74), a condition that is also observed in adult subjects chronically treated with terbutaline, a -adrenergic receptor (-AR) stimulator (22). This indicates the existence of a relevant cAMP-dependent T4-to-T3 conversion pathway in humans that plays a role in energy homeostasis. That this pathway is predominantly through D2 is supported by the fact that its gene is up-regulated several-fold by adrenergic stimulators and cAMP (39). Studies performed with patients receiving l-thyroxine replacement at varying dosages have shown that the BMR correlated directly with free T4 and inversely with serum TSH but, interestingly, not with serum T3 (23). All together, these data indicate that D2-produced intracellular T3 in skeletal muscle might be a significant physiological determinant of energy expenditure in humans.

Studies describing a Dio2 polymorphism in which a threonine (Thr) change to alanine (Ala) at codon 92 (D2 Thr92Ala) are of additional support to a D2 role on energy homeostasis. Of note, in humans, skeletal muscle is the primary site of insulin-dependent glucose disposal (75). Remarkably, this Dio2 polymorphism was associated with a ~20% lower glucose disposal rate in obese women (24). In addition, the frequency of the variant allele was also found to be increased in some ethnic groups, such as Pima Indians and Mexican-Americans, with a higher prevalence of insulin resistance (24).

The possible role of the D2 Thr92Ala polymorphism on insulin resistance was also investigated in patients with Type 2 Diabetes Mellitus (DM2). These patients offer a practical approach to investigate energy expenditure because they require intense metabolic monitoring and are subjected to detailed scrutiny of fuel utilization. In accordance to the previous study in obese individuals, homozygous for the variant allele have increased insulin resistance index as assessed by the HOMA (homeostasis model assessment) index. The increased insulin resistance observed in the DM2 patients homozygous for the Ala allele could be explained by a decrease in D2 activity, as have been found in thyroid and skeletal muscle samples from individuals with this genotype (13). A lower D2 activity would decrease D2-generated T3 in skeletal muscle and create a state of relative intracellular hypothyroidism, decreasing the expression of genes involved in energy utilization, such as GLUT4, leading to insulin resistance.

Supporting this hypothesis, it is remarkable that the UCP1 knock out mouse develops, as a compensatory mechanism, increased D2 activity in white adipose tissue (76), stressing the importance in understanding the D2-generated T3-dependent thermogenic mechanisms. Top

Surprisingly little is known about the cellular and molecular mechanisms mediating the increase in energy expenditure by thyroid hormone. Except for the induction of UCP1, which takes place exclusively in BAT (11), the identity of the set of T3-responsive genes that controls energy expenditure is largely unknown. Regardless of their identity, in skeletal muscle these genes probably encode proteins that (i) decrease the efficiency of ATP synthesis and/or (ii) increase the turnover rate of biochemical pathways that involve ATP breakdown.

Thyrotoxicosis causes mitochondrial uncoupling. In hepatocytes of thyrotoxic rats approximately 50% of the increase in cellular oxygen consumption was accounted for by an increased rate of mitochondrial H+ leak (77). Experiments using JC-1, a mitochondrial membrane potential (m) probe, revealed that hepatocytes from rats with different thyroid status present a decrease in the membrane potential and respiration increase (78). In thyrotoxic human skeletal muscle there is a ~70% increase in the Krebs cycle flux and no increase in ATP synthesis (79). It is not clear, however, if this T3-induced mitochondrial uncoupling is mediated by increased expression of UCPs. Thyrotoxic rats have increased UCP3 mRNA levels in skeletal muscle (80). However, mice with targeted disruption of the UCP3 gene have normal T3-induced metabolic rate (81). In addition, the T3-mediated increase in UCP3 mRNA levels in skeletal muscle could be indirect, resulting from T3-induced lipolysis. Fatty acids are potent stimulators of the UCP3 gene as its mRNA is increased several times in skeletal muscle of fasting animals (82; 83).

It is notable that the turnover of some groups of cyclic reactions that expend relatively large amounts of ATP (involved in the maintenance of ionic and substrate homeostasis) is largely inducible by thyroid hormone, e.g. Na+/K+ transporters in the plasma membrane (84; 85). This is particularly relevant in skeletal muscle, a large tissue in which a small change in the rate of energy expenditure can impact substantially on the total body thermogenesis. In this tissue, a major thyroid hormone-dependent pathway is the calcium (Ca2+) cycle between cytosol and sarcoplasmic reticulum, involved in the contraction and relaxation mechanisms. This cycle consumes a large amount of ATP and is also influenced positively by thyroid hormone (68; 86). Besides regulating the expression of genes coding for different isoforms of myosin heavy chain (MHC) (87), favoring the expression of isoforms with higher catalytic (ATPase) activity (88), T3 stimulates the expression of the sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA) gene, (89-91). As a result, in the thyrotoxic muscle there is an increased number of SERCA units in the sarcoplasmic reticulum, which increases ATP expenditure even under resting conditions. In addition, at every contraction/relaxation cycle the amount of Ca2+ mobilized is larger and, consequently, is the ATP expenditure. More recently, it has demonstrated that thyroid hormone increases the Ca2+-ATPase uncoupled activity, i.e. the activity from ATP hydrolysis that does not results in the Ca2+ accumulation inside of the sarcoplasmic reticulum (92; 93). Consequently, more chemical energy is dissipated into heat. This uncoupled Ca2+-ATPase activity has been shown to be higher for SERCA 1 than for SERCA 2 isoform. Thus, it is notable that thyroid hormone upregulates SERCA 1 gene expression in skeletal muscle (94).

In addition to the Ca2+ cycles, there are a number of similar cycles involving other ions, metabolic intermediates and energy substrates (substrate cycles), that are influenced by thyroid hormone, e.g. fructose 6-phosphate/fructose 1,6-bisphosphate, Cori cycle, lipolysis/lipogenesis (95), glycogenolysis / glycogenesis, proteolysis/protein synthesis, bone formation and resorption (96), and others. It is important to stress that the stimulation of the turnover of these cycles changes very little the sizes of the pools of substrates involved, depending on the intensity and duration of the stimulus. However, all increase ATP expenditure, the metabolic rate and, therefore, heat production. These data, in summary, suggest that intracellular T3 concentration is an important determinant of coupling between energy supply and demand.

Finally, it would not be surprising if there were no single mechanism responsible for the T3-induced increase in ATP turnover, BMR and heat production. The final thermogenic effects of T3 would represent the sum total of small effects spread throughout the metabolic pathways that increase synthesis and hydrolysis of ATP. The complexity of the T3 action has been proposed as a good model for the Top-down elasticity analysis of energy metabolism (97) as a tentative to identify different sites of action of effectors of the energy turnover. Top

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