Download Adaptive Activation of Thyroid Hormone and Energy Expenditure

Bioscience Reports, Vol. 25, Nos. 3/4, June/August 2005 ( 2005)
DOI: 10.1007/s10540-005-2885-6
Adaptive Activation of Thyroid Hormone and Energy
Expenditure
Antonio C. Bianco,1,2 Ana Luiza Maia,1 Wagner Seixas da Silva,1 and Marcelo
A. Christoffolete1
The mechanisms by which thyroid hormone accelerates energy expenditure are poorly
understood. In the brown adipose tissue (BAT), activation of thyroid hormone by type 2
iodothyronine deiodinase (D2) has been known to play a role in adaptive energy expenditure
during cold exposure in human newborns and other small mammals. Although BAT is not
present in significant amounts in normal adult humans, recent studies have found substantial amounts of D2 in skeletal muscle, a metabolically relevant tissue in humans. This
article reviews current biological knowledge about D2 and adaptive T3 production and their
roles in energy expenditure.
KEY WORDS: Deiodinase; brown adipose tissue; skeletal muscle; uncoupling protien.
OVERVIEW
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 [1]. In patients with severe hypothyroidism,
total body energy expenditure can fall as much as ~50%, in thyrotoxic patients, it
can be increased by ~50%, totaling an approximately threefold induction over
hypothyroid baseline. However, given that serum levels of thyroid hormone are
remarkably stable under normal conditions, 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
prohormone and 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 [2]. Because D2-generated T3 has a longer residence time
than D2 within the cell [3], an advantage of this pathway is the possibility of
1
Thyroid Section, Division of Endocrinology, Diabetes, and Hypertension, Department of Medicine
Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, HIM Bldg. #643,
Massachusetts, Boston, MA, 02115, USA.
2
To whom correspondence should be addressed. E-mail: [email protected]
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0144-8463/05/0800-0191/0 2005 Springer Science+Business Media, Inc.
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controlling intracellular T3 concentration and thus T3-receptor (TR) saturation
independently of serum T3. This allows for rapid, tissue-specific D2-mediated
changes in intracellular thyroid status 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 not only for the onset of auditory function
but also for the subsequent maturation of auditory sensitivity, without which severe
hearing impairment develops [2].
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), the 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 [2]. In addition, new evidence
indicates that adaptive T3 production also plays a role in controlling energy
expenditure in a number of mouse models of resistance to diet-induced obesity [4, 5].
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 adult
humans, unlike rodents, express D2 significantly in skeletal muscle [6, 7]. This has
been implicated as a determinant of energy expenditure in adult humans [8–10],
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 the basal metabolic rate
(BMR). In animals the BMR is the energy expenditure necessary to sustain minimal
homeostatic functions as measured at rest, in a 12-hour-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, and
peristalsis; and (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:
ATP synthesis and ATP hydrolysis (Fig. 1). The thermodynamic efficiency of the
ATP synthesis is approximately 65%; i.e., approximately 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
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Fig. 1. Summary diagram of the energy-demanding pathways and heat release during ATP turnover. In this conceptualization, ATP hydrolysis leads to heat production. The ATP hydrolysis
regenerates ADP and Pi, which serve as substrates for oxidative phosphorylation, to activate ATP
production in the mitochondria.
molecule is hydrolyzed 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 approximately 25–30%.
DEIODINASES ALLOW FOR TISSUE-SPECIFIC CONTROL OF THYROID
HORMONE ACTION
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 above, serum T3
concentration is remarkably constant, thus precluding a major role of T3 in the
BMR variations observed after a meal or during sleep. In the last 20 years, light was
shed on this problem by studies demonstrating that in some tissues the cellular
actions of thyroid hormone are determined not only by serum T3.
Thyroid hormone action is initiated through its 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 the affinity of the receptor
for T3 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 an
approximately 50% saturation of TR in most tissues. However, tissues expressing
D2 have an additional source of T3 contributed by the conversion of intracellular
thyroxine (T4) to T3 [11, 12]. As a result, TR saturation can reach as high as 100%,
with more than half of this T3 produced locally [13–15].
While we still do not understand all the intricacies of this system, we do know
that generation of T3 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
[16]. 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
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hand, D3 has recently been found to be localized to the plasma membrane and to
undergo recycling in the endosomes. Remarkably, D3 expression causes cell hypothyroidism as a result of its inactivating effect on thyroid hormone [17], thus creating
a virtual barrier that prevents thyroid hormone from entering 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
(Fig. 2). In addition, because of the plasticity of deiodinase expression, particularly
that of D2, TR saturation of a single cell type might change rapidly and dramatically
Fig. 2. Subcellular localization of the type 1, 2 ,and 3 selenodeiodinases and consequences on
intracellular thyroid status. Confocal microscopy of cells treated with acetone-HEK-293 transiently
expressing Dl-CF (panel a), D2-CF (panel b) and D3C-FLAG (panel c). After fixation and acetone
treatment, cells were incubated with mouse anti-FLAG and anti-mouse-FITC (green). Panels a and b
show a superimposition immunofluorescence images of the same fields costained with goat antiGRP78/BiP (endoplasmic reticulum specific marker) and anti-goat-rhodamine (red). Panel c shows a
superimposition immunofluorescence image of the same fields costained with rabbit anti-FLAG and
mouse anti- Na+/K+ -ATPase a, a typical plasma membrane marker; secondary antibodies were
anti-rabbit IgG FITC and anti-mouse IgG Texas Red-X. Modified from [16, 90] .
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without affecting serum T3. Thus, deiodinase expression in metabolically active
tissues is a potent mechanism by which energy dissipation can be controlled.
MORE HEAT––ADAPTIVE THERMOGENESIS
Most of the time endothermic animals function at higher rates than the BMR
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 by fever. The heat derived from an increase in the metabolic rate is
known as adaptive thermogenesis, which, as opposed to obligatory 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 [18 for review].
Exposure to cold prompts the hypothalamus to initiate a shivering response, the
most important involuntary mechanism of cold-induced adaptive thermogenesis in
adult humans and large mammals. However, shivering inevitably causes convective
heat loss due to body oscillations and is therefore a less economical form of heat
production than mitochondrial uncoupling, particularly in smaller organisms with a
high surface to mass ratio. Thus, adaptive thermogenesis that does not involve
shivering is the most important heat source in human newborns and other small
mammals. This response 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 shortcircuits the proton gradient across the inner mitochondrial membrane, bypassing the
less abundant ATP synthase and thereby uncoupling fuel oxidation from the
phosphorylation of ADP [19–21] (Fig. 3).
The mediators of the SNS in the BAT involve a and b1)3 receptors, which act
synergistically [22–25]. The resulting norepinephrine (NE)-induced increase in cAMP
in brown adipocytes rapidly activates lipolysis, initiating mitochondrial heat production, and increases intracellular conversion of T4 to T3 via D2 by up to ~50-fold
[22]––the human, rat and mouse Dio2 genes contain a highly functional canonical
CREB-binding site in the promoter [26]. This transcriptional mechanism is potentiated by the prolongation of the half-life of D2. 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
[27]. This increases the T3 concentration in BAT four- to five-fold within a few hours
after cold exposure is initiated [28], creating an isolated thyrotoxicosis in the tissue
[29]. 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 [30]. Using the dual-labeling technique, various
investigators have found the level of TR saturation in brown adipocytes fluctuates
primarily according to D2 activity and to plasma T3, the former being a major
determinant of adaptive thermogenesis and survival in the cold [15, 29, 31]. As a
result, the physiological changes that take place during cold and/or NE stimulation
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Fig. 3. Mechanisms of D2-T3 generation in brown adipocytes. Brown adipocytes are stimulated by
norepinephrine (NE) released from sympathetic terminals within the BAT. NE interacts with its receptors
(AR) and activates adenylate cyclase (AC) through stimulatory G-protein, increasing intracellular cAMP
levels. This activates triglyceride (TG) breakdown (lipolysis) and the expression of a series of cAMPdependent genes, namely UCP1, D2, and genes encoding proteins involved in the adrenergic signaling
pathway. Intracellular T3 concentration increases as a result of D2 activation, and there is strong
synergism between T3-dependent actions with cAMP-dependent actions towards an overall increase in
energy expenditure and thermogenesis. Modified from [48].
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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
(Fig. 3). Blockade of D2-catalyzed conversion of T4 to T3 by iopanoic acid blocks
the thermogenic response in T4-treated hypothyroid rats, indicating the essential role
of this enzyme [32].
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 BAT temperature in intact rats
rapidly increases up to ~3 C [33]. This may be explained in part by mechanisms
operating at the UCP1 gene, which is under tight control by NE and thyroid hormones. This has been studied extensively in vivo [31–37] and in freshly dispersed [38]
and cultured brown adipocytes [39]. Cold exposure induces a rapid increase in UCP1
gene expression by transcriptional and post-transcriptional mechanisms [35, 36]. As
a result, levels of UCP1 mRNA are increased three- to four-fold after just 4 h, and
the UCP1 content of mitochondria increase two- to three-fold within four to five
days of cold exposure. Both in vivo and in vitro studies indicate a strong synergism
between T3- and NE-generated signals in stimulating UCP1 gene transcription,
culminating in an approximately eight-fold induction in just a few minutes [38]. The
molecular basis of this synergism relies on two functional TREs and a CRE in the
UCP1 gene promoter [40]. However, after a few hours of cold exposure, UCP1 gene
transcription returns to baseline values and the high levels of UCP1 mRNA during
prolonged cold exposure are sustained by a four-fold increase in its half-life, a
phenomenon that is also thyroid hormone–dependent [36, 38]. 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 [37].
The normal response of UCP1 to cold exposure is blunted in hypothyroid rats
[32, 34, 41] and requires complete saturation of BAT TR [34], as evident from plots
of UCP1 levels in BAT mitochondria against TR occupancy during acute treatment
of cold-exposed hypothyroid rats with thyroid hormone. From the levels of TR
occupancy in hypothyroid animals up to ~70% TR saturation, the response of UCP1
to cold exposure reaches only one-fifth 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 [34]. As with total body O2 consumption,
normalizing the UCP1 response with exogenous T3 requires doses that cause systemic hyperthyroidism [31]. 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 expression of D2 in BAT. D2-catalyzed T4 5¢ deiodination
generates the additional T3 required for adaptive thermogenesis in BAT [22]. Since it
is impossible to generate an acute increase in circulating levels of thyroid hormones,
the brown adipocytes provide the extra T3 required locally, a process we can term
adaptive T3 production.
An additional role played by D2 and thyroid hormones in BAT is to mediate the
three-to fourfold 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 [34, 42]. T3,
in turn, stimulates synthesis of these enzymes, including the expression of Spot-14, a
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lipogenesis-related protein, both in vivo [34, 42] and in differentiating brown
adipocytes [43, 44]. During cold exposure, lipogenesis by BAT is a very active
pathway, accounting for >50% of the de novo fatty acid synthesis in the rat [45].
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 (determined by 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 the absence of D2 activity, 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 [46].
THYROID HORMONE IS ESSENTIAL FOR BAT ADAPTIVE
THERMOGENESIS, BUT WHY?
The lack of a thermal response of BAT to NE infusion in hypothyroid animals was
first attributed to the decreased UCP1 levels in these animals [32, 34, 38]. However,
subsequent studies 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 [33, 47, 48].
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 five- to six-fold
less cAMP in response to adrenergic agents [49–51]. Treatment with T3 in vivo
restores the adrenergic transduction and BAT thermogenesis in 72 h, but the identity
of the T3-dependent gene(s) involved remains unknown [47]. Interestingly, treatment
of hypothyroid mice with the GC-1 compound, a TRb-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 by-product of these studies was the
first demonstration that the two TR isoforms, TRb and TRa, are necessary for the
full normalization of BAT thermogenesis, Stimulation of UCP1 is mediated by TRb,
whereas adrenergic responsiveness is potentiated by TRa [47].
A closer look into the hypothyroid animal model, however, reveals a significant
limitation of this model that arises 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 responsiveness to NE [52]. The increase in the release of
NE causes adrenergic desensitization, ultimately decreasing the responsiveness to NE
[53, 54]. This phenomenon mitigates the suitability of the hypothyroid animal as a
model for investigating 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
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 thyroidadrenergic synergism when most of the adrenergic component is absent.
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A MOUSE WITH TARGETED DISRUPTION OF THE D2 GENE (DIO2)
The recently created mouse with a targeted disruption of the D2 gene (Dio2)/))
constitutes an improved system for studying thyroid-adrenergic interactions [48, 55].
These animals are systemically euthyroid, as their serum T3 level is normal and their
serum T4 level 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 [48].
Non-shivering thermogenesis in BAT is impaired in the Dio2)/) animals, as
documented in vivo and in vitro (Fig. 4). When exposed to cold, the Dio2)/) mouse
develops hypothermia and survives by increasing shivering thermogenesis.
Remarkably, the administration of a single TR-saturating dose of T3 24 h before
cold exposure completely restores brown adipocyte function to normality, demonstrating that the critical role of D2 is to catalyze the production of TR-saturating amounts of T3 in these cells [48]. More recently, near-infrared fluorescence
imaging was used to confirm that Dio2)/) BAT presents a significant deficiency
during cold-induced activation [56]. Thus, contrary to the hypothyroid animal
Fig. 4. Adaptive BAT thermogenesis in Dio2)/) (KO) and wild-type (WT) mice: (a) core temperature during cold exposure; (b) temperature changes in intracellular BAT (IBAT) in response to
NE infusion; (c) O2 consumption in isolated brown adipocytes. All data are expressed as
mean ± SD, n=4–12, *p<0.05 and obtained from (48); (d) near-infrared (NIR) fluorescence
imaging of wild-type mice in response to adaptive thermogenesis. Mice were acclimated to 26C and
treated with saline (c), CL 316,243 – (1 mg/kg) i.p. (CL+) or norepinephrine – ( 0.3 mg/kg) i.p
(NE); (e) same as in d, except that wild-type and knock-out mice were exposed to 4C for 24 h and
the NIR signal was quantified. The ordinate shows the BAT-to-background ratio for each condition, n=3; data are mean ± SEM. Modified from [56].
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model, the Dio2)/) mouse model 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 the
UCP1 gene, is altered in the Dio2)/) brown adipocytes (Fig. 5). 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 UCP1 concentration [48]. 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 for studying 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, CL316,243, and forskolin
concentrations, indicating that the absence of D2 makes the Dio2)/) brown adipocytes relatively hypothyroid and insensitive to catecholamine stimulation.
To bypass its relative adrenergic insensitivity, the Dio2)/) mouse develops a
sustained compensatory increase of approximately nine-fold in BAT SNS stimulation during cold exposure that normalizes the expression of cAMP-dependent genes
and metabolic pathways [57]. 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
Fig. 5. Maximum binding capacity (MBC), sources of T3, and nuclear T3 receptor occupancy in
euthyroid rats during cold acclimation to 4C. Plasma-born T3 is indicated in solid bars and locally
produced T3 via D2 is indicated in empty bars. Modified from [15]. Microarray results obtained
from BAT RNA of Dio2)/) animals treated with saline or 5 lg of T3 16 h before norepinephrine
(NE) treatment. NE (30 lg/100g B.W.) was administered over a 6-h period. The expression data
(signal/reference) of NE+T3-treated Dio2)/) animals is shown as plotted against the expression
data of NE treated Dio2)/) animals. Eighty-one genes, indicated as open and closed circles, were
found to be NE-dependent only when the animals had been treated previously with T3. Differences
in gene expression of approximately twofold lie between the two outside lines.
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depleting the brown adipocytes of their source of fatty acids and impairing adaptive
thermogenesis [57].
SIGNIFICANCE OF D2 TO ADAPTIVE THERMOGENESIS IN HUMANS
The potential role of D2 in human energy homeostasis has been ignored because
human newborns grow less dependent on BAT thermogenesis, and adult humans,
unlike small mammals, do not have substantial amounts of BAT [58]. However,
since the cloning of the human D2 cDNA and the finding of cAMP-inducible D2
mRNA and activity in human skeletal muscle [6, 7], the role of D2 in controlling
human adaptive thermogenesis has been revisited. Thyroid hormone per se is known
to increase energy expenditure in skeletal muscle [for review 59] 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 [60, 61], resulting in increased glucose uptake and utilization
[62, 63]. 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 [64].
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 of humans [65] 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 the serum T3/T4 ratio
[65], a condition that is also observed in adult subjects chronically treated with
terbutaline, a b-adrenergic receptor (b-AR) stimulator [8]. 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 finding that the D2 gene is up-regulated severalfold by adrenergic
stimulators and cAMP [26]. More recently, studies of patients receiving T4
replacement at various dosages have shown a direct correlation of the BMR with
free T4 and inversely with serum TSH but not with serum T3 [9]. Together, these
data indicate that D2-produced intracellular T3 in skeletal muscle might be a
significant physiological determinant of energy expenditure in humans.
Recent studies describing a Dio2 polymorphism in which a threonine (Thr)
change to alanine (Ala) at codon 92 (D2 Thr92Ala) provides is additional support to
a role of D2 in glucose uptake and utilization. Of note, in humans, skeletal muscle is
the primary site of insulin-dependent glucose disposal [66]. Remarkably, this Dio2
polymorphism was associated with an ~20% lower rate of glucose disposal in obese
women than in non-obese women [10]. In addition, the frequency of the variant allele
was found to be increased in some ethnic groups, such as Pima Indians and MexicanAmericans, with a higher prevalence of insulin resistance [10].
The possible role of the D2 Thr92Ala polymorphism on insulin resistance was
also investigated in patients with type 2 diabetes Mellitus (DM2). Studies of these
patients offers a practical approach to the investigation of energy expenditure
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because they require intense metabolic monitoring and are subjected to a detailed
scrutiny of fuel utilization. In accordance with the previous study in obese individuals, individuals homozygous for the variant allele have an 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 has been found in thyroid
and skeletal muscle samples from individuals with this genotype [67]. 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 is the remarkable finding that the UCP1 knock-out
mouse develops, as a compensatory mechanism, increased D2 activity in white
adipose tissue [68], stressing the importance of understanding the D2-generated
T3-dependent thermogenic mechanisms.
THYROID HORMONE-DEPENDENT THERMOGENIC MECHANISMS IN
SKELETAL MUSCLE
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 [32], 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 decrease the efficiency of ATP synthesis and/or 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+ leakage [69]. Experiments
using JC-1, a mitochondrial membrane potential (DYm) probe, revealed that
hepatocytes from rats with different thyroid status present a decrease in the membrane
potential and an increase in respiration [70]. In thyrotoxic human skeletal muscle,
there is an ~70% increase in the Krebs cycle flux and no increase in ATP synthesis [71].
It is not clear, however, if this T3-induced mitochondrial uncoupling is mediated by
increased expression of UCPs. Thyrotoxic rats have increased levels of UCP3 mRNA
in skeletal muscle [72]. However, mice with targeted disruption of the UCP3 gene have
a normal T3-induced metabolic rate [73]. In addition, the T3-mediated increase in
levels of UCP3 mRNA in skeletal muscle could be indirect, resulting from T3-induced
lipolysis. Fatty acids are potent stimulators of the UCP3 gene, as its UCP3 mRNA is
increased several times in skeletal muscle of fasting animals [74, 75].
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), e.g., Na+/K+ transporters in the plasma membrane, is largely
inducible by thyroid hormone [76, 77]. 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, which is involved in the contraction and relaxation mech-
Deiodination and Energy Expenditure
203
anisms. This cycle consumes a large amount of ATP and is also influenced positively
by thyroid hormone [59, 78]. Besides regulating the expression of genes coding for
different isoforms of myosin heavy chain (MHC) [79], favoring the expression of
isoforms with higher catalytic (ATPase) activity [80], T3 stimulates the expression of
the sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA) gene [81–83]. As a
result, thyrotoxic muscle has 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, so 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 result in the accumulation of Ca2+ inside
the sarcoplasmic reticulum [84, 85]. 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 [86].
In addition to the Ca2+ cycles, a number of similar cycles involving other ions,
metabolic intermediates, and energy substrates (substrate cycles) are influenced by
thyroid hormone, e.g., fructose 6-phosphate/fructose 1,6-bisphosphate, Cori cycle,
lipolysis/lipogenesis [87], glycogenolysis/glycogenesis, proteolysis/protein synthesis,
bone formation and resorption [88], and others. It is important to stress that the
stimulation of the turnover of these cycles changes the sizes of the pools of substrates
involved very little, depending on the intensity and duration of the stimulus. However,
all increase the expenditure of ATP expenditure, the metabolic rate, and, therefore,
heat production. These data, in summary, suggest that the intracellular concentration
of T3 is an important determinant of coupling between energy supply and demand.
Finally, it would not be surprising if no single mechanism were 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 [89] as a tentative approach to identify different sites of action of
effectors of the energy turnover.
ACKNOWLEDGMENT
NIH grants DK58538 and DK65655. PEW charitable trusts foundation
fellowship to WSS.
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