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Physiol Rev 86: 435– 464, 2006;
doi:10.1152/physrev.00009.2005.
Thermogenic Mechanisms and Their Hormonal Regulation
J. ENRIQUE SILVA
Baystate Medical Education and Research Foundation, Department of Medicine, Division of Endocrinology,
Baystate Medical Center, Tufts University Medical School, Springfield, Massachusetts
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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
II. Hormonal Regulation of Thermogenesis
A. Thyroid hormone acquires a new role with the advent of homeothermy
B. Thyroid hormone and obligatory thermogenesis
C. Thyroid hormone and facultative thermogenesis
III. Other Hormone Roles in Thermogenesis
A. Insulin, glucagon, and epinephrine
B. Glucocorticoids
C. Leptin
IV. Thermogenesis as a Source of Variability in Energy Expenditure
V. Summary and Conclusions
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Silva, J. Enrique. Thermogenic Mechanisms and Their Hormonal Regulation. Physiol Rev 86: 435– 464, 2006;
doi:10.1152/physrev.00009.2005.—Increased heat generation from biological processes is inherent to homeothermy.
Homeothermic species produce more heat from sustaining a more active metabolism as well as from reducing fuel
efficiency. This article reviews the mechanisms used by homeothermic species to generate more heat and their
regulation largely by thyroid hormone (TH) and the sympathetic nervous system (SNS). Thermogenic mechanisms
antecede homeothermy, but in homeothermic species they are activated and regulated. Some of these mechanisms
increase ATP utilization (same amount of heat per ATP), whereas others increase the heat resulting from aerobic
ATP synthesis (more heat per ATP). Among the former, ATP utilization in the maintenance of ionic gradient through
membranes seems quantitatively more important, particularly in birds. Regulated reduction of the proton-motive
force to produce heat, originally believed specific to brown adipose tissue, is indeed an ancient thermogenic
mechanism. A regulated proton leak has been described in the mitochondria of several tissues, but its precise
mechanism remains undefined. This leak is more active in homeothermic species and is regulated by TH, explaining
a significant fraction of its thermogenic effect. Homeothermic species generate additional heat, in a facultative
manner, when obligatory thermogenesis and heat-saving mechanisms become limiting. Facultative thermogenesis is
activated by the SNS but is modulated by TH. The type II iodothyronine deiodinase plays a critical role in modulating
the amount of the active TH, T3, in BAT, thereby modulating the responses to SNS. Other hormones affect
thermogenesis in an indirect or permissive manner, providing fuel and modulating thermogenesis depending on food
availability, but they do not seem to have a primary role in temperature homeostasis. Thermogenesis has a very high
energy cost. Cold adaptation and food availability may have been conflicting selection pressures accounting for the
variability of thermogenesis in humans.
I. THERMOGENESIS AND BODY
TEMPERATURE HOMEOSTASIS
Core body temperature (TC) is among the bestguarded constants in homeothermic species. This has
resulted from the evolution of mechanisms to regulate the
exchange of heat with the environment and mechanisms
to increase the basal generation of heat by the body as
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well as to produce additional heat in response to colder
environments in which basal heat production and heatsaving mechanisms are not sufficient to maintain TC. The
generation of heat or thermogenesis is thus essential to
homeothermy, but it has an energy cost that is not usually
appreciated. Thermogenic mechanisms are probably
older than homeothermy, but in homeothermic species
some are permanently activated, yet regulated both by
0031-9333/06 $18.00 Copyright © 2006 the American Physiological Society
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J. ENRIQUE SILVA
neural and hormonal signals, the intensity of which is
commensurate with the gap in temperature between the
core and the environment and the overall thermal conductance of the body. This review focuses on thermogenesis and its hormonal and sympathetic regulation. In this
regard, thyroid hormone (TH) plays a pivotal role readily
evident in experimental or clinical conditions associated
with impairment or excess thyroidal secretion.
A. Evolutionary Aspects: Homeothermy and the
Need for Additional Heat Production
B. Regulation of Body Temperature: Obligatory
and Facultative Thermogenesis
The heat normally resulting from sustaining vital
functions is called obligatory thermogenesis, and as men-
FIG. 1. Schematic representation of energy
transformations in biology and differences between poikilothermic and homeothermic species. A: energy transfer from substrates to ATP
and from ATP to support vital functions such
as transmembrane gradients, synthetic processes, growth, and physical activity. Heat is
generated mainly in the synthesis of ATP and
less so in the use of this in some activities. Heat
generation can be increased by accelerating the
use of ATP or by reducing the efficiency with
which a cell captures energy of substrates in
ATP. See text for details. B: comparative rates
of oxygen consumption by a poikilothermic
species (lizard) and a homeothermic one
(mouse) of equal weight and studied at the
same temperature. Data have been normalized
to the corresponding value of the mammal.
[Data from Else and Hulbert (78) and Hulbert
and Else (119).] C: thermodynamic efficiency of
skeletal muscle of poikilothermic and homeothermic species. [Data from Woledge (254).]
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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.
HORMONAL REGULATION OF THERMOGENESIS AND BODY TEMPERATURE
Physiol Rev • VOL
C. Thermogenic Mechanisms
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 increase
ATP 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
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tioned 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-tovolume 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.
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Physiol Rev • VOL
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 lowimpedance 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
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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
HORMONAL REGULATION OF THERMOGENESIS AND BODY TEMPERATURE
439
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).
Until recently, uncoupling of respiration from ATP
synthesis was known to occur in a regulated manner only
in a specialized tissue called BAT. Here, a protein abundantly present in the inner mitochondrial membrane,
called uncoupling protein (UCP), provides an alternative,
low-impedance route for the protons back into the mitochondrial matrix, collapsing the gradient, bypassing the
ATP synthase, producing heat, and accelerating respiration (see Refs. 167, 169 for historical reviews and original
references). Although the intimate mechanism whereby
UCP reduces the proton-motive force is still being debated (93, 101, 134), it has been long known that the
activity of UCP, i.e., its proton conductance, is blocked by
nucleotides and strikingly augmented by fatty acids (168,
186). BAT is found only in mammals (see Ref. 51 for
review), and UCP is found only in this tissue (49), which
was confirmed when it was cloned (34, 35). It appeared
that regulated uncoupling of phosphorylation to produce
heat was a late evolutionary acquisition, present only in
one of the two classes of homeothermic species, mammals. However, parallel independent studies demonstrated the presence of a significant leak of protons
through the mitochondrial inner membrane in several
tissues, not restricted to BAT (38), and subsequent work
provided evidence that it was regulated and could serve a
thermogenic function (reviewed in Ref. 195). This proton
leak was, thus, found to be more active in homeothermic
than poikilothermic species (39) and estimated to acPhysiol Rev • VOL
count for 20 –30% of the basal metabolic rate of the rat
(194) and as much as 50% of muscle resting oxygen consumption (193).
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
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FIG. 2. Mitochondrial respiration and protonmotive force. A: respiratory chain complexes are
represented by the purple circles with their Roman numerals embedded in the inner mitochondrial membrane. As electrons travel down the
respiratory chain, complexes I, III, and IV extrude
protons into the intermembrane space creating a
gradient with the mitochondrial matrix. The energy of this gradient is used by the ATP synthase
complex to produce ATP. B: schematic representation of proton gradient, respiration (oxygen
consumption), and ATP synthesis when a substrate (succinate), ADP, and an uncoupler, p-trifluoromethoxycarbonyl cyanide phenylhydrazone
(FCCP), are added to intact mitochondria in vitro.
For details, see Ref. 144.
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J. ENRIQUE SILVA
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.
In the G3P shuttle, the molecule being reduced is dihydroxyacetone phosphate (DHAP), which results from the
cleavage of fructose-1,6-diphosphate into two 3-carbon molecules, DHAP itself and glyceraldehyde-3-phosphate. These
can be interconverted by a triose phosphate isomerase, and
DHAP can thus continue in the glycolytic pathway or serve
as an acceptor of reducing equivalents transferred from
NADH [and NADPH (84)], in a reaction catalyzed by the
cytoplasmic G3P dehydrogenase (EC 1.1.1.8; cGPD). The
resulting G3P is reoxidized to DHAP by the mitochondrial
glycerol-3-phosphate dehydrogenase (EC 1.1.99.5; mGPD).
This enzyme is a flavin adenine dinucleotide (FAD)-linked
dehydrogenase, located on the outer surface of the inner
mitochondrial membrane, that directly transfers the electrons to the complex III of the respiratory chain, producing
only two ATPs per molecule of H2O formed (64, 133).
By catalyzing the interconversion between DHAP
and G3P, the G3P shuttle constitutes a crossroad between
lipid and carbohydrate metabolism as well as the main
FIG. 3. NADH shuttles. Left: malateaspartate NADH shuttle. Glu, glutamate;
␣-KG, ␣-ketoglutarate. Malate and ␣-KG,
on one hand, and aspartate and glutamate,
on the other, utilize transporters to get in
and out the mitochondria. Right: glycerol3-phosphate (G3P) shuttle. cGPD and
mGPD, cytoplasmic and mitochondrial
G3P dehydrogenases, respectively; DHAP,
dihydroxy-acetone-3-phosphate; UQ, ubiquinone; III, complex III of the respiratory
chain. External mitochondrial membrane
is completely permeable to the substances
involved and is therefore not depicted, and
intermembrane space from this point of
view is contiguous with cytoplasm. See
text and Ref. 144 for more details.
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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
HORMONAL REGULATION OF THERMOGENESIS AND BODY TEMPERATURE
generation. Accordingly, the expression of mGPD is particularly high in the tissues that require rapid generation
of ATP such as the flight muscle of insects, sperm cells,
and pancreatic ␤-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,
FIG. 4. NADH shuttles in the context of glycolysis, gluconeogenesis and triglyceride synthesis, and
hydrolysis. The G3P shuttle is represented by the
thick continuous arrows and the malate-aspartate
shuttle (abbreviated) by the thick dotted arrows. See
Fig. 3 for internal details of the shuttles.
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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).
The activity of these two shuttles and their relative
importance are tissue dependent. In liver, the malateaspartate shuttle is very active, whereas the G3P shuttle is
the main one in skeletal muscle (144). Accordingly, a
mouse genetically deficient in cGPD shows a marked
increase in DHAP and a reduction of G3P in muscle but
not in liver (149). We have found increased levels of G3P
and lactate in muscle but not in liver of transgenic mGPD
⫺/⫺ mice (3, 71) and have made several observations that
support the concept of the different role of the shuttle and
mGPD in muscle and liver, which is illustrated in Figure 5
and discussed in the ensuing paragraphs.
The entrance of reducing equivalents into the respiratory chain via mGPD is a rapid pathway for aerobic ATP
441
442
J. ENRIQUE SILVA
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 G3Pstimulated 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-topyruvate ratio was reduced probably due to large influx of
Physiol Rev • VOL
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).
These results are consistent with the idea that the G3P
shuttle contributes to ObT and that TH utilizes this metabolic pathway (among others) to increase ObT. Further
evidence that the G3P shuttle represents a spend-thrift metabolic pathway is supported by observations we made when
mGPD ⫺/⫺ mice were challenged with high fat or caloric
restriction, in the form of either a short fasting or more
prolonged but partial caloric restriction. Given a high-fat diet
(3), mGPD ⫺/⫺ mice increased weight more rapidly than
WT controls, even though the caloric intake was not different from that of WT. When fasted or caloric restricted,
mGPD ⫺/⫺ mice lost less weight than the WT, as one would
predict from eliminating a spend-thrift metabolic pathway.
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FIG. 5. Effect of substrates on in vitro
respiration by tissue slices (unpublished observations). A: effect of 1 mM succinate or 1
mM glycerol-3-phosphate (G3P) on liver
from wild-type genotype (WT) and mGPD
⫺/⫺ mice. B: effect of same substrates on
skeletal muscle (tibialis anterior) of WT and
mGPD ⫺/⫺ mice. C: effect of aminooxyacetate on glucose-supported respiration of
liver and muscle (tibialis and vastus lateralis). D: stimulation of glucose-supported
respiration by succinate or G3P in muscle,
brown adipose tissue (BAT), or liver.
HORMONAL REGULATION OF THERMOGENESIS AND BODY TEMPERATURE
443
TABLE 1 . Most relevant phenotypic characteristics of mice with targeted disruption of the mitochondrial
glycerol-3-phosphate gene (mGPD⫺/⫺) at 3– 6 mo of age
Compared With Wild Type
Comments
Energy balance
Food intake
QO 2
Core temperature
Weight
⫺10%
⫺(5–10%)
⫺(15–18%)
Unchanged
⫺(5–10%)
Room temperature
Room temperature
Acclimated at 32°C
Room temperature
Apparently no change in body composition
Blood concentrations
Serum T3
⫺14%
Unchanged
⫹84%
⫹45%
⫹22%
Unchanged
⫹18%
⫹10
Room temperature. Males and females
Acclimated at 32°C
Room temperature. Males only
Acclimated at 32°C. Males only
Liver
G3P
Lactate
Pyruvate
Lactate/pyruvate
Unchanged
Unchanged
Increased
Decreased
G3P
Lactate
Pyruvate
Lactate/pyruvate
UCP3 mRNA
Increased
Increased
Decreased
Increased
Increased
Skeletal muscle
Males only
Brown adipose tissue
UCP1
Increased
Plus generalized signs of being more stimulated
High-fat diet
Fasting
Starvation
Obesity
Less weight loss and hypothermia
Less weight loss, reduced QO2, and hypothermia
Responses to challenges
Females only
Females only
Females only
QO2, oxygen consumption; FFA, free fatty acids; G3P, glycerol-3-phosphate; UCP, uncoupling protein. [Data from Alfadda et al. (3) and
DosSantos et al. (71).]
Furthermore, the attenuated weight loss in mGPD ⫺/⫺ was
associated with a greater fall in QO2 and TC, consistent with
the idea that thermogenesis was more reduced in these mice
than in the WT controls in response to food restriction,
ultimately indicating that the G3P shuttle contributes significantly to ObT.
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
Physiol Rev • VOL
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
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Glucose
Serum FFA
Serum triglycerides
Serum G3P
Serum T4
444
J. ENRIQUE SILVA
II. HORMONAL REGULATION
OF THERMOGENESIS
A. Thyroid Hormone Acquires a New Role
With the Advent of Homeothermy
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
Physiol Rev • VOL
FIG. 6. Heat production by muscle as a function of tension developed in a short tetanus in muscle from euthyroid and hypothyroid mice.
A: data from extensor digitorum longus (EDL), a fast-twitch muscle. B:
data from soleus, a slow-twitch muscle. In both cases, heat increased
linearly with mechanical work (tension development). For EDL, slopes
were not affected by thyroid status, but the euthyroid curve was significantly higher, with a y-intercept significantly higher. For soleus, both
slopes and y-intercepts were significantly higher in the euthyroid state.
See text for details and discussion. [Lines represent the regression lines
derived from experimental data by Leijendekker et al (145).]
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).
We have demonstrated in humans that resting energy
expenditure (REE), a close measure of ObT, is remarkably sensitive to TH around the euthyroid state (2), underscoring the physiological relevance of this TH action.
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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.
HORMONAL REGULATION OF THERMOGENESIS AND BODY TEMPERATURE
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.
Physiol Rev • VOL
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
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Thus, in athyreotic patients maintained euthyroid on exogenous thyroxine, minimal changes in their daily dose,
which did not move serum free T4 concentration out of
the normal range, were associated with readily detectable
changes in REE. Except for thyroid stimulating hormone
(TSH), none of the indexes of TH action used clinically
were significantly affected by the dose changes, yet there
was a remarkable negative correlation between TSH and
REE (r ⫽ ⫺0.82; P ⬍ 0.001). The T4 dose changes in these
patients moved TSH between 0.05 and 10 mU/l (normal:
0.4 – 4.5 mU/l), which was associated with a 15% excursion
of REE (2). Moreover, we found that spontaneous fluctuations in free T4 concentration in lean normal males are
also associated with significant changes in REE (29).
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).
445
446
J. ENRIQUE SILVA
Physiol Rev • VOL
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).
It was initially hypothesized that TH increased the
proton leak by changing the phospholipid composition of
the mitochondrial membrane, but such hypothesis did not
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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 slowtwitch 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
HORMONAL REGULATION OF THERMOGENESIS AND BODY TEMPERATURE
find support (44). The cloning of UCP2 and UCP3 (88, 98,
243) raised the expectation that they could mediate not
only a regulated dissipation of the proton gradient across
the inner mitochondrial membrane, as discussed earlier,
but also the portion of the thermogenic effect of TH not
accounted for by mechanisms increasing ATP utilization.
Of the two novel UCPs, it was promptly noted that UCP3,
expressed in muscle, heart, and BAT, responds clearly to
TH and adrenergic stimulation (98), while UCP2 does not
respond to sympathetic stimulation (88) and its response
to TH in vivo is quite variable and inconsistent (see, for
example, Ref. 98). Such findings made UCP3 a more likely
mediator of TH thermogenesis. However, such expectations have not been fulfilled. For one thing, hepatic mitochondria, in which the proton leak had been demonstrated and characterized, do not express either UCP2 or
UCP3. Besides, even though when expressed at high concentrations in mitochondria UCP2 and UCP3 share with
UCP1 the property of reducing the proton-motive force, at
the concentrations normally found in cells they do not
cause a detectable drop in the proton gradient (recently
reviewed in Ref. 196). Changes in either UCP2 or UCP3
associated with manipulations of the thyroid status are
not associated with changes in the proton gradient in
human muscle (10). Lastly, neither ucp2 nor ucp3 knockout mice show any thermogenic defect, and UCP3-deficient mice increase QO2 and body temperature in response to T3 injections as the WT genotype (99, 196).
Altogether, these observations make it unlikely, but do
not exclude, that these proteins mediate the thermogenic
effect of TH, and further studies are necessary to define
their role in TH thermogenesis.
As mentioned earlier, the NADH-G3P shuttle constitutes another mechanism of producing ATP with lower
Physiol Rev • VOL
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.
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FIG. 7. Oxygen consumption (QO2) in hepatocytes from hypothyroid (Hypo), euthyroid (Eu), and hyperthyroid (Hyper) rats. Note that in
the transition from hypothyroid to euthyroid, representing the physiological effect of thyroid hormone, the difference in QO2 is largely due to
the increase in proton leak, whereas that due to ATP synthesis remained
unchanged. Both ATP synthesis and proton leak contributed equally to
the increased QO2 of the hyperthyroid state. [Data from Harper and
Brand (114).]
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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).
1. TH-adrenergic interactions
The SNS and the adrenal medulla are considered part
of a system, centrally controlled at the level of the hypo-
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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 thyrotoxicosisinduced 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
HORMONAL REGULATION OF THERMOGENESIS AND BODY TEMPERATURE
Physiol Rev • VOL
ity 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.
Thermogenesis obviously increases energy needs.
Essential to support thermogenesis is the provision of
extra energy. It makes teleological sense, therefore, that
TH also stimulates food intake and lipogenesis (207, 212
and references therein). The former provides additional
energy and the latter, a mechanism to store it in a high
caloric density form, fat. Hepatic de novo synthesis of
fatty acids and of triglycerides is increased (91), and these
are rapidly mobilized to WAT and muscle, where TH
stimulates lipoprotein lipase (175, 200). In addition, TH
enhances the lipolytic responses to catecholamines in
WAT (116, 191, 246). Increased thermogenesis also demands additional oxygen supply to tissues and TH clearly
stimulates cardiac function, increasing cardiac performance and output as well as the capacity of blood to
transport oxygen (129), acting directly at the end organs,
as well as potentiating the actions of catecholamines on
the heart and peripheral circulation. TH is as essential for
the full responsiveness of the cardiovascular system to
catecholamines as it is for BAT to express all its thermogenic potential in response to SNS stimulation. Likewise,
the stimulation of lipolysis by norepinephrine in white
adipose tissue needs TH (209), as it does the epinephrine
or glucagon stimulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis (113). Where in the signaling pathways and how
is TH necessary to ensure optimal responsiveness to the
sympathoadrenal system is tissue dependent (reviewed in
Refs. 209, 210). Here, the focus will be on how TH affects
the thermogenic function of BAT.
2. TH and BAT thermogenesis
That TH is necessary for a full BAT thermogenic
response in cold adaptation has been long known (see
Ref. 117 for early references). Most of the studies focused
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thalamus 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 activ-
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J. ENRIQUE SILVA
on the diminished or lost responsiveness of BAT to sympathetic stimulation in the hypothyroid state. Mory et al.
(162) noted that BAT of hyperthyroid rats showed morphological changes seen in cold adaptation, yet UCP1 was
not stimulated nor did it respond to cold challenge. Seydoux et al. (206) reported impaired responses of BAT to
sympathetic nerve stimulation or catecholamines that
they attributed to reduced number of ␤-adrenergic receptors (␤-AR), but noted that the responses were reduced to
a much greater extent than the number of ␤-AR, suggesting an additional defect(s) downstream. Sundin et al.
(230) showed reduced respiratory and lipolytic responses
of brown adipocytes to isoproterenol or forskolin, and
again pointed to a more distal effect as neither cAMP
analogs nor forskolin stimulated normally respiration in
hypothyroid brown adipocytes. Altogether these studies,
among others, indicate that TH is essential to maintain the
norepinephrine signaling in BAT, but they also clearly
pointed to additional steps where TH is needed for a
normal response as well as to complex interactions between this hormone and SNS. Thus, while TH was necessary for the response of UCP1 to cold, as assessed by GDP
binding, treatment of rats with doses of T4 greater than
the daily production rate, inducing a significant degree of
thyrotoxicosis, was associated with reductions of GDP
binding or a diminished stimulation of GDP binding by
cold (229, 233), leading to the concept that TH played a
permissive role on BAT function (162, 233).
The finding of D2 and its activation by norepinephrine in BAT encouraged studies to characterize and unravel SNS-TH interactions in regulating the thermogenic
responses of BAT. A synergism between TH and norepinephrine in the stimulation of the UCP1 gene became
promptly evident (18). In the absence of TH, the stimulaPhysiol Rev • VOL
tion of UCP1 by norepinephrine alone, as judged by the
denervation of BAT in hypothyroid rats, is quite modest,
2- to 3-fold, whereas the stimulation is 18- to 20-fold in the
presence of TH. Likewise, the stimulation of UCP1 by T3
on denervated BAT was also quite modest, barely significant (18). A complex TH response element (TRE) was
identified upstream in the rat UCP1 gene (182), included
in a critical enhancer well conserved in both the rat and
mouse genes (54, 137). Such TRE was actually composed
of two TREs in tandem located immediately below a
cAMP response element (CRE) in that upstream enhancer
region of the UCP1 gene (182). The synergism between
cAMP and T3 was demonstrated to occur at the gene level
(181), with TH and its TRE being necessary for the recruitment of an additional CRE downstream, in the proximal 5⬘-flanking region of the gene (reviewed in Ref. 218).
Such multiple-level synergism makes it even more
intriguing that the induction of a hyperthyroid state was
associated with a reduction of the response of the tissue
to adrenergic stimulation (229). The answer is probably
D2 and its responsiveness to adrenergic stimulation and
T4. Thus the synergism between T3 and the SNS makes
BAT responses very sensitive to the removal of either. D2
is highly responsive to endogenous or exogenous norepinephrine stimulation (214 –216), but it is also powerfully
inhibited by T4, reverse T3, or other substrates that facilitate its rapid degradation by proteasomes (226), not only
in BAT but also in other tissues (208, 213, 217). Thus the
reduction of D2 activation by high levels of T4 will substantially reduce the response of UCP1 to adrenergic
stimulation, which is backed by experiments wherein the
enzyme is blocked with iopanoic acid, a competitive inhibitor (20, 21), or its adrenergic activation prevented
with prazosin, an ␣1-AR antagonist (53). In addition to
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FIG. 8. Schematic representation of relationships between BAT facultative thermogenesis and obligatory thermogenesis, sympathetic BAT
stimulation [sympathetic nervous system (SNS) activity], and type II iodothyronine 5⬘-deiodinase (D2) activity as a function of the thyroid status in
rodents acclimated at room temperature. A: BAT thermogenesis and UCP1 (red line), SNS activity (blue line), and D2 activity (green line) expressed
as continued function of hypothyroid and hyperthyroid state departing from the euthyroid condition (Eu), represented by the box on the x-axis. B:
obligatory thermogenesis and BAT facultative thermogenesis (left axis) and BAT SNS activity (right axis) in the euthyroid status (Eu) compared with
severe hypothyroidism (Hpo) or hyperthyroidism (Hper). Numbers along y-axes are relative to the euthyroid state (see text for details and
references).
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roidism have received substantially more attention than
those affecting BAT. In WAT, hypothyroidism causes the
following: reduction in the number of ␤-adrenergic receptors and increased presence of ␣-adrenergic receptors
(22); enhanced inhibitory responses to adenosine (48, 128,
255), probably related to increases in Gi␣ or G␤␥ protein
subunits (147, 157, 176, 184); and augmented phosphodiesterase activity (103). The ␣2-AR, which causes inhibition of the cAMP production through coupling to Gi proteins, is little affected by thyroid dysfunction (66, 189). We
explored each of these possibilities in BAT. We promptly
excluded increased phosphodiesterase activity as a mechanism because inhibition of the enzyme with IBMX increased the accumulation of cAMP by the same factor in
hypothyroid and euthyroid brown adipocytes, without reducing the gap between the two thyroid states (197).
Likewise, we found that increased sensitivity to adenosine or increased Gi␣ or G␤␥ subunits were of limited
relevance, if at all significant, in the defective cAMP response to ␤-AR activation or forskolin stimulation (52).
While intact brown adipocytes from hypothyroid rats consistently show reduced cAMP response to ␤-AR or forskolin, these responses were not reduced but increased in
isolated membranes from hypothyroid brown fat when
assayed for adenylyl cyclase in the standard conditions
(52). Further studies showed that adenylyl cyclase was
exquisitely sensitive to inhibition by Ca2⫹ and affected by
GTP or ATP in a biphasic manner, with stimulation in the
micromolar range and pronounced inhibition at concentrations ⬎0.3– 0.4 mM (52), a profile suggesting that the
predominant isoform of adenylyl cyclase in hypothyroid
brown adipocytes is AC-VI (112). It was proposed that the
reduced capacity of hypothyroid brown adipocytes to
generate cAMP was due to the intracellular conditions
prevailing in these cells, namely, high Ca2⫹ and nucleotides levels, not favorable for the activity AC-VI (52).
However, subsequent studies based on adenylyl cyclase
isoform mRNA measurements found that the predominant form of adenylyl cyclase mRNA in BAT is AC-III(104)
and that this mRNA is increased in hypothyroidism (56).
In addition to adenylyl cyclase activity not being characterized in this latter study, mRNA was extracted from
whole tissue, which contains a large proportion of stromal and vascular cells of BAT (164). Because AC-III
mRNA levels are responsive to sympathetic stimulation
(104), it is possible that the increased abundance of this
mRNA in total BAT RNA resulted from the increased
sympathetic tone of the hypothyroid state on cells other
than brown adipocytes, as vascular cells are also abundantly innervated (164).
Additional evidence of the need of TH for BAT normal responsiveness norepinephrine comes from studying
mice with deletion of the TH receptor ␣ (TR␣). These
mice are cold intolerant and have severely impaired BAT
thermogenic response to norepinephrine. In WT mice,
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inhibiting D2, hyperthyroxinemia is associated with reduced central sympathetic stimulation not only of BAT
but also of heart and other organs (135) so that, even
though there may be some T3 entering BAT from plasma,
there will not be enough cAMP to induce a full response.
Thus, in hyperthyroidism, particularly if induced by T4,
there is a reduction in the central stimulation of BAT as
well as an inhibition of D2 activity, explaining the observations mentioned above (229, 233).
The reduced responsiveness of hypothyroid BAT or
brown adipocytes from hypothyroid rats directed the attention to the effects of TH on norepinephrine signal
transduction (206, 230). While ␤-AR number was found
reduced (206), this latter and subsequent studies showed
the responses were reduced to a much greater extent than
the receptor number (206). It was also observed that the
respiratory responses of isolated brown adipocytes to
forskolin, which directly stimulates adenylyl cyclase, as
well as to free fatty acids were reduced (230). Altogether,
these findings indicated the need for TH at various levels,
not only in the norepinephrine signaling pathway, for a
full BAT thermogenic response. Of note, the authors of
this latter study (230) did not find a reduction in the cAMP
response to forskolin in hypothyroid brown adipocytes,
but subsequent studies have consistently reported a reduction in the cAMP response to both ␤-AR agonists and
forskolin in BAT cells from hypothyroid animals (180,
197). [Such discrepancy was probably due to the short
time of exposure (15 min) to isoproterenol or forskolin in
the study by Sundin et al. (230).]
Besides the poor response of cAMP to forskolin,
additional evidence indicates that a reduction in ␤-AR
number is but a small factor in the limited responsiveness
of BAT to SNS stimulation. Rubio et al. (197) found that
cAMP responses were reduced by more than 60%, but the
normalization of the number of ␤1-AR and ␤2-AR receptors after T3 treatment preceded the normalization of
cAMP generation for nearly 24 h. In another paper, these
authors reported that ␤3-AR are increased in BAT of
hypothyroid rats (whereas they are reduced in WAT)
(198) and that norepinephrine generated a larger fraction
of the cAMP via this receptor in the hypothyroid than in
the euthyroid state (198). Interestingly, ␤3-AR number
was rapidly reduced to the euthyroid levels by treatment
with T3, whereas the cAMP response improved during this
time. Lastly, it is possible that the reduction in ␤1,2-AR
was the result of the desensitization owing to the sustained increase in SNS stimulation of BAT (202, 236),
rather than being the direct result from the lack of TH,
because the difference in BAT ␤1,2-AR number between
hypo- and euthyroid rats became statistically insignificant
within 2 days of acclimation to thermoneutrality
(30°C) (197).
The mechanisms leading to decreased responsiveness or sensitivity to catecholamines of WAT in hypothy-
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Physiol Rev • VOL
sis with mechanisms to adjust FcT responses to ambient
temperature as well as to the level of ObT. When ObT is
reduced, such as in hypothyroidism, SNS stimulation of
BAT is increased and the activation of D2, in this case
amplified by the reduced levels of T4, results in increased
fractional conversion of T4 to T3 in the tissue, maintaining
the tissue levels of T3 needed to sustain the overall thermogenic response to norepinephrine signaling. In the rat,
studies replacing T4 in a graded manner in the hypothyroid state indicate that the enhanced D2 activity allows
the maintenance of thermogenic responses with reductions of circulating T4 down to 30% of the euthyroid levels
(53). Direct measurements of tissue T3 hypothyroid rats
treated with graded infusions of T4 from implanted
minipumps show that BAT and brain can effectively maintain the levels of T3 within a broad range of plasma T4
concentrations (80). We have recently provided another
example of BAT FcT adjustment to reduced ObT in a
transgenic model lacking mGPD (mGPD ⫺/⫺), as mentioned before in this article, as such mice show evidence
of increased BAT stimulation (71). When ObT increases,
as in hyperthyroid states, there is concerted reduction of
SNS stimulation of BAT and of D2 activity, which is
further reduced by the higher plasma T4 concentration so
that the tissue is less stimulated and the concentration of
the active form of TH, T3, is maintained low, disabling the
synergism between norepinephrine and T3. These relationships are schematically depicted in Figure 8A.
3. Is there another site of nonshivering facultative
thermogenesis in mammals?
The evidence that supports BAT as the only site of
FcT comes from observations in rodents (51). This concept has been reinforced by observations made in UCP1
knockout mice. These mice have cold intolerance (79),
which came as a surprise to no one. However, the gradual
exposure of these animals to cold results in improved
cold tolerance. While in WT controls the initial shivering
was rapidly replaced by BAT thermogenic activity during
cold exposure, these authors found persistent muscle
electrical activity during cold adaptation in the UCP1deficient mice, for which they attributed the cold tolerance to a form of “chronic shivering” (96). They argued
that the lack of respiratory response to norepinephrine in
the UCP-deficient mice was an indication of the lack of
BAT FcT or of any other form of nonshivering FcT in the
UCP-deficient mice (96), and they concluded that UCP1
“remains the only protein the activity of which can be
recruited for the purpose of facultative thermogenesis”
(165). While there is good evidence that skeletal muscle is
a site of FcT (nonshivering) in birds (72, 74), there is no
agreement whether muscle, or for that matter other tissues, may be a site of nonshivering FcT in mammals.
Certainly BAT is not prominent in large mammals, at least
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BAT temperature increases linearly with time during a
norepinephrine infusion, which is associated with a parallel increase in TC. In contrast, BAT temperature virtually
failed to increase in TR␣-0/0 mice, suggesting that this
receptor is crucial for the thermogenic response to norepinephrine (155). Apparently, there is a very specific step
in the response that strictly requires TR␣1 for TH stimulation because other responses to SNS stimulation, such
as D2, UCP1, and PGC1␣1 mRNAs were normal. Consistent with these findings, previous studies giving the TR␤selective agonist GC1 to hypothyroid mice show that BAT
thermogenesis was not restored by this T3 analog, even
though it restored the thyroid status of other tissues (187).
In summary, TH appears necessary for a full BAT
thermogenic response. This statement derives largely
from the observed defects in hypothyroidism. Such observations show that ␤1,2-AR are reduced while ␤3-AR are
increased, despite that the generation of cAMP is reduced
in isolated brown adipocytes, even to stimulation by forskolin. Such reduced adenylyl cyclase response does not
seem to be due to a reduction in total enzyme activity but
to the isoform predominantly expressed in the hypothyroid state, probably AC-VI, and to the prevailing intracellular conditions that are not favorable to its activity.
However, there are other defects further downstream that
may derive from the lack of TH at the some critical gene
levels, most notably UCP1. Norepinephrine-stimulated lipolysis is blunted in hypothyroidism, but the respiratory
responses of brown adipocytes to added fatty acids are
also blunted (230), again supporting the idea of multiple
level defects, in this case, a blunted lipolytic response and
a reduced respiratory response probably reflecting the
low levels of UCP1. Moreover, an action of T3 specifically
mediated by TR␣ seems to be critical for the actual heat
production by BAT in response to norepinephrine (155).
In addition, the enhancement of the ␣1-AR pathway in
hypothyroidism (171) may elevate intracellular Ca2⫹ levels that, jointly with increased levels of nucleotides, may
inhibit the activity of AC-VI, which may be compounded
by this isoform being relatively more abundant in the
hypothyroid state. However well-founded some of these
hypotheses may be, they need to be tested rigorously.
Lastly, it is important to keep in mind that some of the
defects in BAT thermogenesis in hypothyroidism are not
necessarily an indication of loci directly affected by TH,
as some of the defects may result from hypothyroidism
elsewhere, such as the downregulation of ␤1,2-AR which is
caused to a significant extent by desensitization due to
centrally mediated sympathetic stimulation of the tissue.
Altogether, the studies reviewed here and others suggest complex interactions between TH and the SNS on
BAT thermogenesis. Such interactions take place at various levels, including the central regulation of the SNS
output to BAT and seem to endow temperature homeosta-
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Physiol Rev • VOL
tion. In the TR␣-0/0 mice reared at room temperature, QO2
was 40 – 45% greater than the appropriate WT controls at
night, when animals are more active, whereas it was only
20% higher during the day, a significantly smaller difference (155). There are mechanisms whereby heat production associated with muscle activity can be increased.
When the gradient of Ca2⫹ between the sarcoplasmic
reticulum vesicles and cytosol increases, the optimal
Ca2⫹ transport-to-ATP hydrolysis coupling ratio decreases from 2 to values close to 1, due to “slippage” of
the pump (123) with the ensuing increase of ATP demands and heat production. Moreover, SERCA can mediate Ca2⫹ efflux following the osmotic gradient with the
resulting heat liberation (this is an exothermic process)
and increased ATP expenditure to pump calcium back
into the sarcoplasmic reticulum (69). In addition, the heat
released in the ATP hydrolysis by the SERCA can vary
over a rather wide range in skeletal muscle (67). As
discussed earlier, TH can increase both resting and tension-associated heat production (reviewed in Ref. 219)
(Fig. 6), and TH stimulates the expression of SERCA1
content predominantly in slow-twitch muscle. Since the
slow-twitch, resistance-endurance motor units are more
frequently active than fast-twitch muscle, the substitution
for SERCA1 in these motor units, by virtue of the higher
catalytic activity of this isoform, will double the rate of
Ca2⫹ turnover, resulting in more ATP turnover, steeper
gradients, and more Ca2⫹ efflux (219). In this context, it is
notable that slow muscle is substantially more abundant
in large mammals (6, 177) than small rodents. Conceivably, the loss of the function of BAT with age and growth,
or for that matter the absence of it in some species,
probably stimulates the development of muscle thermogenesis under the control of TH.
In summary, even though BAT is unquestionably the
major site of FcT in small rodents and bigger mammals
during the neonatal period, muscle and perhaps other
tissues may participate in nonshivering FcT in larger
mammals or even in small ones, when BAT function is
lost, as it occurs in UCP1-ablated or TR␣-deficient mice.
Such may also be a role of muscle in mammals not having
BAT, such as pigs, or during adulthood when BAT thermogenesis becomes less prominent, for example, in humans. Even in small rodents there is evidence suggesting
that muscle may be an alternate site of FcT, although
other evidence suggests this is merely a form of chronic
shivering. There are mechanisms that allow muscle to
produce more heat at rest and during activity, and TH
stimulates such mechanisms. Therefore, whether the
muscle electrical activity seen in the discussed mouse
models of BAT dysfunction is chronic shivering or increased muscle tone, the amount of heat associated with
any form of muscle activity may be increased in response
to sustained cold stimulation. While the separation of FcT
between shivering and nonshivering may be useful, it may
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during adulthood, and it has been proposed on solid
grounds that muscle can take that function (see Ref. 219
for a recent review and additional references).
Even in small rodents, where BAT plays such an
evident role in cold adaptation, there are findings that
could be interpreted as evidence of an alternate form of
nonshivering FcT. Thus, while UCP1 knockout mice were,
as expected, cold intolerant, it was surprising that these
mice were not obese (79). Moreover, when challenged
with a high-fat diet, the UCP1 ablated mice gained less
weight than WT controls. Interestingly, this obesity resistance was ambient temperature dependent because it was
evident when they were reared at room temperature, but
they gained weight faster than the WT, rapidly reaching
their level of obesity, promptly after transferring them to
an ambient closer to TN (148). The authors reasonably
interpreted these findings as the expression of an alternative form of FcT that was more energy demanding than
BAT thermogenesis and thus protected the UCP1 knockout mice from gaining weight when adapted to room
temperature (148). Acclimating these mice to a higher
temperature eliminated the need of this putative form of
FcT and thus made them prone to gain weight on a
high-fat diet. We have made a similar observation in mice
lacking all forms of THR ␣ (TR␣-0/0) that support the idea
of an alternate form of FcT when BAT is nonfunctional.
TR␣-0/0 mice are cold intolerant owing to a failure of BAT
to increase thermogenesis in response to norepinephrine.
Surprisingly, they do not show signs of being coldstressed when reared at room temperature (155), when
BAT normally makes a substantial contribution to overall
thermogenesis (51). Furthermore, despite having a nonfunctional BAT, the metabolic rate (QO2) of TR␣-0/0 mice
was higher at room temperature than that of WT controls
(155). Such difference in QO2 disappeared when TR␣-0/0
mice were acclimated to TN (30°C), indicating that it is
cold dependent, hence by definition the expression of a
form of FcT. It is important to consider in the analysis
that the two models, UCP1-deficient and TR␣-0/0 mice,
congenitally lack BAT function. In this regard, the situation is similar to that in piglets, which normally do not
have BAT (232). In this species, early cold exposure results in the accelerated establishment of a muscle phenotype leading to enhanced thermogenesis (16).
In the light of the observation of persistent muscle
electrical activity in cold-challenged UCP1-deficient mice,
it cannot be excluded that the development of cold tolerance in those mouse models is ultimately due to “chronic
shivering.” However, this was demonstrated at a very low
ambient temperature (96), when an alternative form of
nonshivering FcT may not have been sufficient, and persistent shivering was necessary. Such chronic shivering at
room temperature remains to be demonstrated. On the
other hand, shivering, and for the matter muscle activity,
may be associated with variable amounts of heat produc-
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J. ENRIQUE SILVA
become tenuous when it comes to muscle. Lastly,
whether shivering or nonshivering, the alternate facultative thermogenesis is more costly and less effective than
BAT thermogenesis, as illustrated by the increased QO2 of
TR␣-0/0 mice and the obesity resistance of UCP-ablated
mice when reared at room temperature vis-à-vis their
inability to keep TC when more severely cold-challenged.
III. OTHER HORMONE ROLES
IN THERMOGENESIS
A. Insulin, Glucagon, and Epinephrine
1. Insulin and thermogenesis
Insulin may affect thermogenesis, but it does not
appear to have a primary regulatory role. It is largely
permissive, as one can infer from observation made in
patients with diabetes or insulin resistance, or in experiPhysiol Rev • VOL
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As discussed in previous sections, TH plays a pivotal
role in temperature homeostasis by increasing ObT and
supporting FcT. Such a role appears to be a late evolutionary acquisition because there is no evidence that TH
stimulates thermogenesis in poikilothermic species,
which, however, plays other important functions, shared
with all vertebrates. No other hormone seems to have
such a direct action on thermogenic mechanisms and
temperature homeostasis as TH does. Malfunctioning of
other endocrine organs or altered sensitivity of target
cells to their hormones, e.g., to insulin, is often associated
with disruptions on energy balance and thermogenesis,
but these seem to result from a variety of indirect mechanisms. Most commonly, other hormones play a permissive role, mainly by affecting substrate availability and
partition. Thus epinephrine and glucagon will stimulate
glycogenolysis, gluconeogenesis, and lipolysis, thus increasing circulating levels of glucose and fatty acids. Insulin is essential for fuel storage in the form of glycogen,
in liver and muscle, and fat, in the adipose tissue, but also
for blood glucose availability in skeletal muscle. Of all
hormones, TH excluded, insulin likely plays an essential
role in the so-called thermal effect of food or “specific
dynamic action” of the old German literature (199). Glucocorticoids play an essential permissive role in gluconeogenesis and mobilizing labile proteins and other gluconeogenic precursors but have effects at various levels
geared to reduce energy expenditure and thermogenesis
in situations of stress. Leptin, a comparatively new hormone, stimulates energy expenditure via SNS, acting at
the central level, as probably does insulin as well, while it
also enhances the secretion of TRH, upregulating thyroid
function. Ghrelin, also a recently discovered hormone,
induces satiety but may reduce energy expenditure.
mental models of these. Patients with insulin resistance
progressing into type 2 diabetes have increased basal
metabolic rate, but because of the resistance to insulin
action at the muscle level, the increase in metabolic rate
normally associated with food, the thermal effect of food
(TEF) is reduced in proportion to the insulin resistance at
the muscle level (250). When insulin is given in increasing
amounts, in hyperinsulinemic-euglycemic clamps, there is
a rapid increase in glucose storage and in glucose oxidation, both exhibiting saturation kinetics, but the latter
reaches a plateau at insulin levels ⬍20% of those required
to saturate storage (see Ref. 85 for review and references). The partition between oxidation and storage is
probably variable depending, among other factors, on the
muscle sensitivity to insulin relative to other tissues. This
has the support of observations made on transgenic mice
with tissue-selective deletion of the insulin receptor (IR).
Mice with skeletal muscle deletion of IR oxidize less
glucose and store more as fat (47, 130), in contrast to mice
with deletion of the protein-tyrosine phosphatase-1B, in
which there is an increase in insulin sensitivity in muscle.
These mice are less susceptible to diet-induced obesity
and have increased metabolic rate without change in
UCPs expression, while hyperinsulinemic-euglycemic
clamp studies show an elevated capacity to oxidize glucose (132). Despite PTP-1B being expressed in many tissues, WAT among them, insulin-stimulated glucose uptake was not increased in this latter (132), suggesting that
muscle responsiveness to insulin is a major determinant
in the partition of energy. Furthermore, normal muscle
has the capacity to increase glucose consumption if more
is available, as it occurs in the selective deletion IR in
adipose tissue, which is associated with resistance to
obesity and more glycolysis and glucose oxidation (27). A
corollary of these studies is that even in small rodents
skeletal muscle is important for a form of thermogenesis,
the TEF.
In these latter species BAT also seems to participate
in TEF, with insulin being an essential signal. For one
thing, the selective deletion of IR in BAT is associated
with loss of adipogenic or lipogenic transcription factors
(CEBP, PPAR␥), as well as with loss of lipogenic enzymes
such as fatty acid synthase and malic enzyme and with fat
depletion, but most notably, these animals show agedependent related glucose intolerance (106). Cold exposure is associated with increased glucose uptake by all
tissues, but most markedly by BAT (110-fold) (238), and
on this insulin also plays a permissive role regulating the
expression and activity of glucose transporters (151), as
well as BAT D2 activity. A single injection of insulin
stimulated 8- to 10-fold D2 activity, and this effect was
enhanced by prefeeding carbohydrates or in streptozotocin-induced diabetes (214). Interestingly, a single meal
increased BAT QO2 and weight over appropriate controls
(94) and doubled D2 activity and GDP binding, an expres-
HORMONAL REGULATION OF THERMOGENESIS AND BODY TEMPERATURE
2. Glucagon and epinephrine
Even though the injection of glucagon or epinephrine
is associated with an increase in oxygen consumption, it
is not clear that they play a key role regulating thermogenesis. As mentioned before, they play a permissive role,
by mobilizing stored fat and carbohydrate and stimulating
gluconeogenesis. Epinephrine causes vasodilation in skeletal muscle, augments glucose and oxygen consumption
(222), and has been proposed that it could participate in
thermogenesis in humans (223), specifically in FcT induced by carbohydrates (8). However, the threshold concentration for the thermogenic effect is rather high, for
which this effect is probably of significance in states of
stress, when epinephrine levels rise well over the threshold (63). Despite the earlier belief that glucagon increased
oxygen consumption in muscle (73), this has not been
directly demonstrated (154). Both glucagon and epinephrine increase liver oxygen consumption, but this seems to
be a reflection of the stimulation of gluconeogenesis in
Physiol Rev • VOL
this organ (13, 87). Both epinephrine and glucagon can
also stimulate BAT, but it is not likely that such an effect
is of major importance in temperature and energy homeostasis. Epinephrine will only exceptionally reach concentrations high enough at BAT ␤-AR to stimulate the
tissue, as does the locally, sympathetically released norepinephrine. However, in patients with pheochromocytoma, in whom there is a sustained elevation of circulating catecholamines, predominantly epinephrine, BAT
shows signs of histological and biochemical signs of stimulation, e.g., an increase in UCP1, which may reach levels
comparable to those in cold-exposed rodents (140, 190).
In the case of glucagon, this does not significantly increase GDP binding acutely, but it does after repeated
high doses (23), and although it elicited a thermogenic
response, this could be blocked with propranolol and
occurs equally in rats and hamsters, which do not have
glucagon receptors in BAT (70). Acutely, and again in high
doses, glucagon also increases BAT D2, but this may as
well be an indirect effect (214).
B. Glucocorticoids
Glucocorticoids also have complex effects on thermogenesis and temperature homeostasis. It is not a primary function of these hormones to increase thermogenesis, but rather to coordinate thermogenic responses to
substrate or food availability. For example, in a stress
situation where food is reduced, e.g., illness, glucocorticoids will have an inhibitory effect on BAT, but in cold
stress, the inhibition of BAT thermogenesis would be
counterproductive. Here, substrate mobilization is equally
needed, but not the inhibitory effect on facultative thermogenesis. The stress reaction has many components.
Initial loci are the corticotropin releasing hormone
(CRH)-producing neurons, which activate the adrenal and
thyroid axis, and the locus ceruleus-norepinephrine,
which will activate the branches of the sympathetic nervous system (reviewed in Ref. 235). CRH and products of
the POMC gene will inhibit appetite and activate pathways to increase thermogenesis (235, 253), but the net
effect of these signals will depend on others, such as NPY
and agouti-related protein (AGRP), which in turn depend
on peripheral signals, notably leptin and probably insulin
as well. In the absence of such input, there is a higher
output of NPY and AGRP from the arcuate nucleus that
will antagonize the thermogenic effect (see Ref. 89 for
review). It is interesting that POMC expression stimulation in the hypothalamus will also stimulate TRH secretion, upregulating thyroid function and thermogenesis, an
effect that will obviously be reduced or abrogated in the
presence of signals of limited energy in the periphery,
largely reduced leptin and insulin (141). So, the stimulation of the hypothalamic-pituitary-adrenal (HPA) axis is
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sion of active UCP1 (95), while diabetes or fasting reduced the stimulation of D2 by cold or exogenous norepinephrine (214), underscoring the importance of insulin
for BAT responses to food, but to cold as well.
Another manner for insulin to contribute to thermogenesis and energy expenditure is by increasing sympathetic activity, probably acting at a hypothalamic level
(see Refs. 85, 204 for reviews). Hyperinsulinemic-euglycemic clamps are associated with increased sympathetic
activity in a number of tissues (85), notably in skeletal
muscle, where the sympathetic nerves cause vasodilation
(4, 245). Such an effect facilitates the muscle glucose
uptake and may be an important factor in TEF. Whether
the increased sympathetic activity to the muscle per se
increases glucose oxidation is not clear (245). Independent limiting factors are obviously glucose uptake and
oxidation, which are normally stimulated by insulin but
does not occur in states of insulin resistance, such as
metabolic syndrome and type 2 diabetes (250). The insulin-induced sympathetic activation is believed to result
from direct action of the hormone in the hypothalamus,
where it can pass the blood-brain barrier and reduce
neuropeptide Y (NPY) levels from the arcuate nucleus,
contributing to TEF and satiety, provided concomitant
blood glucose levels are normal or higher (204). In states
of food deprivation, the associated low levels of insulin
may be a factor in reduced sympathetic stimulation of
muscle and reduced thermogenesis as well as increased
hunger (204). On the other hand, studies in a transgenic
model of neuronal insulin receptor deletion suggest even
broader effects for insulin in brain. Expectedly, these
mice have increased food intake and are more susceptible
to diet-induced obesity, but they also show central depression of reproductive function (46).
455
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J. ENRIQUE SILVA
C. Leptin
The role of leptin in regulating energy balance is
dramatically illustrated by the phenotype of the so-called
ob/ob mouse, characterized by hyperphagia and cold inPhysiol Rev • VOL
tolerance (reviewed in Ref. 234). With the cloning of
leptin (259) we learned that this hormone produced by
white adipocytes is a major signal controlling the energy
balance. Its production is a function of the replenishment
of the fat stores, but fasting reduces its levels rapidly,
faster than WAT is depleted of fat (115). Leptin inhibits
food intake and increases thermogenesis via activation of
the sympathetic nervous system, and its reduction is associated with hyperphagia and reduced thermogenesis, as
dramatically illustrated by the ob/ob mouse that has an
inactivating mutation in the gene. On the other hand, the
large volume of research work triggered by the cloning of
leptin has revealed that it not only is a regulator of appetite and thermogenesis, but also plays an important role in
concerting other responses to the nutritional state, notably reproductive and thyroid functions (1, 115, 141). Although leptin receptors and actions in peripheral tissues
have been described, it appears that its thermogenic effect is largely central (152).
IV. THERMOGENESIS AS A SOURCE OF
VARIABILITY IN ENERGY EXPENDITURE
It is well known that basal metabolic rate (BMR) in
humans is closely correlated to lean body mass, yet the
variability around the regression line, for any lean body
mass, is ⬃600 kcal, which represents a huge individual
variability considering that people of 70 kg of lean body
mass have BMRs between 1,600 and 2,200 kcal/day (28,
251). BMR also correlates with body temperature (192),
and a large fraction of BMR variability is due to muscle
thermogenesis (260). Furthermore, such variability is familial, largely genetically based (28, 228; reviewed in Ref.
31), and possibly resulted from varying pressures on our
remote ancestors wherein the need for heat production
was in competition with food availability. As mentioned at
the beginning of this review, homeothermic species have
evolved mechanisms to constantly produce more heat,
facilitating the maintenance of TC in environments much
colder than this temperature. Thus the rate of oxidations
in homeothermic species is more than fivefold higher than
in poikilothermic species, even when measured in similarsized species and at the same ambient temperature, and
whether measured in the whole animal or isolated tissues
(78) (Fig. 1). Not only the metabolism is more active, but
also the thermodynamic efficiency of the homeothermic
machine is lower when the energy cost of certain functions, such as muscle contraction, is considered (254)
(Fig. 1). So, there is significant energy cost attached to
homeothermy.
It follows that food availability may have been a
limiting variable for the amount of heat to be produced for
the sake of maintaining TC during evolution, so in the end,
the need to produce heat and food availability were se-
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primarily associated with reduced appetite and increased
thermogenesis, but the magnitude of this stimulation is
strongly influenced by peripheral signals of abundance.
The resulting effect of the HPA axis is an increase in
adrenal hormones, most importantly glucocorticoids, cortisol in humans, and corticosterone in rodents. By feeding
back negatively at the hypothalamic levels, glucocorticoids will reverse the central effects of activating the HPA
axis described above. This is a very important effect
particularly in the absence of the positive signals of insulin and, especially, leptin. This is supported by the repeated and consistent observation that adrenalectomy is
associated with improvement of obesity and cold tolerance of models of genetic obesity lacking leptin or leptin
signal, such as the ob/ob mouse and the fa/fa (Zucker) rat
(see Refs. 40, 42 for reviews). In addition, glucocorticoids
have multiple peripheral effects that will ultimately affect
thermogenesis and thermogenic responses. In rodents,
these hormones clearly inhibit the function of BAT by
inhibiting UCP1 expression (160, 225). Glucocorticoids
also inhibit the expression of the ␤3-AR in BAT but not in
other tissues such as gastrointestinal tract and WAT (82,
122). However, glucocorticoids may not always inhibit,
but may stimulate, ␤3-AR in vivo, as it occurs in models of
genetic obesity (174). Furthermore, the effect may be
initially inhibitory and then stimulatory in lean C5yBl/6J
mice (82), later found also in normal CD1 mice (9). The
inhibitory defect of glucocorticoids on BAT ␤3-AR is direct and can be demonstrated in isolated brown adipocytes or derived cell lines (9), but even in such cell lines
it is possible to demonstrate a dual, opposing effect (9).
Glucocorticoids induce the CAAT enhancer binding protein beta (C/EBP␤) that stimulates the transcription of
␤3-AR gene (9), and this transcription factor is also stimulated by the sympathetic nervous system (50). Based on
these observations, we proposed a model in which glucocorticoids will directly inhibit ␤3-AR expression by acting on a putative negative CRE, upstream in the gene, but
at the same time they would induce C/EBP␤, the magnitude of which will depend on the sympathetic stimulation,
and if high enough will offset the inhibition (9). As mentioned earlier, ␤3-AR is also differentially regulated by TH
in BAT and WAT (198), suggesting that this receptor is
key to BAT responsiveness to adrenergic stimulation and
is hence involved in the physiological regulation of BAT
thermogenesis. It is possible that ␤3-AR also participate in
signals emanating from the gastrointestinal tract as well
as in mediating central effects on food intake (40).
HORMONAL REGULATION OF THERMOGENESIS AND BODY TEMPERATURE
V. SUMMARY AND CONCLUSIONS
The advent of homeothermy represented an important leap in evolution, as it expanded the niche of species
providing not only a constant internal temperature but
also by setting it at the temperature most conducive to
biochemical and physiological processes inherent to life.
Central to homeothermy is an augmented heat generation
out of biological processes to facilitate the maintenance
of TC in habitats that are usually colder than TC. Not only
is basal heat production greater in homeothermic than in
poikilothermic species, but the former have the capacity
to increase heat production on demand, in a facultative
manner, hence facultative thermogenesis. Interestingly,
the mechanisms used by homeothermic species to generate heat are not new, and they antecede homeothermy, as
evidenced by their presence in lower species, even plants.
Good examples of these are the heater organ of the eye
and brain of certain deep-water fish (24) and the uncoupling proteins, until not long ago believed to be unique to
mammals, but now known to be already present in plants
(30). So, it would appear that nature resorted to preexisting mechanisms to produce heat in concerted and coordinated manner in the homeothermic species. What is
evolutionarily new is then the control and regulation of
this function.
For the purpose of clarity, we have divided thermogenic mechanisms into those that increase ATP demands
and those that increase heat generation during mitochondrial production of ATP. Mechanisms increasing the demands of ATP generate heat by virtue of forcing the cells
to produce more ATP, with its inherent energy loss as
heat. Quantitatively most important among these mechanisms appear to be those that accelerate the movement of
Physiol Rev • VOL
ions against gradients, most notably, sodium and potassium across the cell membrane and calcium across the
sarcoplasmic reticulum membranes. In both cases, mechanisms reducing these gradients seem to be key in forcing
the cell to spend more ATP to maintain it, but as discussed for cytosolic-sarcoplasmic Ca2⫹ exchange, the
coupling of ion exchange to ATP consumption may be
also reduced, forcing more ATP consumption to maintain
the flux of ions needed to keep the gradient up. It is
possible that mechanisms increasing ATP turnover,
clearly more active in homeothermic than poikilothermic
species, are more important for the basal heat production,
that is, are a major component of ObT.
Estimates of the contribution of mechanisms increasing ATP demands suggest that they are insufficient to
explain ObT in homeothermic species. The existence of a
regulated proton leak in the inner mitochondrial membrane, more active in homeothermic species and regulated by TH, has been the main support to the idea that
regulated reductions in ATP synthesis efficiency could be
an important thermogenic mechanism (37). The finding of
uncoupling proteins outside of BAT reinforced the concept that there could be a regulated proton leak or uncoupling mechanism elsewhere. A central thermogenic
role for the novel UCPs, though, has so far not been
supported by studies in transgenic mice with deletion or
overexpression of these proteins. It is possible that nonBAT UCPs play different roles, one of which could be to
support other thermogenic mechanisms, for example, to
reduce the formation of superoxides during increased
ATP synthesis. On the other hand, evidence from polymorphisms in human as well as observations in mGPD
deficient mice, are consistent with UCP3 participating in
ObT without being essential (196). Another mechanism
that could produce ATP with lower efficiency is the
NADH-G3P shuttle, particularly in muscle. G3P is an important fuel for muscle to rapidly produce ATP but with a
lower efficiency. The transgenic deletion of the rate-limiting enzyme in the shuttle, mGPD, results in a reduction
in ObT that is partially compensated by increased FcT in
BAT and possibly by an increase in UCP3 mediated by
increased levels of T3.
Thermogenic mechanisms are regulated in homeothermic species. TH is the major regulator, a new function
that this hormone acquired with the advent of homeothermy. TH not only directly controls critical steps of
thermogenic mechanisms, but also contributes to increase fuel availability by increasing appetite and stimulating lipogenesis in liver and to increase the delivery of
oxygen and fuel to tissues by stimulating cardiovascular
function, lipolysis, and gluconeogenesis. In stimulating
thermogenesis, TH works synergistically with the SNS,
practically at all levels. TH is essential to maximize the
responsiveness to catecholamines acting at the adrenergic receptor level as well as at several postreceptor steps
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lection pressures of variable magnitude that resulted in
biological “machines” of variable fuel efficiency. These
views are supported by observations made on overfeeding or starving humans. The responses are genetically
determined (33, 252), and in general there is correlation
between the increase in energy expenditure with overfeeding and its reduction in fasting, defining a range from
spend-thrift to thrifty phenotypes (252). Many animal
models, notably transgenic with deletion or overexpression of a variety of genes exhibit spend-thrift or thrifty
phenotypes, indicating that many genes may have been
the target of selection. Now, reduced BMR is associated
with an increased risk of obesity (28, 185), as a reduction
in thermogenesis in response to caloric restriction is a
limiting factor in weight loss in obese subjects (41). So,
the understanding of the mechanisms regulating thermogenesis is bound to be enlightening not only in the field of
temperature homeostasis, but it may be also of benefit to
the understanding and treatment of obesity.
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J. ENRIQUE SILVA
Physiol Rev • VOL
Finally, understanding of thermogenesis and temperature homeostasis is not only important for better comprehending these processes but also for getting insight
into the variability in energy expenditure in humans and
into the risk for developing obesity and type II diabetes,
conditions wherein the capacity to burn fuel becomes
limiting. Regardless of the psychosocial and economical
factors behind the excess eating that is plaguing our
society, multiple studies show that the capacity to dispose
of the excess calories varies markedly in humans, on
genetic bases (33). Not only thermogenesis may become
limiting, but also this is promptly reduced in response to
a shortage in the influx of energy, which is important to
survive famine and disease but severely limits the effectiveness of the mainstay of obesity treatment, namely,
caloric restriction. We are well protected against food
restriction, but seemingly not as well against food excess.
ACKNOWLEDGMENTS
I acknowledge the dedicated and enthusiastic work of my
technicians, students, and postdoctoral fellows.
Address for reprint requests and other correspondence: J.
Enrique Silva, Baystate Medical Center, Div. of Endocrinology,
Rm. S2620, 759 Chestnut St., Springfield, MA 01199 (e-mail:
[email protected]).
GRANTS
The work from my laboratory presented in this review has
been supported over the years by the Howard Hughes Medical
Institutes and the National Institutes of Health (while in the
United States) and by the Medical Research Council and Canadian Institutes of Health Research.
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in the catecholamine signaling pathways, particularly
those initiated in the ␤-ARs. The synergistic interaction is
most important and evident on BAT FcT, where the SNS
is sine qua non to activate BAT thermogenesis, but where
the responsiveness of the tissue is dependent on the
presence of high tissue concentrations of the most active
form of TH, T3, for which the SNS activates D2. This
enzyme acts as a gatekeeper for thermogenesis, since the
excess of TH inhibits it, directly and indirectly by reducing SNS stimulation, protecting BAT from the elevated
ambient levels of TH in hyperthyroidism and hence reducing the responses to residual SNS stimulation.
Other hormones’ participation in thermogenesis is
less direct. Insulin is essential to provide fuel for thermogenesis. However, this effect is strongly influenced by the
relative responsiveness of adipose tissue and muscle to its
action, as is revealed by studies in transgenic mice lacking
or overexpressing the insulin receptor in one of these
tissues (131). Prevention of entry of glucose to muscle
results in reduced glucose oxidation, while increased glucose availability, in the presence of insulin, increases
glucose oxidation and thermogenesis as revealed by the
transgenic deletion of the protein tyrosine phosphatase
B1 (132) or the insulin receptor gene selectively in adipose tissue (27). Insulin and leptin are major signals for
the central nervous system to change food intake and
energy expenditure via various mechanisms, most importantly SNS and TH. Being signals of abundance, leptin and
insulin reduce food intake and increase thermogenesis by
using these mechanisms, whereas their suppression by
starvation and diseases is associated with reduced sympathetic activity and thyroid function, hence, reduced
energy expenditure and thermogenesis, and, depending
on other signals, hunger and increased food intake.
An unresolved issue is whether there is FcT in sites
other than BAT in mammals. Skeletal muscle seems an
undisputed site of FcT in birds. In transgenic models with
BAT deficiency, there is evidence of increased energy
expenditure in cool environments but not a thermoneutrality, i.e., by definition a form of FcT, which is more
energy demanding than BAT thermogenesis. While studies in one of these models suggest that this alternate form
of FcT could simply be chronic shivering, independent
evidence suggests that muscle activity, including shivering, may be associated with more or less heat production
in a regulated manner, by TH and possibly by other signals. Such conception makes the line between FcT and
ObT tenuous in the absence of BAT, a situation that
normally occurs in some species such as pigs (232) and in
larger mammals, such humans, where BAT becomes quiescent with adulthood. Thus, in these species a substantial fraction of ObT is normally generated in muscle, but
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