obesity reviews
Mitochondrial uncoupling as a target for drug
development for the treatment of obesity
J. A. Harper1,2, K. Dickinson3 and M. D. Brand1
1
Summary
Cambridge CB2 2XY, UK; 2Department of
Mitochondrial proton cycling is responsible for a significant proportion of basal
or standard metabolic rate, so further uncoupling of mitochondria may be a good
way to increase energy expenditure and represents a good pharmacological target
for the treatment of obesity. Uncoupling by 2,4-dinitrophenol has been used in
this way in the past with notable success, and some of the effects of thyroid
hormone treatment to induce weight loss may also be due to uncoupling. Diet
can alter the pattern of phospholipid fatty acyl groups in the mitochondrial membrane, and this may be a route to uncoupling in vivo. Energy expenditure can be
increased by stimulating the activity of uncoupling protein 1 (UCP1) in brown
adipocytes either directly or through b3-adrenoceptor agonists. UCP2 in a number
of tissues, UCP3 in skeletal muscle and the adenine nucleotide translocase have
also been proposed as possible drug targets. Specific uncoupling of muscle or
brown adipocyte mitochondria remains an attractive target for the development
of antiobesity drugs.
MRC Dunn Human Nutrition Unit, Hills Road,
Biochemistry, University of Cambridge, 80
Tennis Court Road, Cambridge CB2 1GA, UK;
3
Knoll Ltd, Pennyfoot Street, Nottingham NG1
1GF, UK
Received 14 May 2001; revised 12 June
2001; accepted 15 June 2001
Address reprint requests to: Dr Martin Brand,
MRC Dunn Human Nutrition Unit, Hills Road,
Cambridge CB2 2XY, UK.
E-mail: martin.brand@mrc-dunn.cam.ac.uk
Keywords: Uncoupling, Mitochondria, Standard Metabolic Rate, Obesity.
obesity reviews (2001) 2, 255–265
Pharmaceutical interest in uncoupling
Obesity is a disease resulting from a prolonged positive
imbalance between energy intake and energy expenditure
resulting in the storage of fat. The rapidly increasing worldwide incidence of obesity and its association with serious
comorbid diseases means it is beginning to replace undernutrition and infectious diseases as the most significant
contributor to ill health in the developed world (1). Weight
loss, induced by dieting, has been shown to be successful
in reducing the health consequences of obesity but unfortunately >90% of individuals who lose weight through
dietary control eventually return to their original weight
(2). Pharmacological treatment may therefore be desirable
for those patients with associated comorbid conditions
who have been unable to control their obesity through diet
and exercise.
Any treatment for obesity has to reduce energy intake,
increase energy expenditure or combine both effects.
Current therapies for obesity predominantly lead to
decreased energy intake either by acting at satiety centres in the brain (e.g. sibutramine) (3–5) or by reducing
the efficiency of intestinal absorption (e.g. orlistat) (6,7).
In addition to reducing energy intake, sibutramine increases standard metabolic rate (SMR) profoundly in rodents,
but increased energy expenditure appears to be only a
minor component of its activity in humans (8,9). Exercise is the most practical and potentially easiest way
to increase energy output. Studies have shown a sevenfoldincreased risk of the incidence of overweight in those
with a physical activity ratio (total energy expenditure: resting metabolic rate) of <1.8 (10). The main
benefit of exercise is to increase resting metabolic rate,
and overall energy expenditure, by a greater amount
than that resulting directly from the exercise (11).
Pharmacological agents that increase metabolic rate by
increasing uncoupling of mitochondrial oxidative phosphorylation are likely to mimic this beneficial effect of
exercise on resting metabolic rate and could provide
a useful adjunct to agents acting to reduce satiety;
© 2001 The International Association for the Study of Obesity. obesity reviews 2, 255–265
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256 Mitochondrial uncoupling as a treatment for obesity
J. A. Harper et al.
this review will consider how such uncoupling can be
achieved.
As discussed below, uncoupling of mitochondria represents a particularly attractive target, as there is already
excellent proof of concept for this approach in humans
using 2,4-dinitrophenol (DNP) (12) and in animals using
b3-adrenoceptor agonists (13), or overexpression of UCP3
(uncoupling protein 3) (14). However, there are problems
associated with the use of chemical uncouplers like DNP.
Many of these problems may result from inappropriate
activities in critical tissues, and more selective uncoupling
would be desirable.
Skeletal muscle represents a particularly attractive
target for directed uncoupling due to the large muscle
mass, which accounts for approximately 15–20% of SMR
(15,16). The maximal aerobic capacity of a human is
generally estimated to be up to 12 times SMR in untrained
subjects (17). Most of this increase can be directly attributed to skeletal muscle respiratory activity and it is clear
that muscle can greatly increase its metabolic activity.
Doubling metabolic rate by modestly uncoupling skeletal
muscle should produce few adverse side-effects as this
increase would only be equivalent to mild exercise (actually equivalent to approximately the difference between
lying down and standing up (17)). Indeed, support for this
view has been obtained using proteins that may naturally
uncouple mitochondria (uncoupling proteins 1 and 3).
High expression of human UCP3 in mouse skeletal muscle
led to decreased weight gain despite increased food intake
(14), and expression of UCP1 in mouse skeletal muscle led
to improvements in insulin sensitivity and resistance to
obesity on a high fat diet (18).
Standard metabolic rate
Basal metabolic rate (BMR) is the minimal calorific requirement for normal life in an organism in the absence of
external stimulation, work and growth. It is measured
under rigorous conditions at thermoneutrality, in a postabsorptive state. Many physiological processes continue
in this minimal state, including ventilation and blood circulation. At the cellular level, the reactions that make up
BMR include protein turnover, ion cycling across the
plasma membrane, turnover of nucleic acids and lipids, and
proton cycling across the mitochondrial inner membrane
(uncoupling) (19,20). Resting metabolic rate (RMR) is
measured as BMR but not in the post-absorptive state (17).
These measurements are not suitable in animals (particularly ectotherms), therefore standard metabolic rate (SMR)
is used. SMR is measured under defined conditions which
are not necessarily those of BMR; in particular, temperature may vary. SMR varies considerably between species.
It is primarily dependant on body mass to approximately
the 0.75th power (21,22), so mass-specific SMR decreases
obesity reviews
as body mass increases (22,23). For example, the SMR per
gram of a mouse (body mass 0.05 kg) is approximately 10fold higher than the SMR per gram of a horse (body mass
500 kg). The field metabolic rate (FMR) is the metabolic
rate of an animal in its natural environment. Factors such
as digestion, hunting, reproduction, growth and temperature regulation increase the metabolic rate around twofold
in humans and around fourfold in other mammals (20),
irrespective of body size. In other words, around one
quarter of field energy expenditure in mammals is due to
SMR (21,24,25). The differences in mass-specific SMR
between species indicate the existence of regulatory mechanisms that may be amenable to pharmacological manipulation. Some of these differences in SMR may be caused by
differences in mitochondrial proton cycling.
What is uncoupling?
Mitochondria are normally responsible for 90% of cellular
oxygen consumption and the majority of adenosine triphosphate (ATP) production. The flow of electrons from reduced
substrate to oxygen is coupled by a proton electrochemical
gradient across the mitochondrial inner membrane to the
synthesis of ATP from adenosine diphosphate (ADP) and
phosphate (Fig. 1). This process of oxidative phosphorylation can be subdivided into two distinct parts: the generation
of the proton electrochemical gradient by the respiratory
chain and the synthesis of ATP by the Fo-F1 ATP synthase
using the potential energy stored in the gradient. However,
not all of the available energy is coupled to ATP synthesis.
Instead, much is lost by uncoupled reactions when protons
move from the cytosol back into the mitochondrial matrix
via pathways which circumvent the ATP synthase and other
uses of the electrochemical gradient. There are two sorts of
uncoupling. Basal uncoupling is not acutely regulated and is
present in all mitochondria, whereas inducible proton conductance is catalysed by proteins, tightly regulated, and
found in discrete cell types (Fig. 1).
Physiological significance of mitochondrial
proton cycling
Proton cycling is a major contributor to SMR, responsible
for around 26% of the oxygen consumed in resting rat
hepatocytes (26) and about 52% in perfused, resting rat
skeletal muscle (15). Multiplication of these values by the
contribution of each tissue to SMR suggests that proton
cycling accounts for 20–25% of rat SMR (15). This makes
proton cycling the largest single contributor to SMR.
Under field conditions, ATP turnover increases significantly and the ATP synthase competes with proton leak for
the same proton electrochemical energy. It is important to
discover whether proton cycling still represents a significant proportion of energy expenditure when ATP synthe-
© 2001 The International Association for the Study of Obesity. obesity reviews 2, 255–265
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Mitochondrial uncoupling as a treatment for obesity J. A. Harper et al.
257
Figure 1 Chemiosmotic proton circuits
across the inner membrane of isolated
mitochondria. Substrate oxidation consists of
substrate transport, substrate metabolism
and the electron transport chain, and leads
to proton pumping from the matrix to the
intermembrane space, setting up a proton
electrochemical gradient. In the lower
circuit, return of protons is coupled to
adenosine triphosphate (ATP) production via
the ATP synthase. The upper circuits show
uncoupled proton leak through the basal
leak pathway or through an inducible leak
pathway (represented by a protein, such as
an uncoupling protein (UCP)). Uncoupling
by 2,4-dinitrophenol effectively increases the
basal leak pathway whereas uncoupling by
UCPs increases the inducible pathway.
sis is increased. Rolfe and co-workers (16) showed that
when respiration rate was doubled by stimulating ureagenesis and gluconeogenesis in hepatocytes and muscle contraction in perfused rat hindquarters, the rate of proton
cycling stayed fairly constant. Of course, proton cycling
dropped as a proportion of total respiration rate, but only
to 22% in hepatocytes and 34% in muscle (16). Estimates
from these experiments suggest that proton cycling in
animals under more plausibly physiological conditions
uses around 15–20% of total energy consumption and so
remains a substantial proportion of SMR.
SMR varies with body mass, and proton cycling is an
important component of SMR. Does proton cycling change
in parallel with SMR, or does it change less or more than
SMR? Components of SMR such as the urinary excretion
of endogenous nitrogen and sulphur (27) and the rates of
respiration, circulation and renal activity (28,29) remain a
constant proportion of SMR as body mass varies. Only
relatively recently has the contribution of proton cycling to
SMR been examined in animals of different body mass.
Porter and Brand (30) found that proton conductance in
liver mitochondria decreased with increasing body mass in
mammals. However, the proportion of energy expended via
proton cycling was approximately the same in hepatocytes
from all mammals (31,32). Around 70% of the proton
conductance difference was because of less mitochondrial
inner membrane area in cells from larger animals (33). The
remaining difference was some intrinsic property of the
membrane. Because this intrinsic property can be altered
according to body mass it might be modifiable by drugs. If
proton leak could be stimulated in some way, then more
energy would be dissipated during synthesis of ATP. This
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258 Mitochondrial uncoupling as a treatment for obesity
J. A. Harper et al.
partial uncoupling would cause an increase in SMR and an
alteration in the balance between energy input and output
if energy intake did not rise to compensate. At comparable
energy intakes, a 1% change in BMR in humans would
lead to loss or gain of about 1 kg of adipose tissue per year,
so over a long period it could cause or combat obesity.
Mechanism of proton leak: passive diffusion
To consider how to increase mitochondrial uncoupling as
a means to increase BMR and reduce obesity, it would be
helpful to understand how protons re-enter the mitochondrial matrix without passing through the ATP synthase.
Diffusion of protons through the phospholipid bilayer is
one possible mechanism of basal proton conductance. To
judge the physiological importance of this process, phospholipids from mitochondria with different proton conductance were extracted and reformed into liposomes
and the proton conductance of the bilayer was examined
(34). This experiment allowed two issues to be resolved;
first, the percentage of proton conductance that could
be explained by proton diffusion across the bilayer; and
second, the effect of the phospholipid fatty acyl composition of the bilayer on the conductance. The results showed
that passive diffusion in liposomes could account for only
2.5% to 25% of the proton conductance of mitochondria
with the same phospholipid fatty acyl composition, implying that some other property of the membrane, such as the
presence of proteins, was important in determining the
conductance. There was no significant difference in proton
conductance between liposomes with very different phospholipid fatty acyl compositions, despite a known relationship between the phospholipid fatty acyl composition
of intact mitochondria and proton conductance (34).
Perhaps acyl composition is important in mitochondria,
but loss of phospholipid asymmetry or of some specific
protein causes the effect to be lost in liposomes.
Mitochondrial membranes containing a higher ratio of
polyunsaturated fatty acyl groups (PUFA) to monounsaturated fatty acyl groups (MUFA) may allow a higher molecular activity of membrane proteins, so membrane acyl
composition could affect metabolic rate. In particular,
n-3 PUFAs have been strongly linked to SMR (35). As
described above, mammals with a smaller body mass have
higher mass-specific SMR and higher mitochondrial proton
conductance than larger mammals. Surface area and fatty
acyl composition of mitochondria both vary with body
mass (33,35), so either factor could affect proton conductance. Around two-thirds of the difference in proton conductance of mitochondria from mammals of different body
mass is due to the amount of mitochondrial membrane, and
one-third is due to some difference (protein or phospholipid) in membrane composition (33). Dietary fatty acids
can alter the fatty acyl composition of the mitochondrial
obesity reviews
inner membrane (36), and it is conceivable that diet, or
pharmaceutical agents that alter the PUFA : MUFA ratio
of fatty acyl groups in the membrane, could affect mitochondrial uncoupling and hence modify BMR and weight
gain. For example, fish oil high in n-3 PUFA has been suggested to limit obesity (37,38). Oils that contain high levels
of n-3 fatty acids and purport to be beneficial are already
on sale, although evidence that they effect weight loss is
lacking (39).
Artificial uncoupling by dinitrophenol
Uncoupling of mitochondria as a treatment for obesity is not
unprecedented; the artificial uncoupler 2,4-dinitrophenol
(DNP) has been used for this purpose for many years
(12,40). DNP is a lipid-soluble weak acid which acts as a
protonophore because it can cross membranes protonated,
lose its proton and return as the anion, then reprotonate
and repeat the cycle. In this way, it increases the basal
proton conductance of mitochondria and uncouples. The
effect of DNP derivatives was first noted in 1885 when
scientists saw thermogenic effects of Martius Yellow (a
dinitro-alpha-napthol), a coal tar dye used in the 19th
century to give the impression that food was rich in eggs
(41).
DNP was introduced as a drug in the 1930s and used
with considerable success, though reports of side-effects
(cataracts) and some deaths from overdose led to it being
chased off the market by the US Food and Drug Administration (FDA) in 1938. This use of DNP to treat obesity
was stimulated by observations of its toxicity in French
munitions workers during World War I (the French commonly used a mixture of 40% dinitrophenol and 60%
trinitrophenol for their munitions). In animals, it was
shown that the drug promoted a direct stimulation of cellular respiration and a consequent rise in body temperature. It led to the almost immediate onset of rigor mortis
when death was promoted by large doses (42,43).
A series of controlled trials in obese patients were
prompted by these promising mode of action studies. The
most extensive were carried out during the 1930s at Stanford University, CA, USA (44–47), though others also performed careful studies (48). Interpretation of this work can
be complicated, as the doses of DNP had to be optimized
for each patient (due to the steep dose response and patient
to patient variability) and were usually increased as the
studies progressed. This means the DNP-sensitive patients
on low doses of the drug would show enhanced efficacy
compared with the population (and vice versa for patients
on high does of DNP). However, there was a clear dependence of metabolic rate on DNP dose (Fig. 2). There was
an average 11% increase in metabolic rate for each dosage
increment of 0.1 g of DNP (44,48). Doses up to 0.5 g
(about 5 mg kg-1) were generally well-tolerated apart from
© 2001 The International Association for the Study of Obesity. obesity reviews 2, 255–265
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Mitochondrial uncoupling as a treatment for obesity J. A. Harper et al.
Figure 2 Effects of dinitrophenol on metabolic rate and weight loss in
humans. Replotted from data given in (44).
patients almost always reporting a feeling of warmth
together with increased perspiration (45,46,48). Between
about 5–10 mg kg-1, patients reported profuse sweating
but, surprisingly, there was no evidence of increased body
temperature or heart rate. Doses above 10 mg kg-1 led to
increased heart rate, respiration rate and excessive body
temperature rises (45). Single doses of 3–5 mg kg-1 produced an increase in resting metabolic rate of 20–30%
within the first hour. This was maintained for 24 h after
which a gradual fall became apparent. Daily dosing of
3–5 mg kg-1 produced a gradual increase of efficacy: an
average 40% increase in metabolic rate was observed after
a few weeks that was then maintained, with no sign of tolerance, for at least 10 weeks (45).
Weight loss in these studies, where no attempt was made
to control diet, was reported to be variable. Of 170 treated
patients, a mean of 7.8 kg weight loss was recorded, averaging 0.64 kg week-1 (44) (Fig. 2). There were no clearly
reported effects on food consumption. In contrast to the
use of thyroid extract (also in common use at the time
to treat obesity), DNP did not promote urinary nitrogen
excretion, so the assumption was made that weight loss
could be attributed to a specific loss of fat (47). One potential complication in the interpretation of weight loss data
is the ability of the drug to promote oedema. This effect
was reported to account for a common apparent failure to
continue to lose weight in the face of a maintained increase
in metabolic rate (48). For some patients, withdrawing
DNP led to a rapid weight loss that was attributed to loss
of excess body water after which DNP dosing could be
resumed with its former effectiveness. Therapeutic doses of
DNP had no effects on blood pressure or heart rate in
normal patients. Interestingly, a subset of 30 hypertensive
patients exhibited average falls of 9.4% in systolic and
12.6% in diastolic blood pressure (44). These improvements in hypertension were also noted at doses of DNP
insufficient to cause weight loss (48). Some studies were
also performed in diabetic patients with inconsistent results
259
but there did appear to be improvements in glucose tolerance after long-term dosing (48). The reported potency of
DNP to treat obesity (and associated comorbid conditions)
in these early trials compares well with current treatments
for obesity (3,4,6).
This ability of dinitrophenol to produce good reductions
in body weight, without the need for dietary restriction, led
to its widespread use to treat obesity. In the absence of
formal regulatory controls it is not surprising that it was
soon prescribed by inexperienced physicians with no access
to the metabolic rate measurements necessary to determine
optimal doses. DNP was even included in a variety of
‘antifat nostrums’ that could be used by the public without
medical consultation. By 1934 it was estimated that a total
of 100 000 patients had been treated (40).
Given the steep dose dependence of metabolic rate and
the widespread use of DNP it is perhaps not surprising that
a number of people were ‘literally cooked to death’ in the
1930s due to accidental or deliberate overdose (40). It was
argued at the time that the reported deleterious effects of
DNP were remarkably few (when given at the correct therapeutic doses), given the large number of patients who had
taken it. The authors did, however, note a 7% incidence of
severe skin rashes, necessitating discontinuation of treatment. There was no overt liver or kidney damage, but the
same authors voiced some concerns about the incidence of
agranulocytosis though they saw no cases in their own
long-term studies (44). Of more concern, a number of cases
of cataracts were reported in women in 1935 (49,50). In
1938 the FDA acquired more powers to prosecute manufacturers of misbranded therapies and announced that the
use of a variety of patent medicines (including DNP) could
lead to prosecution (12). These threats led to a withdrawal,
in 1938, of the DNP-containing nostrums from the market
as well as an end to the official clinical use of DNP. Interestingly, however, there are reports on the Internet that
describe the use of DNP by US clinics (who avoided illegal
interstate transportation of DNP for human use by synthesizing it within each state), and give detailed protocols
for its use amongst the designer drug community of bodybuilders and those willing to risk self-administration of
DNP to lower body mass.
Given the age of the publications describing the mode
of action in rodents, one of us (KD) recently re-evaluated
the effects of DNP in rats and confirmed the relative insensitivity of this species and the particularly steep dose
response to increase metabolic rate. In contrast to humans,
DNP did not significantly increase metabolic rate in rats
when dosed orally at 10 mg kg-1 (data not shown) but a
good, comparatively long lasting, increase was seen at
30 mg kg-1 that was comparable in magnitude to a rodent
b3-adrenoceptor agonist (Fig. 3) (51). Doses of 100 mg kg-1
produced an escalating hyperthermia that necessitated
killing the animals to avoid distress.
© 2001 The International Association for the Study of Obesity. obesity reviews 2, 255–265
260 Mitochondrial uncoupling as a treatment for obesity
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J. A. Harper et al.
Figure 3 Effect of dinitrophenol (30 mg kg-1)
and the b3-adrenoceptor agonist ZD7114
(1 mg kg-1) on oxygen consumption in female
Wistar rats (mean ± SEM, n = 8). Oxygen
consumption was measured using indirect
closed circuit calorimetry at the thermoneutral
temperature of the rat (29°C). Animals were
temporarily removed from the chambers for
oral dosing with drugs or vehicle at 100 min
(51).
Thyroid hormones
Thyroid hormones have a long history in the treatment
of obesity (52) and are positively correlated with BMR
(53,54). At the cellular level, hepatocytes isolated from
hyperthyroid rats have twice the respiration rate of euthyroid controls. About half of the increase is caused by
increased mitochondrial proton cycling, which is partly
due to greater proton conductance and partly to a greater
driving force (proton electrochemical gradient) (55). At the
subcellular level, mitochondria prepared from the liver of
hyperthyroid animals have increased proton permeability
compared with those from euthyroid animals (56,57).
In the 1950s the respiratory stimulation in isolated
mitochondria was attributed to direct uncoupling by thyroid hormones using the same mechanism as DNP (58).
However, this effect only occurred in vitro with supraphysiological concentrations of hormones (59). There are
now two main hypotheses for the stimulation of respiration and proton conductance in mitochondria and cells by
thyroid hormones (60).
In the first hypothesis, thyroid hormones act directly at
the membrane level, rigidifying the bilayer and leading to
a compensatory change in the fatty acyl composition of the
phospholipids. Alternatively, thyroid hormones could alter
membrane composition through transcriptional regulation
of desaturases and other enzymes. In either case, the change
in phospholipid fatty acyl composition alters the proton
conductance and degree of uncoupling of the mitochondrial inner membrane. In support of this hypothesis, the
polyunsaturation of mitochondrial phospholipid acyl
chains increases in hyperthyroidism (36,60,61), and liver
mitochondrial proton conductance correlates with polyunsaturation of mitochondrial phospholipid acyl chains
(33,62). Hyperthyroid mitochondria also have less cholesterol and increased cardiolipin (63). Changes in the area of
mitochondrial inner membrane are also implicated in the
change in measured proton conductance (57).
In the second hypothesis, thyroid hormones act through
transcriptional regulation and affect proton conductance
through expression of specific proteins (64). For example,
the levels of UCP2 and UCP3 mRNA increase in response
to thyroid treatment (65–69).
Supra-physiological doses of thyroid hormones are
no longer used in the treatment of obesity because of
unwanted side-effects such as tachycardia, increased heart
weight (70,71), thyroid atrophy (72) and a negative nitrogen balance, that is a loss of lean body mass (muscle)
(73,74). They may cause loss of water and muscle rather
than loss of fat and adipose tissue.
Inducible uncoupling catalysed by
specific proteins
Uncoupling protein 1
The ability of hibernators, cold-adapted rodents and
new-born mammals to produce heat and increase SMR by
non-shivering thermogenesis results mostly or only from
the activity of uncoupling protein 1 (UCP1) in mitochondria in brown adipose tissue (BAT) (75,76). The identification of UCP1 as the mediator of uncoupling followed from
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Mitochondrial uncoupling as a treatment for obesity J. A. Harper et al.
the observation of greater uncoupling of BAT mitochondria
than of liver and heart mitochondria (77,78). Importantly,
this increased uncoupling was abolished by albumin and
by purine nucleotides, indicating that it could be regulated
in vivo by free fatty acids and nucleotides (79). The ability
of UCP1 to transport protons was demonstrated by mitochondrial swelling experiments (77,79). A 32-kDa protein
was observed to be specifically and highly expressed in BAT
mitochondria. Photo-affinity labelling identified a 32-kDa
protein as the binding site of purine nucleotides and a putative uncoupling protein was proposed (80). Subsequently,
the 32 kDa protein was purified from hamster and rat
(81,82). The cDNA was sequenced and cloned and the
protein’s proton translocating activity was demonstrated in
transgenic yeast mitochondria (83) and liposomes (84–87).
After these experiments it was generally accepted that
UCP1 was responsible for the regulated uncoupling of BAT.
Further experiments were performed with transgenic
mice (88). A mouse model in which all UCP1 had been
removed by homologous recombination was created by
Enerbäck and co-workers (89). These UCP1-deficient mice
were more sensitive to cold and showed a greatly reduced
effect of b3-adrenoreceptor agonists, indicating reduced
thermogenic capacity. However, when they were fed
normal or high-fat diets they were not obese. There was an
increase in the adiposity of the BAT, as expected if it did
not have to oxidize fatty acids as fast to maintain a normal
proton electrochemical gradient. Interestingly, other studies
in transgenic mice using a diphtheria toxin gene linked
to a UCP1 promoter to genetically ablate BAT did show
obesity linked to loss of BAT activity (90). As the obesity
in this UCP1-DTA model seemed to result predominantly
from hyperphagia it appears that the differences between
the two models resides in other (non-thermogenic) activities of BAT. Other studies using targeted expression of
UCP1 to mouse muscle (18) or white fat (91) demonstrated
a resistance to the development of obesity and an improvement in comorbid conditions consistent with increased
uncoupling activities in these tissues.
b3-Adrenergic receptor agonists
There has been considerable interest within the pharmaceutical industry in developing specific b3-adrenoceptor
agonists that would selectively lead to the activation of
uncoupling through UCP1 in brown fat. The ability to
selectively uncouple should avoid many of the side-effects
that might occur with DNP. The activity of the b3-adrenoceptor to activate lipolysis in both white and brown fat
leading to a consequent activation of UCP1 mediated lipolysis in brown fat is now well understood (92–94). Numerous studies in rodents have shown that b3-adrenoceptor
agonists produce profound improvements in insulin sensitivity in a number of diabetic animal models and lead to
261
substantial weight loss due to selective fat loss (13,95).
These rodent studies provide excellent evidence that activation of uncoupling by brown fat represents an important
potential human therapeutic target. Unfortunately, clinical
studies have yet to provide proof of concept in humans,
though clinical trials are still in progress. It has proved particularly difficult to develop b3-adrenoceptor agonists that
lack activity against b1 and b2-adrenoceptors (13,95). This
difficulty, coupled with the very low levels of brown fat in
adult humans has made the results of the various clinical
trials difficult to interpret. However, if b3-adrenoceptor
agonists prove capable of reactivating dormant brown fat,
or if conversion of white to brown adipose tissue becomes
possible, then a suitably selective agent may show relevant
activity in humans.
Uncoupling proteins 2 and 3
UCP1 is restricted to brown adipocytes, and was originally
thought to be the only transporter capable of uncoupling
mitochondria (96–98). However, UCP1 probes occasionally gave weak signals in other tissues (93), suggesting the
presence of homologues. UCP2 was discovered because of
its relatively high sequence identity to UCP1 (59%). UCP2
mRNA has been found in many tissues, including cardiac
muscle, BAT, white adipose tissue, skeletal muscle, kidney,
non-parenchymal liver cells, lung, placenta, pancreatic b
cells and the immune system. However, mRNA expression
does not necessarily imply protein expression, as UCP2
protein was not detected in heart, skeletal muscle, liver or
BAT (99). UCP3 was also discovered by database searching. Human UCP3 is 59% identical to human UCP1 and
72% identical to human UCP2. UCP3 is restricted to BAT
and skeletal muscle (100), both tissues that make a major
contribution to thermogenesis, making it a more enticing
target for drug development than UCP2.
Evidence that UCP2 and UCP3 are responsible for basal
proton conductance comes from experiments employing
high expression of mammalian UCP2 and UCP3 in yeast
(101–103) and transgenic overexpression of human UCP3
in mice (14). Such high expression lowers mitochondrial
membrane potential and increases state 4 respiration, indicating uncoupling. It inhibits the growth of yeast and
protects against obesity in hyperphagic mice. However,
the normal concentrations of UCP2 (99) and UCP3
(104,105)protein expressed in mammalian tissues are very
low. The amounts expressed in transgenic yeast (104) or
UCP3-overexpressing mice (14) are strongly supra-physiological and there is good evidence that the uncoupling is an
artefact not related to the physiological functions of UCP2
and UCP3 (105,106) (S. Cadenas et al. unpublished data; J.
A. Harper et al. unpublished data). Proteoliposomes have
also been used in studies of the function and regulation of
UCP1 and its homologues (107–110). These studies found
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262 Mitochondrial uncoupling as a treatment for obesity
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different nucleotide sensitivities and low turnover numbers,
but they did identify ubiquinone as an essential cofactor for
uncoupling in vitro by UCP1 and its homologues (111,112).
The UCPs have been knocked out in mice, avoiding interpretational problems resulting from the requirement for
overexpressed protein to be correctly inserted and folded
in membrane. Two studies (113,114) found that UCP2
mRNA was increased in BAT of UCP1 knockout mice with
no increased uncoupling. However, UCP2 protein was not
measured and may not have changed. The proton conductance of skeletal muscle mitochondria from UCP3 knockout mice was decreased (115,116), indicating that UCP3 is
responsible for at least some of the proton conductance in
skeletal muscle. However, we have not been able to confirm
these observations (S. Cadenas et al. unpublished data).
Despite the reported effects in genetically modified mice,
natural changes in UCP2 and UCP3 mRNA and UCP3
protein levels do not alter mitochondrial proton conductance. For example, UCP2 and UCP3 mRNA increase in
muscle from starved rats (117) despite a known depression
in thermogenesis (118). UCP3 mRNA increases fourfold
and UCP3 protein doubles but there is no change in the
mitochondrial proton conductance (119).
Taken together, the evidence suggests that UCP1, UCP2
and UCP3 are not responsible for the basal proton conductance of mitochondria. Clearly, UCP1 causes inducible
uncoupling in BAT mitochondria, and UCP2 and UCP3
may yet be found to catalyse a similar uncoupling that is
induced by unidentified agonists. The UCPs remain important potential targets for specific pharmacological uncoupling as a treatment for obesity. Other members of the
mitochondrial carrier family have also been implicated in
the uncoupling of mitochondria. In particular, the adenine
nucleotide transporter is responsible for uncoupling by free
fatty acids (120,121) and AMP (122). These carriers may
also be potential drug targets.
Is the uncoupling of mitochondria a viable
target for drug development?
Uncoupling mitochondria is an effective way to increase
thermogenesis and basal metabolic rate and it can lead
to a substantial reduction in body weight by loss of fat
deposits. This has been shown in humans taking DNP
orally and in transgenic mice overexpressing human UCP3
or UCP1 in muscle. Proof of concept has therefore been
unambiguously established, showing that uncoupling can
increase energy expenditure without compensatory mechanisms increasing food intake to the extent that they nullify
the uncoupling.
However, using pharmacological agents to uncouple
all mitochondria throughout the body may be a high-risk
treatment, because it might compromise energy homeostasis in tissues such as heart and brain. On the other hand,
obesity reviews
active tissues like these may be less susceptible to mild
uncoupling than less active ones like resting muscle or
resting BAT because proton conductance has much less
control over respiration rate in active mitochondria (123).
The small difference between the effective and the fatal
doses of DNP, as well as side-effects resulting from its nonselective actions, mean that it is not itself a suitable antiobesity drug. Tissue selectivity and safety need to be improved.
The UCP3-overexpressing mouse shows that selective
uncoupling of muscle mitochondria is sufficient for a strong
anti-obesity effect. Specific uncoupling of brown adipose
tissue mitochondria through UCP1 or of muscle mitochondria through UCP3 or in some other way remains a
viable and attractive target for the development of drugs
for the treatment of obesity.
Acknowledgements
JAH was supported by a BBSRC CASE award with Knoll
Ltd.
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