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Bioscience Reports, Vol. 17, No. 1, 1997
REVIEW
The Physiological Significance of
Mitochondrial Proton Leak in Animal Cells
and Tissues
David F. S. Rolfe1 and Martin D. Brand1-2
Received November 1, 1996
Mitochondrial proton leak is an important component of cellular metabolism in animals and may
account for as much as one quarter to one third of the Standard Metabolic Rate of the rat. The
activity of the proton leak pathway is different in a wide range of animal species and in different
thyroid states. Such differences imply some function for proton leak and candidates for this function
include thermogenesis, protection against reactive oxygen species, endowment of metabolic sensitivity
and maintenance of carbon fluxes.
KEY WORDS: Mitochondria; proton leak; Standard Metabolic Rate; thermogenesis; metabolic
sensitivity; reactive oxygen species; carbon flux control.
1. INTRODUCTION
Not all mitochondrial oxygen consumption is coupled to ATP synthesis. The inner
membrane of mitochondria isolated from the major oxygen consuming organs of
the rat (Rolfe et al, 1994) has a significant passive leak for protons (termed
"proton leak"). Proton leak is not an artefact of mitochondrial isolation since it
has been demonstrated in mitochondria within isolated hepatocytes (Nobes et al.,
1990; Brown et al., 1990; Harper & Brand, 1993; Brand et al., 1993), thymocytes
(Buttgereit et al., 1992), lymphocytes (Buttgereit et al., 1991), perfused skeletal
muscle (Rolfe & Brand, 1996) and intact heart (Challoner, 1968). This review
summarises research on the significance of the proton leak pathway to the
Standard Metabolic Rate of the adult rat and on the differences in proton leak in
a wide range of animal species. It ends with a discussion of the possible functions
of this pathway including a possible role for proton leak in controlling the
production of reactive oxygen species.
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW,
United Kingdom.
2 To whom correspondence should be addressed.
1
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0144-8463/97/020O0009$ 12.50/0© 1997 Plenum Publishing Corporation
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Rolfe and Brand
2. MEASUREMENT OF PROTON LEAK IN CELLS AND TISSUES
There is evidence that mitochondrial oxygen consumption and proton
pumping are obligatorily coupled under physiological conditions (Brown, 1989;
Zolkiewska et al., 1989; Hafner & Brand, 1991; Brand et al., 1994a, 1994b;
Kesseler & Brand, 1995; Porter & Brand, 1995a), so oxygen consumption in the
absence of phosphorylation may be used as an indirect measure of proton leak
activity (Nicholls, 1974). Inhibition of oxidative ATP production will result in a
drop in the oxygen consumption rate and an increase in the mitochondrial
membrane potential. Because of the strong dependence of the mitochondrial
proton leak on membrane potential the leak will increase, so measurement of
oxygen consumption after complete inhibition of oxidative phosphorylation will
result in an overestimate of the proton leak flux. For an accurate measurement,
the mitochondrial potential must be returned to its resting value by slight
inhibition of the mitochondrial respiratory chain. The remaining oxygen consumption is a measure of the original oxygen consumption not coupled to ATP
synthesis and may include a significant contribution from non-mitochondrial
oxygen consuming reactions. This non-mitochondrial oxygen consumption can be
determined by completely inhibiting mitochondrial respiration, and subtracted to
allow the original proton leak flux to be calculated.
3. CONTRIBUTION OF PROTON LEAK TO RAT STANDARD
METABOLIC RATE (SMR)
Experiments (of the type described above) to estimate the proton leak rate
have been carried out using isolated rat liver cells and perfused rat skeletal
muscle, and indicate that proton leak accounts for about 26% of oxygen
consumption in resting rat liver cells (Nobes et al., 1990; Brown et al., 1990;
Harper & Brand, 1993; Brand et al, 1993; reviewed in Brand et al., 1994a) and
about 52% in resting rat skeletal muscle (Brand et al, 1994; Rolfe & Brand,
1996). The experiments of Challoner (1968) provide an upper limit estimate of
12% for the contribution of proton leak to the respiration rate of the intact,
beating heart.
Estimates of the contribution to rat SMR of skeletal muscle range from
13-42% (mean 30%), and estimates of the contribution of liver range from
10-20% (mean 15%) (see Rolfe & Brand, 1996). The contribution of heart is
around 3% (Jansky, 1965). From these values and those in the paragraph above,
the contribution of proton leak in liver, skeletal muscle and heart to rat SMR can
be estimated to be, on average, 20% (30% of 52% + 15% of 26% + 3% of 12%)
with a range of 10% to 27%. The contribution of proton leak flux in other tissues
has not been estimated, although the proton leak of mitochondria isolated from
brain and kidney has similar characteristics to that found in isolated liver
The significance of mitochondrial proton leak
11
mitochondria (Rolfe et al, 1994). If the contribution of proton leak to the
respiration rate of these organs is similar to that of liver cells, then the
contribution of proton leak to the SMR of a rat would be 25% (range 15-33%).
The assays of mitochondrial proton leak mentioned above were conducted
with resting liver cells and muscle so they may give an upper limit to proton leak
activity in the intact animal. Skeletal muscle and liver may have a greater ATP
demand in the standard state in vivo than in vitro, for example due to muscular
contraction and additional liver work functions. However, the oxygen consumption rate of isolated hepatocytes may be scaled to allow comparison with the in
vivo rates in whole liver (Berry et al., 1991). The respiration rate of whole liver in
vivo is 2.84 /z,mol O 2 /min/g liver (Jansky, 1965). The respiration rates of resting
hepatocytes isolated from fed rats incubated at 37°C in Krebs-Henseleit bicarbonate buffer are between 33 and 79% (mean 58%) of this figure (Rabkin & Blum,
1985; Porter & Brand, 1995b; Nobes et al., 1990; Brown et al., 1990; Harper &
Brand, 1993). Similarly the respiration rates of perfused muscle preparations are
within the same range as estimates of the oxygen consumption rate of skeletal
muscle in vivo. These calculations suggest that resting cells and tissues in vitro
may not be very different in respiration rate from the corresponding tissues in
vivo. In support of this, proton leak still accounts for 20% of liver cell respiration
rate even when the measurements are made at high rates of gluconeogenesis
(Price, S. J. & Brand, M. D., unpublished observations). Thus perfused muscle
and liver cells may be reasonable models for the whole tissue in vivo, and the
experiments outlined above show that proton leak may be an important
contributor to standard metabolic rate in rats.
4. PROTON LEAK IN ANIMALS WITH HIGHER OR LOWER
STANDARD METABOLIC RATES
Proton leak appears to be a significant contributor to the SMR of a rat. Is
proton leak equally important in determining the metabolic rate of the tissues of
other animals? This question has been addressed by measuring the proton leak of
liver mitochondria and the contribution of proton leak to the respiration rate of
liver cells isolated from a number of animal species of different body mass and
phylogeny. These species show marked differences in their Standard Metabolic
Rate—for example the mass-specific metabolic rate (rate per unit mass) of a 20 g
mouse is roughly 10-fold greater than that of a 500 kg horse and a rat has a roughly
5-fold higher standard metabolic rate than does a lizard of the same body mass at
the same temperature. Liver mitochondria were isolated from these species and
their proton permeabilities were determined. The contribution of proton leak to
the respiration rate of liver cells isolated from these animals was also measured. If
the proton leak activity of the isolated mitochondria showed a positive correlation
with the oxygen consumption rate of the isolated cells this would indicate that
proton leak was a variable property of the mitochondrial inner membrane and
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Rolfe and Brand
that it had some important function. If the leak had no function, it might be
expected to be low and constant in mitochondria from all species.
4.1. Proton Leak in Mammals of Different Body Mass
The proton permeability of liver mitochondria isolated from mammals of
widely ranging body mass (20 g - 750 kg) is different (Porter & Brand, 1993). In
general, the smaller the mammal, the higher the proton permeability of its
isolated mitochondria. The same is true of the respiration rate of isolated liver
cells: smaller animals yield cells with faster respiration rates (Porter & Brand,
1995b). However, the proportion of hepatocyte respiration used to drive proton
leak (around 20%). ATP turnover (around 65%) and non-mitochondrial respiration (around 15%) showed no dependence on body mass (Porter & Brand,
1995c). It therefore appears that the activity of the proton leak pathway is
different in different animals but a balance is maintained between proton leak and
the other cellular pathways driven by oxygen consumption.
4.2. Proton Leak and Phytogeny
The Standard Metabolic Rate of a homeotherm (rat) is roughly 5-fold higher
than that of a poikilotherm (lizard) of the same body mass at the same
temperature, and the proton permeability of the isolated liver mitochondria is 4-5
fold greater in the homeotherm (Brand et al, 1991). A comparison of the isolated
liver cells of the rat and the lizard has shown that although the cells of the
homeotherm had a 4-fold greater respiration rate, the proportions of oxygen
consumption attributed to non-mitochondrial oxygen use, the mitochondrial
proton leak, and total ATP turnover are similar in the two cell types (Brand et al.,
1991).
The studies in sections 4.1 and 4.2 indicate that a balance between the rates
of the various cellular processes is maintained even though the metabolic rate of
the cells differs widely. This balance would preserve a particular pattern of the
distribution of control over oxidative metabolism. Analysis of the distribution of
control over respiration in mitochondria isolated from the livers of different
animals (from data in Porter & Brand, 1995c), from different tissues of the same
animal (Rolfe et al., 1994) and even from plants (Kesseler et al., 1992; Diolez et
al., 1993) indicate that the distribution of control over mitochondrial respiration
(and other variables) is a conserved property in different tissues and species. This
might be taken to indicate the importance of this pattern of control. The need to
conserve a certain control pattern presumably reflects the importance of certain
key points in a pathway that are subject to in vivo regulation, in order to effect
short-term changes in metabolic rate which are produced by alteration of the
activity of only some points in a metabolic pathway. Proton leak has significant
control over the mitochondrial respiration rate of resting liver cells (22-25% of
the control; Brown et al., 1990, Harper & Brand, 1993) and resting skeletal
The significance of mitochondria] proton leak
13
muscle (38% of the control; Rolfe & Brand, 1996) and maintenance of the
balance between proton leak and other metabolic pathways may therefore
indicate some function for the leak, rather than it being the unfortunate and
unwanted consequence of having a membrane with a very high protein content.
5. MITOCHONDRIAL PROTON PERMEABILITY AND LEAK FLUX
DEPENDS ON THYROID HORMONES
Liver cells isolated from hypothyroid rats have a 15% lower respiration rate
compared to those taken from euthyroid controls (Harper & Brand, 1993).
Hepatocytes taken from hyperthyroid rats have a 2-fold greater respiration rate
compared to euthyroid controls (Harper & Brand, 1993). The proton permeability of mitochondria isolated from liver is 7-fold greater in hyperthyroid rats
compared with those from hypothyroid animals (Hafner el al., 1988).
The lower oxygen consumption rates of liver cells isolated from hypothyroid
rats compared with euthyroid controls were the result of reductions in the rates of
non-mitochondrial respiration (-31%) and mitochondrial proton leak (-24%),
whilst the ATP turnover rate remained unaltered. In liver cells isolated from
hyperthyroid rats, the increase in oxygen consumption in comparison with
euthyroid controls resulted from a stimulation of proton leak flux (+100%) and
processes contributing to ATP turnover (+75%), whilst the non-mitochondrial
oxygen consumption rate remained unaltered (Harper & Brand, 1993). The
differences in proton leak rates were caused partly by differences in the activity of
the leak pathway and partly by differences in the driving force (Ap).
These results indicate that proton leak is dependent on thyroid status. If
thyroid hormone was dominant in controlling proton leak activity, one might
expect a positive correlation between the proton permeability of liver mitochondria and the thyroid activity of the animals they were isolated from. The total (i.e.
bound + free) serum T4 level in reptiles (Hulbert & Williams, 1988) is much
lower than it is in mammals (Larsson et al., 1985) and this correlates with the
difference in the proton permeability of mitochondria isolated from the rat and
the lizard (section 4.2). However, the total T4 levels of mammals (Larsson et al.,
1985) do not correlate with body mass even though the proton permeability of
liver mitochondria isolated from these mammals does (section 4.1). Thus, unless
some other explanation is invoked (e.g. small mammals have a greater ratio of
free: bound thyroid hormones or have greater tissue sensitivity to a given level of
hormones) it appears that differences in thyroid hormone levels are not the
principal cause of the differences in proton permeability seen in mammals of
different body mass.
Nevertheless, thyroid hormone is generally considered (Girardier, 1977) to
be the main hormone controlling obligatory thermogenesis (i.e. Standard Metabolic rate). The results quoted in this section indicate that mitochondrial proton
leak flux depends on thyroid hormone and this therefore indicates a potential role
for proton leak in obligatory thermogenesis. This, and other possible roles for
proton leak are discussed overleaf.
Rolfe and Brand
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6. FUNCTIONS OF MITOCHONDRIAL PROTON LEAK
Several different functions could be suggested for proton leak. These include:
(1) production of heat to maintain body temperature, (2) endowment of increased
sensitivity of key metabolic reactions to effectors, (3) reduction of harmful free
radical production and (4) regulation of carbon flux.
6.1. Proton Leak as a Means of Heat Production
It is possible that the mitochondrial proton leak flux in mammals is
determined by the necessity of maintaining a constant body temperature, and thus
a rate of heat production which matches the rate of heat loss. Mammals need to
maintain a constant body temperature of between 33°C and 39°C, and at
thermoneutral environmental temperatures the required heat is provided by
standard metabolism. The fact that the proton leak activity of mitochondria
isolated from a poikilotherm is much lower than that of a homeotherm of
equivalent body size and preferred body temperature might indicate that proton
leak is involved in thermoregulation. However, the fact that the balance between
leak and other oxygen consuming processes is the same in liver cells isolated from
both the rat and the lizard indicates that heat production is not the only, or even
the primary function of proton leak.
6.2. Proton Leak Increases the Potential for Regulation
The presence of futile cycles (coupling and uncoupling processes) in cellular
metabolism confers on metabolism: (a) decreased transition times to new steady
states, (b) a higher sensitivity to effectors, and (c) more sites at which control is
exerted, as first proposed for substrate cycles by Newsholme and Underwood
(1966). High activity of the processes producing and consuming an intermediate
relative to the net rate of production also means that relatively small changes in
the production or consumption result in large changes in the net rate, and thus
increase the sensitivity of control. Mitochondrial proton leak, which uncouples
mitochondrial ATP synthesis, may increase sensitivity and decrease response time
to changes in ATP utilisation in the cell. Thus proton leak may provide a
mechanism for increasing the sensitivity and rate of response of oxidative
phosphorylation to effectors.
6.3. Proton Leak as a Means of Reducing Harmful Free Radical Production
A major function of proton leak might be to decrease the production of
harmful free radicals, either by the mitochondria themselves, or by nonmitochondrial processes (Skulachev, 1996). Mitochondria are a major source of
cellular superoxide and hydrogen peroxide (Halliwell, 1992). The rate of
generation of superoxide free radicals is related to the concentration of oxygen
within the cell (Halliwell & Gutteridge, 1989) and the degree of reduction of the
species which donate electrons to O2 to form superoxide, which in mitochondria
The significance of mitochondrial proton leak
15
appear to be the semiquinone of the Q cycle (Turrens et al, 1985) and possibly
also complex I (Ambrosio et al, 1993). One might therefore suppose that
production of reactive oxygen species would become most problematic in the
resting cell, since such a cell would consume less oxygen and have a higher
concentration of reduced electron-transport-chain components which could donate an electron to oxygen to form superoxide. This might be overcome by
maintaining a relatively high mitochondrial proton permeability which would (a)
decrease tissue oxygen levels, and (b) decrease the reduction level of the
mitochondrial respiratory chain. This model could explain why oxygen consumption to derive the proton leak is always a constant proportion of liver cell
respiration rate.
6.4. Regulation of Carbon Fluxes by Proton Leak
Under certain conditions (e.g. in the resting cell or tissue) levels of reduced
cofactors such as NADH may rise. This rise may compromise the biosynthesis of
certain compounds, for example the synthesis of essential amino acids from
oxaloacetate. Oxaloacetate production (and hence synthesis of amino acids from
oxaloacetate) requires NAD*. Similarly, ketogenesis requires continued oxidation of fatty acids under conditions where little of the NADH generated is needed
for ATP production. A possible function for proton leak in these cases might
therefore be to keep the cell NAD + /NADH ratio sufficiently high to allow the
required flow of carbon to continue.
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