1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5; and 2 Pennington Biomedical Research Center, Baton Rouge, Lousiana 70808
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ABSTRACT |
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Mice deficient in mitochondrial uncoupling protein (UCP) 1 are cold sensitive, despite abundant expression of the homologues, Ucp2 and Ucp3 (S. Enerbäck, A. Jacobsson, E. M. Simpson, C. Guerra, H. Yamashita, M.-E. Harper, and L. P. Kozak. Nature 387: 90-94, 1997). We have analyzed characteristics of mitochondrial proton leak from brown adipose tissue (BAT) of Ucp1-deficient mice and normal controls and conducted the first top-down metabolic control analysis of oxidative phosphorylation in BAT mitochondria. Because purine nucleotides inhibit UCP1 and because UCP2 and the long form of UCP3 have putative purine nucleotide-binding regions, we predicted that proton leak in BAT mitochondria from Ucp1-deficient mice would be sensitive to GDP. On the contrary, although control over mitochondrial oxygen consumption and proton leak reactions at state 4 are strongly affected by 1 mM GDP in mitochondria from normal mice, there is no effect in UCP1-deficient mitochondria. In the presence of GDP, the overall kinetics of proton leak were not significantly different between Ucp1-deficient mice and controls. In its absence, state 4 respiration in normal BAT mitochondria was double that in its presence. Leak-dependent oxygen consumption was higher over a range of membrane potentials in its absence than in its presence. Thus proton leak, potentially including that through UCP2 and UCP3, is GDP insensitive. However, our measurements were made in the presence of albumin and may not allow for the detection of any fatty acid-induced UCP-mediated leak; this possibility requires investigation.
oxidative phosphorylation; uncoupling protein(s); thermogenesis
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INTRODUCTION |
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THE FINDING THAT MICE having gene-targeted inactivation
of uncoupling protein (UCP) 1 are lean and become no more obese on a
high-fat diet than do controls on the same diet was unexpected (16). It
was anticipated that the
Ucp1-deficient mice would be obese,
based on the well-recognized importance of brown adipose tissue (BAT)
toward energy balance and the development of obesity in rodents (30,
35, 38). Ucp1-deficient mice are,
however, characterized by a cold-sensitive phenotype, and they have an abnormally low response in resting metabolic rate to acute treatments with the well-studied
3-adrenergic agonist CL-316,243
(16).
To assess the metabolic significance and control of proton leak in BAT mitochondria from Ucp1-deficient mice and controls, we have used top-down metabolic control analysis (11, 22) and its extension, top-down elasticity analysis (5, 8, 28). Mitochondrial proton leak in tissues other than BAT has been studied for over 10 years, and its possible role in establishing resting metabolic rate has been proposed (4). However, the mechanisms responsible were unknown, and only very recently was it realized that proteins might be involved (10). Coincident to this was the identification of two new forms of the long-known UCP1, which occurs exclusively in mature brown adipocytes. These forms, UCP2 and UCP3, are both expressed in BAT and in various other tissues, such as white adipose tissue, skeletal muscle, and heart (1, 16, 17, 19, 41). These, and perhaps other yet to be cloned UCPs, have been proposed to mediate the mitochondrial proton leak (17, 18, 24).
Brand and colleagues (3-9) have shown that the proton leak in tissues other than BAT contributes significantly to cellular energy expenditure. As studied in intact hepatocytes, thymocytes, and in mitochondria from a variety of tissue types, the proton leak has been estimated to account for ~25-35% of total cellular oxygen consumption rate or 35-45% of mitochondrial oxygen consumption rate (3, 4). Brand et al. (3) have estimated that up to 38% of resting metabolic rate in the rat may be caused overall by the leak.
To assess the metabolic significance and control of proton leak in BAT mitochondria from Ucp1-deficient mice and controls, we have used top-down metabolic control analysis (11, 22) and its extension, top-down elasticity analysis (5, 8, 28). Previous applications of these approaches include those used to characterize thyroid hormone regulation of mitochondrial proton leak and oxidative phosphorylation in liver mitochondria and hepatocytes (23, 25-27) and to investigate the sites of action of fatty acids in hepatocytes (37) and of glucagon (9) in liver mitochondria.
In the original paper describing the characteristics of the Ucp1-deficient mice, it was shown that Ucp2 mRNA is increased fivefold in BAT compared with the message level in BAT of control mice (16). No significant changes in Ucp2 expression were detected in any of the other tissues studied, including epididymal and inguinal white fat, liver, and muscle. Subsequent analyses show that there were no significant changes in the levels of expression of Ucp3 mRNA in any of the tissues studied (Kozak, unpublished results). If UCP2 is functionally analogous to UCP1, as well as structurally homologous, one might predict that the fivefold elevation in BAT Ucp2 mRNA would provide a mechanism for maintaining body temperature when mice were exposed to the cold. That this protection did not occur suggests that UCP2 and UCP3 cannot compensate for UCP1 with respect to the role of the latter in the regulation of body temperature on an acute exposure of mice to the cold. Accordingly, a role for either UCP2 or UCP3 in the regulation of either body temperature or body weight has not been established. With this in mind, we have investigated the characteristics of the proton leak and respiration in mitochondria isolated from Ucp1-deficient and control mice during its modulation by GDP. We wished to ascertain whether the residual UCPs in Ucp1-deficient mice are subjected to regulation by GDP at concentrations and conditions known to inhibit UCP1 activity.
In this report, we describe the metabolic characteristics and control of mitochondrial proton leak in mitochondria isolated from BAT of Ucp1-deficient mice and heterozygous controls. Results show that the leak remaining in BAT mitochondria of Ucp1-deficient mice is insensitive to GDP, whereas under identical experimental conditions the leak in BAT mitochondria from controls was, as expected, partially inhibited by GDP. These results support the conclusion that the leak in UCP1-deficient mitochondria from BAT is insensitive to GDP. The leak may be mediated by UCP2 and UCP3, and our findings reveal that this leak is regulated distinctly from that mediated through UCP1.
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MATERIALS AND METHODS |
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Treatment of animals. Male 6-mo-old
Ucp1-deficient (/
;
UCP1tm1) mice and male
heterozygous controls (+/
) of a hybrid C57BL/6J and 129/SvPas
genetic background (1) were obtained from the research colonies of
Leslie P. Kozak. The mice were group housed (3/cage), given free access
to Purina 5001 chow (4.5% fat by weight) and water, and kept at
23°C with light from 0700 to 1900. Mice used in this study were
cared for in accordance with the principles and guidelines of the
Canadian Council on Animal Care and the Institute of Laboratory Animal
Resources (National Research Council).
Isolation of mitochondria. Mitochondria were isolated from interscapular BAT depots of 12 Ucp1-deficient and 12 control mice. Mice were killed by decapitation before removal of BAT. BAT was dissected free of other adhering tissues and homogenized in 3.5 ml of buffer containing 250 mM sucrose, 1 mM HEPES, and 0.2 mM EDTA (pH 7.2 with KOH) using a glass-Teflon Potter-Elvehjem tissue grinder. Fractionation of homogenate was carried out by spinning at 1,500 g at 4°C for 10 min. The supernatant was then poured through a 250 µM Nitex screen and respun at 16,000 g for 14 min at 4°C to obtain a mitochondrial pellet. The pellet was resuspended (on ice) in 175 µl of a suspension medium containing 120 mM KCl, 20 mM sucrose, 3 mM HEPES, 2 mM MgCl2, 2 mM EGTA, and 0.5% BSA (pH 7.2 with KOH). Resuspension was carried out in both the presence and absence of 1 mM GDP. Stock 9% BSA was defatted by the method of Chen (13) and dialyzed against 153 mM NaCl and 11 mM KCl. Protein concentration of the mitochondrial suspension was assayed by the biuret method (21) using BSA as the reference standard.
Measurement of oxygen consumption. The
respiration rate of BAT mitochondria was measured using a Hansatech
(Norfolk, UK) Clark-type oxygen electrode. Suspensions of mitochondria
in the incubation chamber were maintained at 37°C and magnetically
stirred. Each rate was assessed by incubating enough
mitochondria in 1.0 ml of suspension medium (120 mM KCl, 20 mM sucrose,
3 mM HEPES, 2 mM
KH2PO4,
2 mM MgCl2, 2 mM EGTA, 0.5% BSA;
pH 7.2) to give ~1.0 mg mitochondrial protein/ml in the electrode
chamber. All respiration rates were determined simultaneously and in
parallel with measurements of protonmotive force (p).
State 3 respiration rate was defined as the oxygen consumption rate in the presence of 10 mM succinate, 0.75 U/ml hexokinase, and 10 mM ADP. Oxidation of any endogenous substrate
during incubations was inhibited with 5 µM rotenone. State 4 oxygen consumption was
determined in the presence of maximal amounts of the ATP synthase
inhibitor oligomycin (6 µg/ml). It was confirmed that ATP synthase
was completely inhibited in each experimental condition by additional
oligomycin, which caused no further inhibition of oxygen consumption
and no further increases in
p.
Measurement of mitochondrial p. The
p was determined using a methyltriphenylphosphonium
(TPMP+)-sensitive electrode that
was constructed using the methods of Kamo et al. (31). The pH component
of the electrochemical gradient converted to voltage units by
incubating mitochondria in the presence of 80 ng/ml of nigericin. The
outputs from the TPMP+ electrode
and the oxygen electrode were transferred to two voltmeters, the
reference sockets of which were connected together; data were then fed
in a data analysis software package that allows real-time monitoring
and recording on a personal computer.
The calibration of the
TPMP+-sensitive electrode,
determination of mitochondrial matrix volumes, and calculation of p
from TPMP+ electrode data were
carried out as described by Brand (6). The
p is calculated using the
Nernst equation as
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Application of top-down elasticity analysis and top-down control analysis. To quantitatively determine the effects of knocking out UCP1 on oxidative phosphorylation processes in BAT mitochondria, we used metabolic control analysis and top-down elasticity analysis, as described by Brand and co-workers (5, 8, 28).
Briefly, while metabolic control analysis allows the identification and quantitative description of the important sites of control within metabolic pathways, top-down elasticity analysis renders additional data describing pathway regulation. Elasticity analysis can be valuable in identifying the sites of metabolic effects in pathways of the insertion of a transgene, of gene knockout, or of the treatment with hormones or drugs. Some of the useful parameters determined in an elasticity analysis include "elasticity coefficients," "flux control coefficients," and "concentration control coefficients." An elasticity coefficient (often referred to simply as an "elasticity") describes the responsiveness of a branch of a metabolic pathway to changes in the amount of an intermediate in that pathway. If the elasticity for that branch differs between the transgenic and the control preparations being compared, then one site of action of the drug is located within the reactions circumscribed by that branch. Other parameters include flux control and concentration control coefficients, which describe, respectively, the relative proportion of control by branches of the pathway over the flux rate of the pathway and over the amounts of intermediates within the pathway. Several useful reviews on both approaches have been published recently (e.g., see Refs. 7 and 28).
The oxidative phosphorylation system can be defined as the tripartite
system shown in Fig. 1. We have determined
the overall elasticities to changes in p of the reactions that
produce
p (i.e., substrate transport, the tricarboxylic acid cycle,
and the electron transport chain, referred to herein simply as
substrate oxidation reactions) and two blocks of reactions that consume it (i.e., ATP synthesis and consumption reactions and the mitochondrial proton leak reactions). The kinetic response of the
p producers to
p was measured by titrating the
p consumers with oligomycin (1-6 µg/mg protein). The kinetic response of the leak to
p
was assessed by completely inhibiting proton return through ATP
synthase by use of oligomycin (6 µg/mg protein) and titrating the
substrate oxidation block of reactions with malonate (0.20-2.0
mM), a competitive inhibitor of complex II of the respiratory chain.
The elasticity of the phosphorylating subsystem to
p was measured by
titrating
p producers with malonate alone (0.2-2.0 mM). All
titrations were performed both in the presence and in the absence of 1 mM GDP.
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Statistical analysis. Data were analyzed using Student's t-tests or ANOVA, which was followed by Tukey's post hoc tests. Linear regression lines were compared by analysis of covariance using Prism 2 for Windows. A P value of <0.05 was considered statistically significant. Unless otherwise stated, results are presented as means and SE.
Materials. Oligomycin, malonate, valinomycin, BSA (fraction V), TPMP, succinate, nigericin, and rotenone were from Sigma. TPMP bromide was from Aldrich. 3H2O, [86Rb]Cl, [14C]sucrose, and [3H]TPMP bromide were from NEN. Water-insoluble compounds were dissolved in DMSO.
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RESULTS |
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Overall kinetics of the three blocks of reactions
comprised by the oxidative phosphorylation system. As
described in the introduction, the approach that we have used to study
metabolic differences in BAT mitochondria isolated from
Ucp1-deficient and control mice is
referred to as top-down elasticity analysis. In Figs.
2-5, the overall responsiveness, or elasticity to changes in p for the
three blocks of reactions of the oxidative phosphorylation (as depicted
in Fig. 1), are shown. The graphs in Fig. 2 show the overall kinetics
of the leak, substrate oxidation reactions, and phosphorylation plus
leak reactions. Results from BAT mitochondria of
Ucp1-deficient mice are shown in Fig.
2A, whereas those of control mice are
shown in Fig. 2B. Mitochondria were
incubated in the absence of GDP but in the presence of 0.5% BSA to
limit fatty acid-activated uncoupling and extend the time over which the mitochondria respired. Fatty acids are known to activate uncoupling through UCP1 (20, 32, 33) and may activate acutely other UCPs. In Fig.
2, A and
B, the furthermost point on the right
shows the state 3 respiration rate and
p values; comparison of values for UCP1-deficient and control
mitochondria shows that the presence of UCP1 in control mitochondria
causes an ~70% increase in "state 3" respiration rate. It should also be noted that
this state 3 rate in control
mitochondria, in the absence of GDP, is higher than that which is
achieved in its presence. This results presumably from the situation
where UCP1 is not inhibited by GDP, protons leak into the matrix
through it, and the chain responds by oxidizing substrates at a higher
rate to sustain
p.
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State 4 (i.e., nonphosphorylating)
respiration (highest p values and located at the top of the proton
leak curves) is ~100% greater in mitochondria from control BAT than
in those from UCP1-deficient mitochondria; again, this is due to the
activity of UCP1 in control mitochondria. The respiratory control
ratios (i.e., state
3/state 4 respiration
values) were 2.0 and 1.6 for control and UCP1-deficient mitochondria, respectively.
Overall kinetics of the mitochondrial proton leak and their sensitivity to the purine nucleotide, GDP. The data in Fig. 3 clearly show that state 4 (maximal leak-dependent) oxygen consumption in BAT mitochondria from Ucp1-deficient mice is insensitive to GDP (Fig. 3A), whereas the leak in BAT mitochondria from control mice is inhibited by the presence of GDP (Fig. 3B). Technical details are provided in the legend to Fig. 3.
The data presented also allow the comparison of the overall kinetics of
the leak reactions over a range of mitochondrial membrane potentials
between UCP1-deficient and control BAT mitochondria. The
overall kinetics of the leak in UCP1-deficient mitochondria (Fig. 3A) in the presence and
absence of 1 mM GDP are virtually identical. These data were
unexpected; 1 mM GDP is well known to inhibit the activity of UCP1 in
BAT mitochondria (15, 36). Because a fivefold increase in mRNA for
Ucp2 in BAT was observed in
Ucp1-deficient mice compared with
heterozygous controls, it was hypothesized that an increased activity
of UCP2 protein compensated to some degree for the loss of UCP1 and
this, in turn, contributed to the lean phenotype. These data show that,
over a wide range of p values, there is no increase in the oxygen
used to balance the leak of protons back into the matrix. This is
evident when the oxygen used to balance the leak is compared over a
range of
p between controls in the presence of GDP (Fig.
3B) and UCP1-deficient mitochondria
in the presence or absence of GDP (Fig.
3A); oxygen consumption is roughly
equal at any given
p.
The overall kinetics of substrate oxidation and
phosphorylation reactions in the presence and absence of
GDP. The results in Figs. 4 and 5 show the overall
kinetics of substrate oxidation and phosphorylation reactions,
respectively. In Fig. 4 the kinetics of the substrate oxidation
reactions in the presence of GDP are compared with those in the absence
of GDP. In UCP1-deficient mitochondria, there is no difference between
the kinetics of the substrate oxidation system in the presence and
absence of GDP (P > 0.05 by analysis of covariance). These results show that, at any given value of p,
the amount of oxygen used to balance the activity of the substrate oxidation reactions is not significantly affected by the presence of
GDP. In mitochondria from control mice, however, the presence of GDP
had a marked effect on the kinetics of substrate oxidation reactions
(P < 0.002). These results show
simply that, when UCP1 is functional, the activity of the substrate
oxidation reactions increases to fuel this leak and to maintain a
relatively normal
p (i.e., by increasing the activity of electron
transport chain proton pumps).
The graphs shown in Fig. 5 depict the kinetics of the phosphorylation
reactions. As indicated in the legend to Fig. 2, and as described
previously (22), to obtain the kinetics of the phosphorylation
reactions alone (i.e., in the absence of proton leak reactions), the
oxygen used to support leak reactions at each mean data point must be
subtracted. The data from mitochondria of
Ucp1-deficient mice shown in Fig.
5A indicate that there is no
significant effect of GDP on the kinetics of the phosphorylation reactions. At any given value of p, the amount of oxygen used to
support ATP turnover reactions is not significantly different in the
presence and absence of GDP. In control mitochondria, there is however
a significant difference between the kinetics of the phosphorylating
system in the presence of GDP compared with in its absence. When GDP is
present, the amount of oxygen used (at similar
p values) to balance
phosphorylation reactions is much greater than it is when GDP is absent
(e.g., compare rates at a
p value of 100 mV). This is intuitive;
when GDP is present and UCP1 is inhibited, the proton gradient is used
to fuel the activity of ATP synthase; in its absence the gradient can
be rapidly dissipated through the UCP1-mediated leak.
Application of top-down metabolic control
analysis. As well as being useful in the identification
of differences in the activities of blocks of reactions in a metabolic
pathway, top-down elasticity analyses provide all of the data needed
for a top-down control analysis of the pathway (11, 22). The latter
provides extensive data describing the distribution of control by the
blocks of reactions over the overall flux through the system (e.g.,
mitochondrial oxygen consumption, as in the present study) and over
each of the other blocks of reactions in the pathway or system being
studied. The elasticity coefficients, describing the
responsiveness of the three blocks of reactions to p in
state 3 and state
4 respiration, are shown in Table
1.
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Flux control coefficients and concentration control coefficients of the
three blocks of reactions over the rate of each of the subsystems are
shown in Tables
2-5.
The concentration control coefficients of the three subsystems over the
intermediate in the system, p, are given in Table 5. The flux
control coefficients in Table 2 show that the greatest proportion of
control over state 3 mitochondrial
oxygen consumption (equivalent to flux through the substrate oxidation
subsystem) is by the substrate oxidation reactions themselves,
regardless of the presence or absence of 1 mM GDP. This is similar to
the high degree of control over state 3 respiration in liver mitochondria (e.g., see Ref.
11). The data in Table 2 also show that the presence of GDP in the
incubation medium has effects on the distribution of control over
state 3 respiration in both
UCP1-deficient and control BAT mitochondria. For example, in control
mitochondria, there is an increase in the proportion of control by the
substrate oxidation reactions and slight decreases in the control by
the phosphorylation and leak reactions. In UCP1-deficient mitochondria,
the effect of GDP is different; there is a decrease in the proportion
of control by the substrate oxidation reactions, no change in control
by the phosphorylation reactions, and increased control by the leak.
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The data in Table 3 describe the distribution of control by the three blocks of reactions over the oxygen used to support the phosphorylating reactions in state 3. As no ATP synthesis occurs during state 4 respiration, there are no data for state 4 in Table 3. The control over phosphorylating reactions in state 3 is held to a large extent by the substrate oxidation reactions; however, the degree of control by the phosphorylating reactions is also relatively high. These data also show that the shifts in the control in the presence of GDP compared with its absence are similar to those described for the control over mitochondrial respiration (Table 2). The flux control coefficients shown in Table 4 describe the distribution of control between the three blocks of reactions over the oxygen used to support the mitochondrial proton leak. In state 3, the control over the small rate of proton leak is shared in most instances roughly equally between the three blocks of reactions. In control mitochondria in the presence of GDP, the control by substrate oxidation and phosphorylation reactions is greater than in the absence of GDP; the amount of control by leak remains approximately the same. In contrast, in UCP1-deficient mitochondria in the presence of GDP, the control by substrate oxidation and phosphorylation reactions is lower than in the absence of GDP; the amount of control by leak again remains approximately the same.
The distribution of control over the three subsystems during state 4 respiration, in both the presence and absence of GDP, is also described in Tables 2-4. Again, as there is no synthesis of ATP during state 4 respiration, there are no control coefficients for the phosphorylating subsystem (Table 3). These data show clearly that there are differences in the control over state 4 mitochondrial oxygen consumption in the presence and absence of GDP in control BAT mitochondria only. GDP does not alter the distribution of control in UCP1-deficient mitochondria.
In Table 5 the concentration control coefficients are presented. These
values describe the distribution of control over the level of p by
the substrate oxidation, phosphorylating, and leak reactions. At
state 3, these results generally show
that most of the variation in
p is controlled by the substrate
oxidation subsystem, and the remainder of the control is through the
p consumers (i.e., proton leak and the phosphorylating reactions). In state 4, control over
p is
shared equally between the producers and the consumers of
p (i.e.,
the substrate oxidation and proton leak subsystems). These results are
thus similar to results from intact hepatocytes of hypothyroid,
euthyroid, and hyperthyroid rats (12, 26, 27).
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DISCUSSION |
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The overall purpose of this study was to analyze the metabolic control and characteristics of proton leak in mitochondria from BAT of Ucp1-deficient mice and of heterozygous controls. The proton leak that remains in these mitochondria may be mediated through the activity of UCP2 and/or UCP3. The levels of the proteins themselves have not as yet been quantified, as antibodies that are unequivocally specific for one or the other are not available. An important finding of this study is that the leak in these mitochondria is insensitive to GDP at concentrations and incubation conditions that cause leak in mitochondria from control mice to be cut in half (Fig. 3, A and B). The flux control coefficients show that the control over mitochondrial oxygen consumption and proton leak reactions is affected strongly by the presence of 1 mM GDP in control mitochondria, whereas in UCP1-deficient mitochondria there is little effect of the purine nucleotide. These findings are discussed below, after a short discussion of some of the characteristics of UCP2 and UCP3.
The first UCP1 homologue, UCP2, was reported in March 1997 (17). This protein has 59% amino acid identity to UCP1. Several protein motifs known to be important in UCP1 function are conserved in UCP2. Similar to UCP1, there are three mitochondrial carrier motifs, consistent with ion transport activity. The putative purine nucleotide-binding sequences are also conserved. The amino acid sequences of mouse and human UCP2 are 95% identical; UCP1 is only 79% identical between mouse and human forms. Northern blot analyses for Ucp2 show widespread expression (17, 19). The highest levels are found in white adipose tissue in both mice and humans (19). It is also highly expressed in muscle in humans and in BAT in mice; this may reflect the relative importance of these thermogenic tissues (19).
The evidence that UCP2 is, in fact, capable of uncoupling oxidative phosphorylation includes that yeast transfected with UCP2 grow more slowly than controls [as reported earlier for UCP1 (2, 17)]. Second, mitochondrial membrane potential, as estimated by flow cytometry, is lowered in yeast transfected by either UCP1 or UCP2 (17). Third, there are data that suggest that the expression of UCP2 confers some protection from the development of obesity in mice. Fleury et al. (17) found higher levels of Ucp2 mRNA in white adipose tissue of the obesity-resistant mouse strain, A/J, than the obesity-prone strain, C57BL/6J. Subsequently, Surwit et al. (40) showed that, in the obesity-resistant strains A/J and C57BL/KsJ, Ucp2 expression in white fat increases roughly twofold in response to 2 wk of a high-fat diet, whereas no diet effect was observed in C57BL/6 mice. In BAT, only the expression of Ucp1 was increased by high-fat feeding. Overall, these authors conclude that the consumption of a high-fat diet selectively regulates Ucp2 expression in white fat and Ucp1 expression in brown fat and that resistance to obesity is correlated with this early selective induction of Ucp1 and Ucp2 and is not associated with changes in expression of Ucp3. Insofar as there is increased expression of Ucp2 (16), but not of Ucp3 in tissues of the Ucp1-deficient mouse, our results support their findings. The fact that we do not observe any increase in leak-dependent oxygen consumption (either in the presence or absence of GDP) in BAT mitochondria from Ucp1-deficient mice shows that the relative levels of Ucp2 mRNA do not correspond to any differences in uncoupling.
Two groups reported the cloning of a third UCP, UCP3 (1, 41). One group screened a human skeletal muscle cDNA library and isolated UCP3L and UCP3S, as well as UCP2 (1). They found that UCP3 is highly skeletal muscle specific and is 57 and 73% identical to UCP1 and UCP2, respectively, at the amino acid level. The potential purine nucleotide-binding domain is found in the long form, UCP3L, but not in the short, UCP3S. The other group (41) showed that human UCP3 is 71% identical to human UCP2 and 57% identical to human UCP1. Human UCP3 is expressed abundantly and preferentially in skeletal muscle; in rodents, expression is in skeletal muscle and BAT. As these tissues are important sites of energy expenditure, UCP3 may be an important thermogenic mediator (41).
At this point, it is not understood how the activities of UCP2 and UCP3 are controlled. Sequences consistent with purine nucleotide binding appear to be conserved in UCP2 and in the UCP3L (1, 17). However, after it was discovered almost 20 years ago that purine nucleotides bind and inhibit UCP1 (15, 29), GDP binding was assessed in mitochondria from a variety of tissues and found to be low to negligible. As a result, purine nucleotide binding has been a key factor in identifying UCP1 activity. In addition, the loose coupling observed in skeletal muscle mitochondria is insensitive to GDP, unlike that in BAT mitochondria (Monemdjou and Harper, unpublished observation and Ref. 14). Gimeno et al. (19) hypothesize on the control of UCP2: because a single amino acid mutation in the inhibitory nucleotide-binding site of UCP1 (268Phe to Tyr) creates a UCP that has higher uncoupling activity (2) and because both mouse and human UCP2 naturally contain Tyr at the equivalent position (270Tyr), UCP2 may be less susceptible to inhibitory effects of purine nucleotides. This hypothesis is congruent with our findings showing that the mitochondrial proton leak remaining in BAT mitochondria of Ucp1-deficient mice is GDP insensitive under our incubation conditions. In addition, results from metabolic control analyses of oxidative phosphorylation show that proton leak in liver, kidney, and skeletal muscle may be controlled independently of ATP turnover. Control over leak by phosphorylation reactions is very low (<10%); ~80% of control lies in the leak mechanism itself (3, 26). However, it has recently been shown using rhodamine-123 uptake in rat liver nonparenchymal cells, which express UCP2, that GDP raises relative membrane potential (34). Moreover, in a recent study, Simonyan and Skulachev (39) found that, in oligomycin-treated heart mitochondria of cold-exposed rats, the addition of GDP caused an increase in membrane potential; this effect was not observed in mitochondria from room temperature-acclimated rats. Cold exposure caused an increase in palmitate-induced mitochondrial uncoupling, and induction was GDP insensitive. Our measurements were made in the presence of albumin and may not allow for the detection of any fatty acid-induced UCP-mediated leak. The latter possibility requires examination.
Our findings also suggest that adaptive thermogenesis may occur in other tissues of the Ucp1-deficient mouse, allowing the mice to maintain a normal resting metabolic rate, feed efficiency, and phenotype as lean controls during the feeding of a high-fat diet (16). The obvious tissue to analyze in relation to this hypothesis is skeletal muscle, and this is one aspect of our current investigations.
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ACKNOWLEDGEMENTS |
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This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to M.-E. Harper), from the National Institutes of Health (to L. P. Kozak), and Pfizer (to L. P. Kozak).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: M.-E. Harper, Dept. of Biochemistry, Microbiology and Immunology, Univ. of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada K1H 8M5 (E-mail: mharper{at}uottawa.ca).
Received 27 October 1998; accepted in final form 23 February 1999.
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