1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada; and 2 Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808
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ABSTRACT |
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Mice having targeted inactivation of
uncoupling protein 1 (UCP1) are cold sensitive but not obese
(Enerbäck S, Jacobsson A, Simpson EM, Guerra C, Yamashita H,
Harper M-E, and Kozak LP. Nature 387: 90-94, 1997).
Recently, we have shown that proton leak in brown adipose tissue (BAT)
mitochondria from UCP1-deficient mice is insensitive to guanosine
diphosphate (GDP), a well known inhibitor of UCP1 activity (Monemdjou
S, Kozak LP, and Harper M-E. Am J Physiol Endocrinol
Metab 276: E1073-E1082, 1999). Moreover, despite a fivefold
increase of UCP2 mRNA in BAT of UCP1-deficient mice, we found no
differences in the overall kinetics of this GDP-insensitive proton leak
between UCP1-deficient mice and controls. Based on these
findings, which show no adaptive increase in UCP1-independent leak in
BAT, we hypothesized that adaptive thermogenesis may be occurring in
other tissues of the UCP1-deficient mouse (e.g., skeletal
muscle), thus allowing them to maintain their normal resting metabolic
rate, feed efficiency, and adiposity. Here, we report on the overall
kinetics of the mitochondrial proton leak, respiratory chain, and ATP
turnover in skeletal muscle mitochondria from UCP1-deficient and
heterozygous control mice. Over a range of mitochondrial protonmotive
force (p) values, leak-dependent oxygen consumption is higher in
UCP1-deficient mice compared with controls. State 4 (maximal
leak-dependent) respiration rates are also significantly higher in the
mitochondria of mice deficient in UCP1, whereas state 4
p is
significantly lower. No significant differences in state 3 respiration
rates or
p values were detected between the two groups. Thus the
altered kinetics of the mitochondrial proton leak in skeletal muscle of
UCP1-deficient mice indicate a thermogenic mechanism
favoring the lean phenotype of the UCP1-deficient mouse.
uncoupling protein(s); thermogenesis; oxidative phosphorylation; top-down elasticity analysis; obesity
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INTRODUCTION |
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UNCOUPLING PROTEIN 1 (UCP1) is a 32-kDa protein found in the mitochondrial inner membrane of mature brown adipocytes. The sequence of events comprising the discovery of UCP1 is now occurring in reverse order with regard to the recently identified UCPs. In the latter case, description of the physiological function, nonshivering thermogenesis, preceded the identification of the 32-kDa protein, which in turn preceded the cloning of the gene. In the case of the novel UCPs, the opposite has occurred; the genes have been cloned, and the search is on for the physiological phenomena that their proteins impart. There are now five putative UCPs. UCP2 is expressed in most tissues of humans and rodents (12, 13). UCP3 is expressed preferentially in skeletal muscle of humans; in rodents it is also expressed in brown adipose tissue (BAT) (3, 32). UCP4 is expressed exclusively in the brain (22). The fifth putative UCP is brain mitochondrial carrier protein 1 (BMCP1), and it is expressed mainly in the brain but also to a very minor extent in other tissues (31). The true physiological functions of the novel UCPs are largely unknown.
It was anticipated that mice having gene-targeted inactivation of UCP1
would become obese and diabetic at a young age compared with mice
having normal levels of UCP1. This hypothesis and the motivation to
create a transgenic mouse that was deficient specifically in UCP1 were
based on the findings that overexpression of Ucp1 reduced
adiposity in both genetic (20) and dietary
(21) obesity. Thus the findings of Enerbäck et al.
(9) that showed that UCP1-deficient mice were lean and
became no more obese than controls on either a high- or a low-fat diet
was surprising (9), despite abundant expression of the
remaining isoforms, UCP2 and UCP3. UCP1-deficient mice are, however,
characterized by a cold-sensitive phenotype, and they have an
abnormally low response in resting metabolic rate (RMR) to acute
treatments with the 3-adrenergic agonist, CL316,243
(9).
UCP1-deficient mice also have elevated levels of Ucp2 expression (9). We previously tested the hypothesis that the elevated expression of Ucp2 increases mitochondrial proton leak in their brown adipose tissue (BAT) mitochondria, thereby compensating for the loss of UCP1-derived proton leak. To do this, we investigated the characteristics of the proton leak by using top-down elasticity analysis (4, 6, 17) in the context of oxidative phosphorylation in mitochondria isolated from BAT of UCP1-deficient and control mice (25). More specifically, we aimed to ascertain whether or not the residual uncoupling proteins (UCP2 and UCP3) in the BAT mitochondria of UCP1-deficient mice were subject to control by guanosine diphosphate (GDP) at concentrations and conditions known to inhibit UCP1 activity. Results showed that the leak remaining in BAT mitochondria of UCP1-deficient mice is insensitive to GDP (25). In addition, our finding that there are no differences in the total activity and overall kinetics of the GDP-insensitive proton leak between UCP1-deficient mice and controls refuted our initial hypothesis that UCP2 and/or UCP3 provided compensatory thermogenesis in BAT.
These findings (25) and those describing the expression of Ucp2 and Ucp3 in various tissues (3, 12, 13, 32) led us to speculate that perhaps adaptive or compensatory UCP-dependent thermogenesis might be occurring in tissues other than BAT, allowing the mice to maintain their normal observed RMR, feed efficiency, and lean phenotype (9). Skeletal muscle, by virtue of its high metabolic activity on a per-gram basis and by virtue of the large proportion of total body weight that it represents, contributes significantly to RMR (10, 11, 29). Moreover, mitochondrial proton leak in skeletal muscle is substantial; Rolfe and Brand (28) demonstrated that the proton leak accounts for 52% of the oxygen consumption rate of the resting respiration rate of perfused rat hindlimb, and they estimated a contribution of 16-31% to the basal metabolic rate of the rat (28, 30).
In this report, as with our studies with BAT mitochondria, we used
top-down elasticity analysis to assess the metabolic significance and
control of proton leak in skeletal muscle mitochondria from UCP1-deficient mice and controls. Results herein show that oxygen consumption used to support the leak in skeletal muscle mitochondria from UCP1-deficient mice is greater than that in controls. In UCP1-deficient mice compared with controls, maximal leak-dependent oxygen consumption (state 4 respiration) was found to be higher, whereas mitochondrial protonmotive force (p) was lower in skeletal muscle mitochondria. These results support the conclusion that altered
proton leak in skeletal muscle mitochondria of UCP1-deficient mice is a
thermogenic mechanism favoring the lean phenotype of these mice.
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EXPERIMENTAL PROCEDURES |
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Treatment of animals. Male 6-mo-old UCP1-deficient (-/-)mice and heterozygous controls (+/-) on a hybrid C57BL/6J and 129/SvPas genetic background (9) were obtained from the research colonies of Leslie P. Kozak. The mice were group housed (3/cage), given free access to Charles River 5075 rodent chow (4.5% fat by weight) and water, and kept at 23°C with lights on 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).
Northern blots for Ucp2 and Ucp3.
Hindlimb muscle tissue was removed and frozen in liquid
nitrogen. RNA was isolated with TRIzol reagent
according to the manufacturer's instructions. Ten milligrams of total
RNA were separated on a 1% agarose gel containing 5% formaldehyde and
were blotted onto a nylon membrane. Probes were randomly labeled with
-[32P]dCTP. The probe used for Ucp2 has
been used previously (9). The Ucp3 probe was a
350-bp Kpn1 fragment of the Ucp3 coding region. The Ucp3 clone was a kind gift of Dr. B. B. Lowell.
Isolation of mitochondria from skeletal muscle. Mitochondria were isolated from hindlimb and forelimb skeletal muscles of nine UCP1-deficient and eight control mice by the method of Bhattacharya et al. (1). Specifically, muscles included those of the lower leg (gastrocnemius), thigh (vastus lateralis, rectus femoris, quadratus femoris, adductor brevis, semimembranous, gluteus maximus, gluteus minimus, and gluteus medius), and shoulder (triceps longus and medius, biceps brevis and longus). Protein concentration of the mitochondrial suspension was assayed by the Biuret method (16) with bovine serum albumin as the reference standard.
Measurement of mitochondrial oxygen consumption.
The respiration of skeletal muscle mitochondria was measured with the
use of a Hansatech Clark-type oxygen electrode whose incubation chamber
was 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, 20 mM glucose, 10 mM
KH2PO4, 5 mM HEPES, 2 mM MgCl2, and
1 mM EGTA, pH 7.2 with KOH) to give ~0.5 mg mitochondrial protein/ml
in the electrode chamber. All respiration rates were determined
simultaneously and in parallel with measurements of protonmotive force.
Titrations were done in the presence of 10 mM succinate, 80 ng
nigericin/ml to convert pH to voltage units, and 5.0 µM
rotenone to prevent the oxidation of any endogenous NAD-linked
substrates. State 3 respiration rate was defined as the oxygen
consumption rate in the presence of saturating amounts (1.3 units/ml)
of hexokinase, 100 µM ADP, and 100 µM ATP. State 4 oxygen
consumption was determined in the presence of maximal amounts of the
ATP synthase inhibitor, oligomycin (8 µg/mg mitochondrial protein).
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 increase in
protonmotive force. Analyses of proton leak were initially performed in
the presence and absence of GDP in experiments with three
UCP1-deficient and three control mice. After it was established that
GDP had no effect, it was no longer used, and the results were pooled
with subsequent experiments conducted in the absence of GDP.
Measurement of mitochondrial protonmotive force.
Protonmotive force (p) was determined by means of a
methyltriphenylphosphonium (TPMP+)-sensitive electrode,
which was constructed with the methods of Kamo et al.
(19). The outputs from the TPMP+ electrode and
the oxygen electrode were transferred to two voltmeters, whose
reference sockets were connected together; data were then fed into a
data analysis software package that allows real-time monitoring and
recording on a personal computer.
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Application of top-down elasticity analysis.
To quantitatively determine the effects of "knocking out" UCP1 on
oxidative phosphorylation processes in skeletal muscle mitochondria, we
used the top-down elasticity approach (4, 6, 17).
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. The oxidative
phosphorylation system was defined as the tripartite system, as
described previously (25); the system is essentially
divided into three distinct blocks of reactions, or subsystems, which
consist of the reactions that produce p (i.e., substrate
oxidation reactions) and the two blocks of reactions that consume
p
(ATP turnover reactions and the mitochondrial proton leak reactions).
Serum glucose and nonesterified fatty acid measurements.
Blood was collected from mice at the time they were killed and was
stored on ice for a minimum of 1 h. Samples were then centrifuged at 3,000 g for 15 min. Serum was placed in small cryovials,
immediately frozen in liquid nitrogen, and then stored at 80°C.
Glucose levels were measured with Sigma Diagnostics glucose assay kits
(Glucose Trinder 100 Sigma, St. Louis, MO). Nonesterified fatty acid
(NEFA) levels were assessed with the NEFA C assay kit (Wako Chemicals, Richmond, VA).
Statistical analysis. Two sample comparisons were conducted using Student's t-tests. Linear regression lines were compared by analysis of covariance using Prism 3 (San Diego, CA) for Windows. A P value <0.05 was considered significant. Unless otherwise stated, results are presented as means ± SE.
Materials. Oligomycin, malonate, valinomycin, bovine serum albumin (fraction V), TPMP+, succinate, nigericin, rotenone, and glucose assay kits were from Sigma. 3H2O, [86Rb]Cl, [14C]sucrose, and [3H]TPMP-I were from Mandel Du Pont NEN (Guelph, ON, Canada). NEFA C assay kits were from Wako Chemicals. Water-insoluble compounds were dissolved in dimethyl sulfoxide.
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RESULTS |
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Northern blots for Ucp2 and Ucp3.
There were no significant differences in the levels of expression of
Ucp2 and Ucp3 in hindlimb muscle tissue of
UCP1-deficient (-/-), heterozygous (+/-), and wild-type control
(+/+) mice (Fig. 1).
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Comparison of kinetic responses of mitochondrial proton leak,
substrate oxidation, and phosphorylation subsystems to p in
mitochondria from UCP1-deficient and control mice.
Maximal leak-dependent oxygen consumption (state 4 respiration) rates
were found to be significantly higher (P < 0.02) in the mitochondria of UCP1-deficient mice than in controls. Values were
146.3 ± 7.1 nmol O · min
1 · mg
protein
1 (n = 9) and 121.9 ± 7.1 nmol O · min
1 · mg protein
1
(n = 8) in mitochondria of UCP1-deficient and control
mice, respectively (Fig. 2; furthermost
points on the right of each curve). At state 4,
p was significantly
lower (P < 0.002) in UCP1-deficient mice compared with
controls. Values were 168.4 ± 3.2 mV (n = 9) and 181.9 ± 2.6 mV (n = 8) in UCP1-deficient mice and
in controls, respectively (Fig. 2). No significant differences were
detected in state 3 respiration or state 3
p values between the two
groups (Figs. 3 and 4; furthermost points
on the right of each line).
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Serum glucose and NEFA levels. No significant differences were observed between serum glucose levels of UCP1-deficient and control mice (data not shown). However, the level of serum NEFA in UCP1-deficient mice was found to be significantly higher than in controls (P = 0.04). Values were 0.643 ± 0.077 mM (n = 3) and 0.470 ± 0.030 mM (n = 3) in UCP1-deficient and control mice, respectively.
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DISCUSSION |
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The main objective of this study was to examine the
characteristics of the proton leak in skeletal muscle mitochondria of UCP1-deficient mice to test our hypothesis that skeletal muscle is a
site where adaptive thermogenesis occurs in these mice and contributes
to their lean phenotype. The finding of major interest from this study
is that mitochondrial proton leak-dependent oxygen consumption is
higher in mitochondria of UCP1-deficient mice compared with controls.
For any given value of mitochondrial p, more oxygen is used to
support the proton leak reactions in mitochondria of the UCP1-deficient
mice compared with controls (Fig. 2). In addition, maximal
leak-dependent oxygen consumption (state 4 respiration) rates were
found to be significantly higher in the mitochondria of UCP1-deficient
mice than in controls, whereas
p at state 4 is significantly lower
in UCP1-deficient mitochondria compared with controls. Although we
found no significant differences in state 3 respiration or state 3
p
values between the two groups, the overall kinetics of the respiratory
chain show that respiratory chain activity is more sensitive to changes
in
p in control mitochondria compared with UCP1-deficient
mitochondria (Fig. 3).
Further examination of the results presented in Figs. 2 and 3 reveal
some interesting differences in the responsiveness of the mitochondrial
proton leak and substrate oxidation reactions to changes in p. For
example, at a protonmotive force value of 150 mV, an intermediate value
between state 3 and state 4, where mitochondria would predominantly
function, leak-dependent oxygen consumption in skeletal muscle
mitochondria of UCP1-deficient mice is twice that of controls (~76
vs. 38 nmol O · min
1 · mg mitochondrial
protein
1). As
p increases from 150 to 160 mV, the
increase in leak-dependent oxygen consumption in UCP1-deficient mice is
approximately twice that of controls (32 vs. 16 nmol
O · min
1 · mg mitochondrial
protein
1). Similar comparisons can be made in examining
the overall kinetics of the substrate oxidation reactions shown in Fig.
3. As
p increases, e.g., again from 150 to 160 mV, the decrease in
substrate oxidation-dependent oxygen consumption in mitochondria from
UCP1-deficient mice is approximately one-half that in control
mitochondria (13 vs. 26 nmol
O · min
1 · mg mitochondrial
protein
1). Thus the absolute change in oxygen consumption
that is induced by alterations in
p is greater for the proton leak
reactions than for the substrate oxidation reactions in the
UCP1-deficient mice (32 vs. 13 nmol
O · min
1 · mg mitochondrial
protein
1). These results show clearly that oxygen
consumption in skeletal muscle mitochondria of UCP1-deficient mice is
affected more by changes in proton leak than by changes in reactions
involved in substrate oxidation. The reverse is true in the
mitochondria from control mice. Interestingly, the responsiveness to
changes in
p varies by a factor of two in both branches of the
oxidative phosphorylation system between the two groups of mice.
We also found that the level of serum NEFA in UCP1-deficient mice was
significantly higher than in controls. It has recently been shown that
increasing circulating levels of free fatty acids (FFAs) by severe food
restriction (90%) for 24-48 h in rodents (2, 14, 33)
or for 5 days in humans (24) increases UCP3 mRNA
expression in muscle. FFAs have also been proposed to play a role in
UCP activation. UCP1-mediated uncoupling has been thought for many
years to be activated by FFAs (8, 27). However, the recent
findings of Matthias et al. (23) show that fatty acids
activate uncoupling in UCP1-deficient mouse BAT mitochondria just as
well as they do in normal control BAT mitochondria. Although it is
still possible that the fatty acid effect is mediated by UCP2 and/or
UCP3 and/or another mitochondrial carrier protein, their results
support the idea that the effect is not due to UCP1. There is recent
evidence to suggest that fatty acids may activate UCP2 and UCP3
uncoupling activity. In liposomes containing Escherichia coli-expressed and -reconstituted UCP2 or UCP3, Jabrek et
al. (18) have shown that fatty acids induce UCP2 and
UCP3-catalyzed electrophoretic proton flux. In relation to our data,
and on the basis of the above evidence, FFAs may play a role in
increasing UCP2 and UCP3 activity in muscle mitochondria of
UCP1-deficient mice, thereby contributing to the differences in their
mitochondrial proton leak kinetics compared with controls. To date,
however, we have observed no significant increased mRNA levels for UCP2 or UCP3 in muscle of UCP1-deficient mice compared with controls (Fig.
1). Moreover, our immunoblotting of UCP3 has not revealed any
differences in the amount of protein per milligram of mitochondrial protein between UCP1-deficient mice and controls (our unpublished observations). Although we are able to specifically identify
UCP3 protein through the use of mitochondria from UCP3-deficient mice (15) and recombinant proteins, immunoblots of UCP2 that
unequivocally identify it in tissues that express more than one UCP are
not yet possible.
These results indicate that proton leak is increased in skeletal muscle mitochondria in UCP1-deficient mice and thus that skeletal muscle mitochondrial proton leak is a potential adaptive thermogenic mechanism in these lean yet cold-sensitive mice. The factor(s) underlying the increased proton leak, however, remain(s) to be determined.
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ACKNOWLEDGEMENTS |
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This research was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada (M.-E. Harper) and the National Institutes of Health (HD-08431 to L. P. Kozak).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M.-E. Harper, Dept. of Biochemistry, Microbiology and Immunology, Univ. of Ottawa, 451 Smyth Rd., Ottawa, ON K1H 8M5, Canada (E-mail: mharper{at}uottawa.ca).
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. Section 1734 solely to indicate this fact.
Received 23 February 2000; accepted in final form 12 June 2000.
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