In Vivo Effects of Uncoupling Protein-3 Gene
Disruption on Mitochondrial Energy Metabolism*
Gary W.
Cline
§,
Antonio J.
Vidal-Puig¶
,
Sylvie
Dufour**,
Kevin S.
Cadman
,
Bradford B.
Lowell¶, and
Gerald I.
Shulman
**
From the
Department of Internal Medicine and
the ** Howard Hughes Medical Institute, Yale University School of
Medicine, New Haven, Connecticut, 06520 and the ¶ Division of
Endocrinology, Department of Medicine, Beth Israel Deaconess
Medical Center and Harvard Medical School, Boston, Massachusetts
02215
Received for publication, March 21, 2001
 |
ABSTRACT |
To clarify the role of uncoupling protein-3
(UCP3) in skeletal muscle, we used NMR and isotopic labeling
experiments to evaluate the effect of UCP3 knockout (UCP3KO) in mice on
the regulation of energy metabolism in vivo. Whole body
energy expenditure was determined from the turnover of doubly labeled
body water. Coupling of mitochondrial oxidative phosphorylation in
skeletal muscle was evaluated from measurements of rates of ATP
synthesis (using 31P NMR magnetization transfer
experiments) and tricarboxylic acid (TCA) cycle flux (calculated
from the time course of 13C enrichment in C-4 and
C-2 of glutamate during an infusion of [2-13C]acetate).
At the whole body level, we observed no change in energy expenditure.
However, at the cellular level, skeletal muscle UCP3KO increased
the rate of ATP synthesis from Pi more than 4-fold under
fasting conditions (wild type, 2.2 ± 0.6 versus knockout, 9.1 ± 1.4 µmol/g of muscle/min,
p < 0.001) with no change in TCA cycle flux rate
(wild type, 0.74 ± 0.04 versus knockout, 0.71 ± 0.03 µmol/g of muscle/min). The increased efficiency of ATP production may account for the significant (p < 0.05)
increase in the ratio of ATP to ADP in the muscle of UCP3KO mice
(5.9 ± 0.3) compared with controls (4.5 ± 0.4). The data
presented here provide the first evidence of uncoupling activity by
UCP3 in skeletal muscle in vivo.
 |
INTRODUCTION |
Uncoupling protein-1
(UCP1)1 has been shown to
regulate non-shivering thermogenesis in brown adipose tissue by
allowing proton flux across the inner mitochondrial membrane to bypass
ATP synthase (1, 2). The high sequence homology (57%) of UCP1 to UCP3, a protein expressed primarily in muscle, is suggestive of a common physiological function of the two proteins (3, 4). However, the results
of in vitro and in vivo studies of UCP3 casts
doubt on the hypothesis that the primary role of UCP3 is the regulation of energy efficiency in muscle (3-6). Hence, the functional role of
UCP3 in the regulation of mitochondrial oxidative phosphorylation in
skeletal muscle remains uncertain. For example, the up-regulation of
UCP3 protein levels during fasting, when energy efficiency should be
increased, is inconsistent with UCP3 acting as an uncoupling protein
in vivo. Also, studies of energy metabolism in UCP3 knockout mice (UCP3KO) were unable to demonstrate any phenotypic changes in
whole body energy metabolism (3, 4). These mice showed no differences
in body composition, growth characteristics, exercise tolerance, fatty
acid oxidation, or cold-induced thermogenesis. The clearest evidence to
date that UCP3 might have the capacity to act as an uncoupling protein
comes from measurements in isolated mitochondria. These studies
established for the first time that oxidative ATP production in
mitochondria isolated from the muscle of UCP3KO mice were more coupled
compared with normal littermates (3, 4) and that proton leak
over a range of mitochondrial membrane potentials was decreased (4).
Conversely, overexpression of UCP3 in muscle uncoupled oxidative
phosphorylation in isolated mitochondria (7). However, because these
studies were performed in vitro under artificial
concentrations of ADP and substrates, it is difficult to extrapolate
these results to describe the physiologic function of UCP3.
To determine whether the lack of UCP3 in muscle causes changes in the
coupling of skeletal muscle mitochondrial oxidative phosphorylation
in vivo, we performed NMR saturation-transfer experiments to
estimate rates of muscle ATP synthesis and isotopic labeling
experiments to estimate rates of TCA cycle flux. In addition, we used
the doubly labeled water method to determine total energy expenditure
over days in the free moving mice (8). The ratio of ATP synthesis rates
and TCA cycle flux rates provides an index of mitochondrial coupling
in vivo (9, 10). Deletion of UCP3 in muscle may result in
any one or a combination of several potential physiological
perturbations. If UCP3 ablation increases the efficiency of
mitochondrial ATP production, then ATP concentration could be
maintained constant by a corresponding reduction in TCA cycle flux
rate. However, if TCA cycle flux rate remains unchanged, then ATP
production rates should increase, which must then be matched by an
increase in ATP turnover. Finally, if UCP3 has no uncoupling function
in vivo, then neither ATP synthesis nor TCA cycle flux rate
would be affected. We show here that deletion of muscle UPC3 does
result in significant perturbations of mitochondrial energy production
in vivo.
 |
EXPERIMENTAL PROCEDURES |
Animals
Age- and weight-matched wild-type and UCP3KO mice were
studied at 5-7 months of age. The generation of UCP3KO mice has been described previously (3). Animals were housed four per cage in a
temperature-controlled room with a 12-h light/dark cycle with food and
water available ad libitum unless noted otherwise. All
experiments were conducted in accordance with the National Institutes
of Health Guidelines for the Care and Use of Laboratory Animals, and
the protocol was approved the Yale Animal Care and Use Committee.
Doubly Labeled Water Method for Measurement of Whole Body
Metabolic Rate
Dosage and Sampling--
Mice were injected intraperitoneally at
8 a.m. with a solution of D218O (15 µl/g
of mouse, 80% D2O, and 20% H218O,
150 mM sodium chloride). Tail tip bleeds were taken at 24-h intervals over the course of a week.
Analysis for Deuterium Enrichment--
The deuterium atom
percent enrichment of plasma water was determined by reacting 10 µl
of the plasma with calcium carbide to form acetylene gas and analyzed
by GC-MS (electron impact ionization) (11). A small amount (10-15 mg)
of freshly ground calcium carbide was added to a dry crimp-top vial and
capped with a rubber septum. The plasma sample was injected into the
vial through the septum and allowed to react for 5 to 15 min. A 5-µl
sample of the gas phase within the vial was drawn into a
Hamilton glass syringe and injected into the GC-MS. The GC temperature
was isothermal at 180 °C, and ions m/z 26 and
27 were selectively monitored.
Analysis for Deuterium and 18O
Enrichment--
Deuterium and 18O isotopic enrichment of
plasma water was determined by isotope ratio mass spectrometry
(Metabolic Solutions, Inc., Nashua, NH) and are expressed as
V-SMOW
(i.e. the difference from Vienna-standard mean ocean water).
There was no difference in the kinetics of deuterium water turnover as
determined by either method.
Calculations--
Total energy expenditure was determined
from the difference in the rates of loss of the deuterium and
18O isotopes following a bolus injection of doubly labeled
water (i.e. D2O/H218O).
Deuterium is lost as water, whereas 18O is lost either as
water or as C18O2, and the difference in the
rates of isotopic loss (ko = rate of loss of
18O; kh = rate of loss of deuterium)
will be the rate of CO2 output, which is proportional to
whole body metabolic rate (8).
The rate of CO2 loss, rCO2, was calculated
as
|
(Eq. 1)
|
where N is total body water calculated as the mean of
the corrected isotope dilution spaces of D2O and
H218O at the time of the bolus injection.
Total energy expenditure (TEE) was calculated as
|
(Eq. 2)
|
Because it has previously been shown that there were no changes
in the respiratory exchange quotient (RQ) as the result of UCP3
disruption in either the fed or fasted states (3, 4), we used a mean RQ
of 0.79 to calculate total energy expenditure (3). The mean RQ of 0.79 may tend to overestimate the proportion of fat to carbohydrate
metabolized over the 7-day period that total energy expenditure was
estimated but will not mask any differences in total energy expenditure
between the groups.
ATP Flux Rates in Skeletal Muscle in Vivo
All in vivo NMR experiments were performed on
a Bruker (Billerica, MA) Biospec 7.0T system (horizontal/22-cm-diameter
bore magnet). 31P NMR measurement of ATP flux was measured
at 121.66 MHz using a 1-cm diameter inner coil (for 31P)
and a 3-cm outer coil tuned to the proton frequency for scout imaging
and shimming.
On the day of the NMR experiment, overnight-fasted mice were secured in
a Plexiglas tube restrainer that allowed the right hind limb to be
secured to the outside of the tube and centered over concentric surface
coils. The mouse and probe assembly were then placed in the magnet isocenter.
Unidirectional rates of ATP synthesis from Pi are the
product of the rate constant of ATP synthesis and the concentration of
intracellular Pi. The unidirectional rate constant for ATP synthesis from Pi was measured using the
saturation-transfer experiment (9, 10). The steady-state Pi
magnetization, Mz, was measured with selective
irradiation of the
-ATP resonance, and compared with the equilibrium
Pi magnetization, Mo, with the selective
irradiation placed symmetrically down field from the Pi
frequency. T1*, the spin lattice relaxation time
for Pi when ATP is saturated, was measured using a modified
version of the inversion recovery experiment
(180°-
-90°-acquire-ID) in the presence of a steady-state
saturation of
ATP during the interpulse delay (ID) of
4 s and during the variable delay,
.
Pi concentration was calculated from the
ratio of integrated peak intensities (T1 corrected) of
Pi and
-ATP. The concentration of ATP was determined by
HPLC as described below.
TCA Cycle Flux Rates in Skeletal Muscle
Isotopic Measurement of TCA Cycle Flux Rates--
The mice were
studied 4 days after a catheter was surgically implanted into their
jugular veins. Following an overnight fast, the mice were placed in
tube restrainers, and an infusion of [2-13C]acetate at a
rate of 4 µg/g of mouse/min was begun. Blood samples for
determination of acetate concentration and isotopic enrichment were
obtained immediately prior to the end of the experiment by tail tip
bleeds. TCA cycle flux was calculated from the time course of
13C enrichment in glutamate from perchloric acid
extracts of freeze-clamped muscle sacrificed at a various times after
the start of the [2-13C]acetate infusion. At the end of
the infusion period (variable from 2 to 90 min), the mice (controls,
n = 12, UCP3KO, n = 12) were sacrificed
and the muscles freeze-clamped in situ for the determination
of glutamate concentration and 13C enrichment.
Analysis of [2-13C]Acetate and
[13C]Glutamate--
Plasma acetate concentration and
13C isotopic enrichment were determined by GC-MS as
described in detail elsewhere (12). Briefly, 50 µl of plasma was
spiked with an equal volume of an internal concentration standard
containing 50 µM sodium acetic-d3 acid. To
this solution was then added 50 µl of 0.1 M HCl and ~50
mg of solid NaCl. The acetate was then coupled to 2,4-difluoroaniline (100 µl of reagent, 0.2 M in hexane) using
1,3-dicyclohexylcarbodiimide (100 µl of reagent, 0.2 M in
toluene). The solution was capped and mixed for 1 h. After 1 h, 1 ml of 1 M sodium bicarbonate was added, and the
acetyl-2,4-difluoroanilide was extracted (3×) into ethyl acetate (1 ml). The organic layer was dried over sodium sulfate and concentrated
under a stream of dry nitrogen gas for GC-MS analysis. 13C
and d3 isotopic enrichments were corrected using standard
curves at similar concentrations and processed as described above for the plasma samples. Analysis of the d3-acetate internal
standard yielded a background acetate concentration of 8.0 ± 0.04 nM in the blank, which was less than 8% of the
measured plasma acetate concentration. The plasma concentrations given
are corrected for the acetate concentration in the blank.
Analyses were carried out using a Hewlett-Packard (H-P) 5973MSD
interfaced to an H-P 6890 gas chromatograph. The samples were then
analyzed using electron ionization at 70 eV by monitoring ions
m/z 171, 172, and 174.
Glutamate concentration and 13C enrichment in the muscle
were determined as follows. 100-200 mg of muscle was weighed,
homogenized in ice-cold perchloric acid (0.9 M), and
neutralized with KOH. Glutamate concentration was measured in a 10-µl
aliquot with a 2700 STAT Plus analyzer (Yellow Springs Instruments,
Yellow Springs, OH) using a glutamate ion-specific electrode. The
enrichment of glutamate at C-2, C-3, and C-4 was determined following
purification from any glutamine by anion exchange chromatography as
follows. The pH of the supernatant was adjusted to ~8, and the sample
applied to a 1-ml mini-column of anion exchange resin (AG1-X8, Bio-Rad) in a pasture pipette. Glutamine was eluted with 5 ml of water, and
glutamate was then eluted with 5 ml of acetic acid (0.5 M). The glutamate fraction was freeze-dried and reconstituted in 400 µl
of D20 for analysis of concentration and enrichment. The
relative enrichment at each carbon position was determined by
13C-NMR spectroscopy and total enrichment by GC-MS (CI
ionization with isobutane) as its trifluoroacetyl
n-butyl ester derivative (m/z 356, 357, 358) (13).
Calculation of TCA Cycle Flux Rates--
Rates of TCA cycle flux
were calculated from the time course of 13C isotopic
enrichment of plasma acetate and muscle glutamate C-2 and C-4 by
iterative fitting of metabolic simulations to the data using the
program Cwave (15). Mass and isotope balance equations were derived
using the model of TCA cycle as shown in Scheme
1. The isotopic enrichment and
concentrations of plasma acetate, glucose, and free fatty acids are
used as input drivers. Using these input parameters, flux rates are
determined that provide the best fit to the observed time course of
enrichment in [2-13C]- and
[4-13C]glutamate.

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Scheme 1.
Intramuscular label flow resulting from
[2-13C]acetate administration. Vac,
flux of acetate into acetyl-CoA; Vdil, combined flux
of glucose and fat into acetyl-CoA; Vglu, flux of
a-ketoglutarate to glutamate; Voaa, flux of
oxaloacetate to aspartate; VTCA, TCA
cycle flux.
|
|
The mass balance equations used were as follows.
|
(Eq. 3)
|
|
(Eq. 4)
|
|
(Eq. 5)
|
The isotope balance equations used were as follows,
|
(Eq. 6)
|
|
(Eq. 7)
|
|
(Eq. 8)
|
|
(Eq. 9)
|
|
(Eq. 10)
|
|
(Eq. 11)
|
|
(Eq. 12)
|
|
(Eq. 13)
|
where FE is the fractional enrichment calculated as the quotient
of the mass of 13C-labeled molecules and the total mass of
labeled and unlabeled molecules. The position of the
13C-label is denoted in brackets. Ac is plasma acetate,
AcCoA is acetyl-CoA, Cit is citrate, Glu is glutamate,
KG is
-ketoglutarate, ffagluc is the combined pools of plasma free fatty
acid and glucose, and Vsubscript is the flux through the
pathways as shown in the schematic, where Vdil is the
combined flux into acetyl-CoA from free fatty acids and glucose.
Determination of ATP, ADP, and AMP Concentrations in Skeletal
Muscle--
The concentrations of ATP, ADP, and AMP in the muscle of
overnight-fasted UCP3KO mice and control mice were determined at the
end of the acetate infusion or NMR experiments. Muscles were freeze-clamped in situ and then extracted with 0.9 N ice-cold perchloric acid. The concentrations of
nucleotides in the supernatant were then determined by HPLC using a
modification of the method described by Sabina et al.
(14). ATP, ADP, and AMP in a 20-µl sample of the muscle
extract were separated on a Supelcosil SAX1 (25 cm × 4.6 mm × 0.5 µm) column using a gradient of 5 mM ammonium phosphate, pH 2.8 (buffer A) and 750 mM ammonium phosphate,
pH 3.9 (buffer B) at a flow rate of 1 ml/min. A linear gradient was developed over 14 min at 0% buffer B to 9% buffer B, then from 14 to
32 min from 9% buffer B to 100% buffer B. A Rainin HPXL solvent
delivery system (two pumps) with a Rainin Dynamax UV-1 absorbance
detector (254 nm) controlled by Rainin Dynamax HPLC Method Manager was
used for solvent programming and data collection. Peak identification
was assigned by comparison of retention times to known external
standards (AMP ~ 5.5 min, ADP ~ 25.7 min, ATP ~ 29.0 min). Nucleotide concentrations were calculated from the concentration standard curves of absorbance for the external standards.
 |
RESULTS AND DISCUSSION |
Whole Body Metabolism--
During the 7 days that the mean rates
of total energy expenditure were measured, the mice had free access to
food and water and unrestricted movement. Deuterium (normal
mice, 3.5 + 0.4; UCP3KO, 3.1 + 0.1) and 18O (normal
mice, 0.88 ± 0.05; UCP3KO, 0.78 ± 0.03) fractional
enrichments of plasma water at t = 0 were determined
from extrapolation of the semilog plots. From the deuterium enrichment
of plasma at t = 0 and the bolus injection of doubly
labeled water, the body water was calculated to be 64 ± 9% in
the controls and 65 ± 8% in the UCP3KO (p = NS (not significant)). From the difference in the rates of decay of
deuterium and 18O enrichments (Fig.
1), we calculated mean rates of
CO2 production of 37 ± 2 ml/kg/min for the normal
mice and 37 ± 1 ml/kg/min for the UCP3KO mice. From the mean
rates of CO2 production, we calculated free range total
energy expenditure of 325 ± 8 kcal/kg/day in the normal mice and
321 ± 15 kcal/kg/day in the UCP3KO mice. In agreement with
previous results (3, 4), a lack of UCP3 in skeletal muscle did not
cause any detectable phenotypic changes in energy metabolism at the
whole body level.

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Fig. 1.
Decay curves (semi-log) for loss of
D2O (triangles) and
H218O (circles) from normal
(upper plot) and UCP3KO (lower plot)
mice following a bolus intraperitoneal injection of doubly labeled
water. The fractional enrichments (FE) are those
obtained by ISRMS and are expressed in relative /million
units using Vienna-standard mean ocean water as the standard. The mice
were unrestrained and had free access to food and water. Isotopic
enrichment in the body water was determined from plasma obtained by
tail tip bleeds. Each point is the mean of 6 mice. Standard deviations
are shown but are masked by the data point symbol.
|
|
ATP, ADP, and AMP Concentrations--
Mean concentrations of ATP,
ADP, or AMP in the UCP3KO mice were not significantly different from
their littermates (Table I). However, the
mean ratio of ATP to ADP determined for each individual mouse was
significantly higher in the UCP3KO mice compared with the controls. As
shown previously (3), the deletion of UCP3 in skeletal muscle increased
the proportion of ATP to ADP in the tissue.
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Table I
Phosphorous metabolites in skeletal muscle of wild-type (WT) and UCP3KO
(KO) overnight-fasted mice
Concentrations (µmol/g of tissue) were determined as described under
"Experimental Procedures." Unpaired Student's t test
was used for the statistical analysis. Results are expressed as the
mean ± S.E. For ATP, ADP, and AMP: WT, n = 5; KO,
n = 11. For Pi: WT, n = 7, KO,
n = 7.
|
|
Coupling of ATP Synthesis and TCA Cycle Flux--
In comparison
with normal mice, mitochondria isolated from the muscle of UCP3KO mice
have a lower rate of respiration under state 4 conditions (3) and
decreased proton leak (4), indicating that UCP3 functions as an
uncoupler of oxidative phosphorylation. However, the up-regulation of
UCP3 protein in skeletal muscle under fasting conditions is
counterintuitive to the need to increase the efficiency of energy
production. To determine whether UCP3 functions as an uncoupler of
mitochondrial ATP production in vivo, we used a combination
of NMR and isotopic labeling methods to assess the role of UCP3 in the
regulation of skeletal muscle mitochondrial energy production.
A representative 31P-NMR saturation-transfer experiment to
determine mitochondrial ATP synthesis rates is shown in Fig.
2, and the mean results are presented in
Table II. From these experiments, we determined that the rate
constant of skeletal muscle ATP synthesis from Pi was
~2-fold higher (p < 0.001) in the UCP3KO mice
compared with the control mice. We
observed no difference in the T1 relaxation time
for Pi. From the observed rate constants and Pi
concentrations, we calculated that the rate of skeletal muscle of ATP
synthesis under fasting conditions is ~4-fold higher in the UCP3KO
mice compared with normal mice (Table II). A conservative estimate of
flux rates, using the mean Pi concentration (0.64 ± 0.11 µmol/g of tissue), gave a 2-fold increase in the ATP synthesis
rates in the UCP3KO mice (0.120 ± 0.10 µmol/g of muscle/s)
compared with normal mice (0.059 ± 0.10 µmol/g of muscle/s).

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Fig. 2.
Typical 12-min spectra of the
31P-NMR saturation-transfer experiment for determination of
unidirectional Pi to ATP flux in the hind limbs of awake
mice.
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Table II
Saturation-transfer measurements of ATP synthesis rates in the hind
limb muscle of overnight-fasted wild-type (WT) and UCP3KO (KO) mice at
rest
The magnetization-transfer (WT, n = 8; KO,
n = 12) and T1 (WT,
n = 4; KO, n = 3) measurements were
made as described under "Experimental Procedures."
Mz/Mo, ratio of steady-state to
equilibrium magnetization; k, rate constant for
unidirectional synthesis of ATP from Pi. Unpaired Student's
t test was used for the statistical analysis. Results are
expressed as the mean ± S.E.
|
|
Steady-state plasma [2-13C]acetate enrichments (normal
mice, 21.1 ± 4.6; UCP3KO, 18.7 ± 2.4, p = NS) and concentrations were reached within 5 min. The mean basal plasma
acetate concentrations were slightly but not significantly higher in
the UCP3KO mice (controls, 0.087 ± 0.006 mM; UCP3KO,
0.108 ± 0.006 mM). During the infusion of
[2-13C]acetate, acetate concentrations rose to 0.109 ± 0.001 mM in the controls (p = 0.02 compared with basal) and to 0.124 ± 0.015 mM
(p = NS compared with basal). Steady-state plasma
acetate concentrations were not significantly different in the controls
compared with the UCP3KO mice. Glutamate 13C enrichments
reached a plateau of 8% within 30 min. From the time course of
glutamate C3 and C4 enrichment, we calculate similar TCA cycle flux
rates in both groups of mice (normal mice, 0.74 ± 0.04 µmol/g/min; UCP3KO, 0.71 ± 0.03 µmol/g/min).
The ratios of the rates of ATP synthesis to TCA cycle flux provide an
in vivo index of mitochondrial coupling in the muscle (Fig.
3). Our results indicate that, under
fasting conditions, the coupling of oxidative phosphorylation was
2-4-fold higher in the skeletal muscle of UCP3KO mice compared with
wild-type mice. These results are consistent with earlier studies in
rats in which an increase in UCP3 expression, induced by T3 treatment (9) or fasting (10), was associated with an increase in mitochondrial uncoupling. However, in contrast to these earlier studies where the
observed increased mitochondrial uncoupling was the result of an
increased TCA cycle flux (i.e. the denominator), the change in coupling that we observed in the UCP3KO mice was the result of
changes in ATP synthesis rates (i.e. the numerator).

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Fig. 3.
Rates of unidirectional ATP synthesis
(upper plot), TCA cycle flux (TCA,
middle plot), and the coupling index (lower
plot) calculated as the ratio of the rates of ATP synthesis
and TCA cycle flux in normal and UCP3KO mice after an overnight
fast.
|
|
The qualitative difference in the regulation of mitochondrial coupling
due to these perturbations may indicate that the regulation of
mitochondrial function is dependent on other factors in addition to the
demand for ATP. An increase in UCP3 content (such as those caused by T3
treatment (9), fasting (10), or transgenic overexpression of UCP3 (7)
or UCP1 (16) in muscle, etc.) would tend to decrease the membrane
potential and subsequently the rates of ATP synthesis. However, because
cellular energy demands are not decreased, substrate oxidation must
increase to maintain the membrane potential necessary for adequate ATP
synthase proton flux. In contrast, ablation of UCP3 leads to an
increase in membrane potential while not inhibiting substrate
oxidation. If the mitochondrial response of the UCP3KO mice was to
simply reduce substrate oxidation to maintain constant ATP synthesis
rates, then the NADH/NAD ratio could increase to abnormally high levels
and profoundly disrupt rates of numerous cellular
oxidation-reduction reactions.
Under the steady-state conditions of our experiment, the increased rate
of ATP synthesis must be balanced by an equivalent rate of ATP
hydrolysis. Metabolic control theory provides one of the best tools
currently available to describe the complex interactions that multiple
pathways exert on each other to regulate metabolite concentrations and
enzymatic flux rates, supporting the possibility that the rate of ATP
hydrolysis is determined by the rate of ATP synthesis (17). Analysis of
the factors responsible for regulating the rate of ATP consumption
reveals that the rate of ATP consumption is not controlled exclusively
by the reactions of ATP hydrolysis but that control is shared to a
similar degree by the reactions of ATP production (i.e.
mitochondrial phosphorylation and oxidation) and ATP hydrolysis (17).
It is likely that the UCP3KO mice maintain ATP concentrations constant
by increasing the rate of one of the ATP-consuming futile cycles in the
muscle, the major ones being Na+-K+-ATPase,
Ca2+-ATPase, and protein turnover (18). Whether increased
ATP utilization is occurring in all reactions that utilize ATP via a
mass action effect, or is occurring in a subset of reactions involving
systems that are designed to sense ATP (e.g.
KATP channels, membrane depolarization, or ion
pumping), remains to be determined.
In conclusion, the results presented here provide the first direct
evidence for alterations of energy metabolism in UCP3KO mice in
vivo. UCP3 disruption in skeletal muscle resulted in a doubling of
the ATP synthesis rate without any increase in TCA cycle flux rate,
under fasting conditions, implying an increased degree of mitochondrial
energy coupling. Therefore, these data suggest an important role for
UCP3 in the regulation of skeletal muscle mitochondrial energy
metabolism in vivo.
 |
FOOTNOTES |
*
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.
Present address: University of Cambridge, Depts. of Clinical
Biochemistry and Medicine, Addenbrooke's Hospital, Box 232, Cambridge CB2 2QR, UK.
§
To whom correspondence should be addressed: Dept. of Internal
Medicine, P. O. Box 9812, 295 Congress Ave., New Haven, CT 06510. Tel.: 203-785-5934; E-mail: gary.cline@yale.edu.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M102540200
 |
ABBREVIATIONS |
The abbreviations used are:
UCP, uncoupling
protein;
UCP3KO, uncoupling protein-3 knockout;
GC-MS, gas
chromatography-mass spectrometry;
RQ, respiratory exchange quotient;
HPLC, high pressure liquid chromatography;
TCA, tricarboxylic
acid.
 |
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