1 Section of Neurobiology, The molecular basis
for variations in resting metabolic rate (RMR) within a species is
unknown. One possibility is that variations in RMR occur because of
variations in uncoupling protein 2 (UCP-2) and uncoupling protein 3 (UCP-3) expression, resulting in mitochondrial proton leak differences.
We tested the hypothesis that UCP-2 and -3 mRNAs positively correlate
with RMR and proton leak. We treated thyroidectomized and sham-operated
mice with triiodothyronine (T3)
or vehicle and measured RMR, liver, and skeletal muscle mitochondrial nonphosphorylating respiration and UCP-2 and -3 mRNAs.
T3 stimulated RMR and liver UCP-2
and gastrocnemius UCP-2 and -3 expression. Mitochondrial respiration
was not affected by T3 and did not
correlate with UCP-2 and -3 mRNAs. Gastrocnemius UCP-2 and -3 expression did correlate with RMR. We conclude
1)
T3 did not influence intrinsic mitochondrial properties such as membrane structure and composition, and 2) variations in UCP-2 and -3 expression may partly explain variations in RMR. One possible
explanation for these data is that
T3 stimulates the leak in vivo but
not in vitro because a posttranslational regulator of UCP-2 and -3 is
not retained in the mitochondrial fraction.
proton leak; thyroid hormone; mitochondria; uncoupling protein
EIGHTY-FIVE TO NINETY PERCENT of whole body oxygen
consumption is attributable to the mitochondria (54). In liver and
skeletal muscle, proton leak is an important contributor to
mitochondrial respiration (55). Brand et al. (3) have reported that
26% of isolated rat hepatocyte and 50% of perfused rat hindlimb
skeletal muscle mitochondrial respiration occur to support proton leak in these tissues. Moreover, liver and skeletal muscle mitochondrial proton leak has been estimated to account for 20% of rat resting metabolic rate (RMR; Refs. 53, 54). Thus the well-documented effects of
thyroid hormone on RMR may involve changes in proton leak. In support
of this, Hafner et al. (21) and Harper and Brand (22, 24) have observed
higher rates of proton leak in isolated mitochondria as well as in
mitochondria within isolated hepatocytes from hyperthyroid compared
with euthyroid rats; they have also reported lower hepatocyte
mitochondrial proton leak rates in hypothyroid compared with euthyroid rats.
Thyroid hormone status can affect the total surface area of the inner
mitochondrial membrane as well as its phospholipid composition (5, 12,
28, 31, 32, 56). Because phospholipid bilayers are inherently leaky to
protons (7-9, 18), a change in inner membrane surface area is one
mechanism by which triiodothyronine (T3) may influence proton leak.
Phospholipid composition may also be important because the degree of
mitochondrial proton leak correlates with the number and type of
unsaturated fatty acids found within mitochondria (4, 52). However,
liposomes of differing fatty acid compositions (prepared from
mitochondria differing in composition) do not exhibit differences in
proton leak (6). Although this indicates that compositional differences
do not affect the inherent leakiness of lipid bilayers, this does not
exclude the possibility that such differences influence proton leak in
intact mitochondria via interactions with membrane structure or
integral membrane proteins (6, 7). Thus the inner mitochondrial
membrane phospholipid bilayer may be one target of thyroid
hormone-induced changes in proton leak.
A third possible mechanism by which thyroid hormone may affect proton
leak is by inducing expression and synthesis of one or more proteins
that facilitate proton transport across the inner mitochondrial
membrane. This effect may involve uncoupling protein 2 (UCP-2) and/or
uncoupling protein 3 (UCP-3) because these proteins, when expressed in
yeast or myoblast cells, reduce mitochondrial membrane potentials (1,
17, 19, 20, 41) and are upregulated at the mRNA level by relatively
high concentrations of T3 (20, 38,
39, 42). However, there is no direct evidence that UCP-2 or UCP-3
facilitates proton leak in mammalian tissues. Furthermore, integrating
studies of proton leak with those of UCP expression in an effort to
define the role of these proteins in the thermogenic action of thyroid
hormone is difficult because 1) no
study to date has examined the effect of thyroid hormone on whole body oxygen consumption, mitochondrial nonphosphorylating respiration (an
index of proton leak), and UCP-2 and UCP-3 expression in the same
animal; 2) studies that have
addressed the effects of thyroid hormone on proton leak have primarily
been confined to liver, whereas those that have addressed the effects
of thyroid hormone on UCP-2 and UCP-3 expression have focused on heart,
skeletal muscle, and adipose tissue; and
3) the
T3 concentrations used in these
studies were variable and of different durations.
Thus the goal of our study was to measure whole body and mitochondrial
oxygen consumption as well as UCP-2 and UCP-3 expression from the same
animal treated with a relatively low concentration of
T3 to approximate physiological
levels (49). In this way, we could test the hypothesis that UCP-2 and
UCP-3 expression positively correlates with RMR and with mitochondrial
proton leak. For this, we treated hypothyroid and euthyroid mice for 6 days with 2.5 µg T3 per 100 g
body mass or vehicle and subsequently measured whole animal RMR, liver
and skeletal muscle mitochondrial oxygen consumption, and UCP-2 and
UCP-3 expression in these tissues as well as in heart.
Animals.
Thyroidectomized-parathyroidectomized
(Tx) and sham-operated male
C57BL/6 mice were obtained from Taconic (Germantown, NY). Surgeries
were performed when the mice were ~1 mo old. The mice were delivered
to us within 3 days after the surgery. They were housed four per cage
for 5-8 wk at 22°C on a 14-10 light-dark cycle (lights on
0600) with free access to food (Purina 5001 chow) and water. Because
the parathyroid gland was also removed,
Tx mice received 2% calcium
lactate in their drinking water. At the beginning of the experiment,
mice were housed two per cage according to their surgical category. One
mouse per cage received T3
injections at 2.5 µg/100 g body mass (in 100-140 µl), whereas
the other received vehicle (5 mM NaOH; 100-140 µl). Injections
were given intraperitoneally once a day for 6 consecutive days. Mice
were weighed daily and allowed free access to chow and water during the
treatment. Pooled serum thyroid-stimulating hormone levels averaged 964 ng/ml in Tx + vehicle
mice (range 1673-255), 208 ng/ml in
Tx + T3 mice (range 218 to 198), 223 ng/ml in sham + vehicle mice (range 232-213), and 196 ng/ml in
sham + T3 mice (range
199-193). Mouse thyroid-stimulating hormone RIA was developed and
performed by Dr. A. F. Parlow, Scientific Director, National Hormone
and Pituitary Program, Harbor-University of California Los Angeles
Medical Center (Torrance, CA).
RMR measurements. Immediately after
the sixth and final injection of
T3 or vehicle (given between 1130 and 1200), mice were transported to the laboratory and kept overnight
in a quiet room having the same light cycle and environmental
temperature as previously described. Between 0630 and 0700 the day
after the final injection, mice were placed into individual 220-ml
metabolic chambers after measurement of their colonic temperatures and
body masses. The chambers were positioned in a water bath to maintain
chamber temperatures at 28 ± 1°C, which is near the
30-32°C thermoneutral zone for mice. The rate of dessicated
air delivered to each chamber was held constant at 300 ml/min with mass
flow controllers calibrated to a single gas flow gauge. The air exiting
each chamber was passed through dessicant (Drierite) and
CO2 absorbent (Baralyme) before the fractional oxygen content was measured with an Ametek oxygen analyzer. Over the course of the measurements, drift in the oxygen analyzer was checked periodically by sampling air exiting an empty chamber. Any drift in the oxygen analyzer over the time period of
measurement was assumed to be linear. Measurements were documented with
a strip chart recorder. Oxygen consumption was calculated as described
by Hill (27).
On any given trial day, two mice were measured. They were acclimated to
the chambers for ~4 h before oxygen consumption measurements were
started, which were made over the next 4-5 h. RMR was assessed from oxygen consumption measurements during periods in which the mice
were inactive. At the end of the measurements, mice were killed by
decapitation and colonic temperatures were immediately measured. Liver, hindlimb skeletal muscle, and heart were
rapidly removed for mitochondrial isolation and/or RNA analysis. One
liver lobe was saved for RNA analysis, and the rest of it was
immediately used for isolation of mitochondria. The gastrocnemius and
soleus muscles from one leg were removed and saved for RNA analysis, and the rest of the hindlimb skeletal muscle on both legs was immediately used for isolation of mitochondria.
RMR changes with body mass, and this change is described by a power
function (RMR = aMb, where a is
the mass coefficient, M is body mass, and b is the mass exponent).
Although there has been debate as to the value of b, Heusner has
provided both theoretical and empirical evidence that b = 2/3 (25, 26).
That is, when b = 2/3, the variation in RMR that can be explained by
the variation in body mass is removed, and any difference remaining in
the RMR values can be attributed to intrinsic differences in the
animals being studied. Because the body masses of the sham-operated
mice in this study were significantly greater than those of the
Tx mice, we wanted to
express RMR on a mass-independent basis so that we could ask the
question: would RMR differ between the groups if all the mice had the
same weight? We found that 25% of the variation in RMR of all mice
could be accounted for by variations in body mass, as evidenced by
regression analysis (data not shown). Differences in RMR manifested
after dividing by body mass to the power 2/3 suggested that thyroid
status affected one or more intrinsic properties of the mass itself.
Changes in body composition and in the metabolic activity of the mass
(including proton leak) are two primary factors dictating the intrinsic
nature of the mass.
Mitochondrial isolations and measurement of
nonphosphorylating respiration. Liver and hindlimb
skeletal muscle (excluding the gastrocnemius and soleus muscles on one
leg, which were used for RNA isolation) were homogenized in (mM) 250 sucrose, 50 HEPES, 10 EDTA, 1 EGTA, and 0.2 phenylmethylsulfonyl
fluoride, pH 7.1, with a Tekmar Tissumizer. After two centrifugations
for 15 and 20 min at 750 and 1000 g,
respectively, mitochondria were pelleted by centrifugation for 12 min
at 10,000 g. The pellets were washed of loosely packed material by swirling with 5 ml of homogenization buffer. After the pellets were resuspended in 30 ml of buffer, the
mitochondria were pelleted and washed as before. The final pellet was
resuspended in the same buffer lacking EDTA. Once the tissues were
removed, all procedures were carried out at 0-5°C.
Nonphosphorylating respiration of 0.2-0.7 mg liver mitochondrial
protein or 0.12-0.28 mg skeletal muscle mitochondrial protein was
measured at 37°C with a Clark type electrode in air-saturated buffer containing (final concentration) 120 mM KCl, 5 mM NaCl, 5 mM
KH2PO4/K2HPO4,
5 mM EGTA, 2 mM MgCl2, 0.1 mM Mg
acetate, 50 mM HEPES, 5 µM rotenone, and 3.3 µg/ml oligomycin B
(final pH 6.9). We found no correlation between the amount of protein used to measure respiration rates and the rates of oxygen consumption expressed per milligram of protein. Thus the preparation-to-preparation variation in the oligomycin-to-protein ratio had no effect on the
measured rates of nonphosphorylating respiration. Oligomycin B was
added to inhibit ATP synthesis; rotenone was added to inhibit oxidation
of NADH via complex I of the respiratory chain. Respiration was
initiated by addition of succinate to 5 mM. Nonphosphorylating respiration was measured over 5-10 min. Aliquots of each
preparation were saved for quantification of total protein with the
bicinchoninic acid assay (Pierce, Rockford, IL) with BSA as standard.
Northern blot analysis. Total RNA was
extracted from liver, gastrocnemius, soleus, and heart with Trizol
reagent (Gibco, Gaithersburg, MD) according to the instructions of the
manufacturer. Excluding soleus samples, 20 µg of total RNA from each
tissue were fractionated by electrophoresis on a 1% agarose gel
containing 2.2 M formaldehyde. Preliminary experiments indicated that
the amount of RNA obtained from each soleus was generally insufficient
for individual determinations of transcript levels, so the RNA samples
within a group were pooled in pairs. The final soleus RNA preparations
from Tx + vehicle mice (where
n = 5) were the exception, consisting
of two pooled and one individual sample. Ethidium bromide was added to
each electrophoresed sample so that RNA integrity and loading could be
verified. On a given gel, three samples from each treatment group were
present. RNA was transferred to nylon membranes (Duralon-UV; Stratagene, LaJolla, CA) by overnight capillary transfer and then ultraviolet cross-linked.
Mouse UCP-2 and UCP-3 cDNA clones were obtained from the IMAGE
consortium (GenBank access no. W71569 for UCP-2 and AA062091 for
UCP-3). We confirmed the identity of these clones by direct sequencing
with the ThermoSequenase radiolabeled terminator cycle sequencing kit
(Amersham Life Science, Cleveland, OH). Clones were excised by
EcoR
1/Not 1 digestion and gel purified
with the Qiaquick gel extraction kit (Qiagen, Santa Clarita, CA).
Labeled probes were generated by random priming (Rediprime kit,
Amersham, Buckinghamshire, UK) with
Blots were prehybridized 30 min at 68°C in Express Hyb solution
(Clontech, Palo Alto, CA) and then hybridized for 60 min in the same
solution containing labeled cDNA at 2 × 106
counts · min Statistics. Unless otherwise stated,
data were analyzed by two-way ANOVA, with surgery and treatment as main
effects. The level of significance was
P < 0.05. When significant main
effects were observed, they were followed with a one-way ANOVA with the Tukey's post hoc test to discern individual group differences.
Before beginning injections (and 5-8 wk after surgeries), sham
mice weighed significantly more than did
Tx mice (24.66 ± 0.31 vs.
22.82 ± 0.49 g; P < 0.01 by
t-test).
T3 treatment significantly increased body weight gain (Tx + T3: 1.01 ± 0.44 g and sham + T3: 1.34 ± 0.47 g vs.
Tx + vehicle: 0.33 ± 0.52 g
and sham + vehicle: 0.11 ± 0.19 g;
P = 0.037 for main effect of
treatment). Tx mice tended to have
lower colonic temperatures (P = 0.07)
despite no effect of thyroidectomy on RMR (Table
1). Mice treated with
T3 had significantly greater RMR
than those treated with vehicle (Table 1). Although
T3 treatment did not have a
significant effect on colonic temperatures measured at the beginning of
the experiment (0700, 1 h after lights on), the treatment significantly
attenuated the fall in rectal temperature observed in all mice over the
7-8 h they were in the metabolic chambers (Table 1). Final colonic temperatures at this time positively correlated with RMR (Fig. 1).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P-dCTP (3000 Ci/mmol,
Amersham). Unincorporated nucleotides were removed with NucTrap columns (Stratagene).
1 · ml
1.
After the blots were washed [twice at room temperature in
2× saline sodium citrate (SSC)/0.05% SDS; twice at
60-65°C in 1× SSC/0.1% SDS], they were exposed to
a storage Phosphor screen (Eastman Kodak, Rochester, NY) overnight;
signal intensities were quantified with ImageQuant software (Molecular
Dynamics, Sunnyvale, CA). Quantification of 18S ribosomal RNA
(DECAprobe template, Ambion; Austin, TX) was performed in a similar
manner to control for variations in the total amount of RNA loaded into
each lane. The data are expressed as the ratio of UCP-2 or UCP-3 signal
to 18S signal. Two blots were made to accommodate all samples from a
given tissue; these blots were probed in the same hybridization buffer
to avoid variations due to differences in probe specific activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Table 1.
Effects of surgery and treatment on colonic temperature, resting
metabolic rate, and nonphosphorylating mitochondrial oxygen consumption
View larger version (11K):
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Fig. 1.
Colonic temperature at time of death
(TC final) as a function of
resting metabolic rate (RMR). Immediately after RMR measurements by
indirect calorimetry (see MATERIALS AND
METHODS), TC was
measured as mice were killed by decapitation. Data represent all 23 mice in 4 experimental groups. , Thyroidectomized
(Tx) + vehicle (V) group;
,
Tx + triiodothyroinine
(T3) group;
, sham (Sh) + V
group;
, Sh + T3 group. A line
was fitted to data with simple linear regression analysis. Correlation
coefficient
(r2) was 0.187 (P = 0.044).
TC taken before RMR measurements
(TC initial, within 1 h of lights
on) was not correlated with RMR (P = 0.91). BM, body mass.
Nonphosphorylating respiration rates of hindlimb skeletal muscle
mitochondria isolated from Tx mice
were significantly lower than those from sham mice; liver mitochondrial
respiration rates from Tx mice
tended to be lower than sham mice, but this was not significant (Table
1). Mitochondrial nonphosphorylating respiration rates from both
tissues were unaffected by T3
treatment. Surprisingly, liver mitochondrial respiration negatively
correlated with RMR (Fig. 2), whereas
skeletal muscle mitochondrial respiration did not correlate with RMR
(r2 = 0.054, P = 0.43; data not shown).
|
To determine if expression of UCP-2 and UCP-3 was correlated with
mitochondrial proton leak, we quantified steady-state mRNA levels in
the same tissues from which mitochondrial respiration measurements were
made. T3 treatment significantly
increased gastrocnemius UCP-2 and UCP-3 steady-state mRNA levels (Figs.
3 and 4), with the effect being greater in Tx
mice (63 and 60% for UCP-2 and UCP-3, respectively) than in sham mice
(27 and 46% for UCP-2 and UCP-3, respectively), although the surgery × treatment interaction was not significant. Gastrocnemius UCP-2
and UCP-3 mRNA levels significantly correlated with RMR (Fig.
5) but did not significantly correlate with
muscle mitochondrial nonphosphorylating respiration (UCP-2:
r2 = 0.058, P = 0.41; UCP-3:
r2 = 0.119, P = 0.23; data not shown). Soleus
UCP-2 and UCP-3 levels were unaffected by surgery or treatment (data
not shown). We were unable to determine if soleus UCP-2 and UCP-3
levels were correlated with RMR or mitochondrial respiration because
soleus samples were pooled for Northern analysis.
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Liver UCP-2 mRNA levels were affected by surgery and treatment (Fig.
6); the levels were stimulated by
T3 to a similar extent in
Tx (60%) and sham (71%) mice
compared with their respective controls. However, UCP-2 levels did not
significantly correlate with RMR
(r2 = 0.137, P = 0.09; data not shown) or with
liver mitochondrial nonphosphorylating respiration
(r2 = 0.022, P = 0.51; data not shown). Heart UCP-2
was unaffected by surgery or treatment (Fig.
7A),
although Tx mice given
T3 had levels 39% greater than
controls. Similarly, T3 treatment
tended to upregulate heart UCP-3, with this effect just failing to
reach statistical significance (Fig.
7B; P = 0.06). As was the case with gastrocnemius UCP levels,
T3 tended to stimulate heart UCP-3 expression more in Tx (57%) than
in sham (27%) mice. Heart UCP-3 levels significantly correlated with
RMR, whereas the levels of UCP-2 were positively, but not
significantly, correlated with RMR (Fig.
8).
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DISCUSSION |
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A major finding of this study is that T3 stimulates RMR but not isolated liver and skeletal muscle mitochondrial nonphosphorylating respiration. Because nonphosphorylating respiration is an index of proton leak activity (2), we conclude that the stimulatory effect of T3 on RMR is not accompanied by greater proton leak of isolated liver and skeletal muscle mitochondria. This conclusion, although requiring further scrutiny by determining the proton leak kinetics as described by Brand (2), suggests that either liver and skeletal muscle mitochondrial proton leak do not contribute to the stimulatory effect of T3 on RMR or the leak in vitro does not reflect that which occurs in vivo. We believe the latter alternative is important to consider given studies reporting that liver and skeletal muscle mitochondrial proton leak accounts for 20% of rat RMR (53, 54) as well as those suggesting that differences in proton leak partly explain the differences in mass-specific metabolic rates of animals having different body masses (50-52).
Another finding of this study is that T3 stimulates expression of liver UCP-2 and gastrocnemius and heart UCP-2 and UCP-3. Moreover, gastrocnemius UCP-2 and UCP-3 expression, and to a lesser extent, liver UCP-2 expression, positively correlate with RMR but not with isolated skeletal muscle and liver mitochondrial nonphosphorylating respiration. Heart UCP-2 and UCP-3 also positively correlate with RMR (we did not measure heart mitochondrial respiration because of limited amounts of this tissue). There are several possible reasons to account for the discordance between UCP-2 and UCP-3 expression and in vitro mitochondrial proton leak activity. These include 1) T3 stimulates the total leak capacity (i.e., the leak per mitochondrion) but not the specific leak activity (i.e., the leak per protein); 2) T3 stimulates UCP-2 and UCP-3 expression, but a required cofactor or allosteric activator (whether constitutively present or upregulated by T3) is removed during mitochondrial isolation; 3) UCP-2 and UCP-3 mRNA levels do not reflect functional mitochondrial protein levels; and 4) UCP-2 and UCP-3 do not facilitate proton leak. There is insufficient evidence in the literature to negate any of these possibilities.
The possibility that T3 stimulates the total leak but not the specific leak requires that T3 1) stimulates UCP-2 and UCP-3 expression to the same extent as other mitochondrial proteins while having no effect on their specific activities or 2) increases the total area of the inner mitochondrial membrane without affecting the surface area-to-protein mass ratio. Regardless of the mechanism, this effect would result in a greater total leak activity in mitochondrial fractions from T3-treated mice. Two-way ANOVA of our data revealed no main effect of T3 treatment on total leak activity of the mitochondrial fractions (data not shown). Assuming equivalent recoveries of mitochondria from T3- and vehicle-treated mice, our data do not support the hypothesis that T3 stimulates the total leak per mitochondrion.
Allosteric regulation of UCP-2 and UCP-3 is a reasonable possibility given that the activity of UCP-1 is activated by fatty acids and inhibited by purine nucleotide di- and triphosphates (47). The COOH-terminal region of UCP-1 thought to interact with fatty acids is not highly conserved in UCP-2 (6 of 18 amino acids conserved) or UCP-3 (7 of 18 amino acids conserved), which indicates that a putative activator may be something other than a fatty acid. Amino acids comprising the purine nucleotide-binding domain in UCP-1 are well-conserved in UCP-2 (12 of 16 conserved) and UCP-3 (13 of 16 conserved), although contradictory evidence has been reported on whether mitochondrial proton leak is inhibited by purine nucleotide di- and triphosphates (46 vs. 10, 34, and 44).
The possibility that UCP-2 and UCP-3 mRNA levels do not reflect functional mitochondrial protein levels is of interest because UCP-1 exists in inactive and active forms. Brown fat mitochondria from rodents acutely cold exposed or aroused from hibernation show greater GDP binding than do controls (14, 15, 30). Because the time frame is too short for an increase in the amount of UCP-1 via transcription and translation, the increase in GDP binding has been thought to reflect the unmasking/activation of preexisting inactive UCP-1 proteins (14, 15); this effect is primarily mediated by catecholamines (14, 15). As shown here and elsewhere (20, 38, 39, 42), UCP-2 and UCP-3 expression and presumably protein levels are stimulated by T3 (although the assumption of parallel changes in expression and protein levels is not true for UCP-1 under certain conditions; see Ref. 36). However, changes in one or more additional factors may be required to activate the preexisting and newly synthesized proteins. Whatever factors might be involved, this possibility, as with the previous one, suggests that UCP-2 and UCP-3 are posttranslationally regulated.
The possibility that UCP-2 and UCP-3 do not facilitate proton leak in mammalian cells needs further evaluation. The primary evidence supporting an uncoupling effect of UCP-2 and UCP-3 derives from yeast transfection experiments (17, 19, 20, 41). These experiments show that yeast expressing UCP-2 and UCP-3 have a greater proportion of mitochondria with lower membrane potentials than those transfected with vector alone. Recent evidence (57) suggests that this is not a nonspecific effect of a protein being inserted into the inner mitochondrial membrane because yeast transfected with the oxoglutarate carrier do not exhibit reduced membrane potentials. Because UCP-1 is a known uncoupler in mammalian mitochondria and has effects in yeast similar to those of UCP-2 and UCP-3, the evidence to date indicates that UCP-2 and UCP-3 are likely to be uncouplers in mammalian mitochondria. Whether or not they uncouple by directly facilitating proton leak or as a consequence of some other activity remains to be determined.
Many experiments investigating the relationship between thyroid hormone status and proton leak have focused on the liver because it is rich in thyroid hormone receptors, an important contributor to basal metabolism, and easily homogenized for preparation of mitochondria. Yet, the liver is not an ideal tissue to examine the relationship between UCP-2 expression and proton leak because UCP-2 mRNA is normally present at low levels in whole liver RNA preparations (11, 29, 40); presumably, the protein is also present at low levels in mitochondria isolated from whole liver preparations. This is one explanation to account for our finding that liver UCP-2 expression did not correlate with mitochondrial respiration (P = 0.51). However, because we found that liver UCP-2 expression positively correlates, albeit not significantly (P = 0.09), with RMR, it is possible that low expression of UCP-2 contributes to hepatic and thus resting metabolism.
Skeletal muscle mitochondria were isolated from the gastrocnemius and soleus of one leg along with most of the other muscle groups from both legs. Our results show that T3 regulation of UCP-2 and UCP-3 expression (determined from the remaining gastrocnemius and soleus) is dependent on fiber type and/or muscle group. Expression in the soleus (slow-red oxidative) was insensitive to T3, whereas expression in the gastrocnemius (a mixed muscle, but predominantly fast-white glycolytic) was induced by T3. Despite the fact that the gastrocnemius is a mixed muscle, we cannot state with certainty that the mitochondria isolated from most of the hindlimb muscles would be representative of the mitochondria isolated from the gastrocnemius. This could be a potential source of variation leading to our observation that gastrocnemius UCP-2 and UCP-3 mRNA levels are not correlated with mitochondrial respiration from a mixed hindlimb preparation.
Lanni et al. (37) recently reported that state 4 skeletal muscle mitochondrial respiration correlated with UCP-3 expression in rats of different thyroid hormone status. Several possible reasons for the discrepancy with our data are noted. First, their correlation involved three points (the means for hypo-, eu-, and hyperthyroid conditions) due to the fact that UCP-3 expression and mitochondrial respiration were not measured in the same animals. Thus their data do not reflect the individual variability occurring among as well as within the groups. Second, the physiological responses to thyroid state apparently differ between rats and mice (see below). Third, hyperthyroidism was induced with a sixfold higher concentration of T3 than we used (see below). Fourth, hypothyroidism was induced over 3 wk by injections of propylthiouracil and iopanoic acid, which inhibit not only thyroid hormone synthesis but also peripheral deiodinases; thus peripheral metabolism and clearance of thyroid hormones are markedly different in propylthiouracil-iopanoic acid-treated animals compared with those that have undergone thyroidectomy.
In contrast to our finding that UCP-2 and UCP-3 expression positively correlates with RMR, Schrauwen et al. (58) found that skeletal muscle (vastus lateralis) UCP-3, but not UCP-2, expression positively correlated with sleeping metabolic rate in Pima Indians, whereas Millet et al. (43) found no correlation between RMR of lean or obese Caucasians and vastus lateralis UCP-2 and UCP-3 expression or subcutaneous white adipose tissue UCP-2 expression. These differences indicate that species and/or genetics may influence the contribution of UCPs to RMR. Alternatively, these differing results could indicate that UCP-2 and UCP-3 expression is not causally connected to RMR.
Liver and skeletal muscle mitochondria from rats injected with higher concentrations of thyroid hormone (10 injections of 15 µg T3/100 g body mass over 10 days or 20 µg T4/100 g body mass over 21 days) exhibit greater proton leak and basal respiration than do mitochondria isolated from euthyroid and hypothyroid rats (21-23, 37, 60). This effect also occurs in mitochondria of isolated hepatocytes, demonstrating that changes in the leak of isolated mitochondria can qualitatively reflect the changes that occur in situ (22). For liver mitochondria, much of the change has been ascribed to two factors that could alter the nonspecific leak of protons across the inner mitochondrial membrane. One is the inner mitochondrial membrane surface area-to-protein mass ratio, and the other is the inherent permeability of the phospholipid bilayer (5). Hypothyroidism decreases and hyperthyroidism increases these two factors (5). Because we saw no effect of T3 on mitochondrial respiration, we conclude that the T3 treatment in this study did not affect either of these variables.
We offer two potential explanations to account for the fact that the surface area per mass protein and the inherent bilayer permeability were not affected by T3. First, the sixfold lower concentration of T3 used in our study vs. that of Hafner et al. (21), Brand et al. (5), and Lanni et al. (37) may be insufficient to induce these changes. [The fact that we used 6 injections over 6 days as opposed to 10 injections over 10 days (5, 21) is unlikely to have contributed to the absence of in vitro changes in mitochondrial respiration because Tata et al. (60) have shown that a single injection of T3 into rats at ~7-15 µg/100 g body mass (our estimation of their dose) will produce liver and skeletal muscle mitochondria with higher rates of basal respiration 48-72 h later.] The second factor that may be important in accounting for the difference in our results is the species used. Liver mitochondrial proton leak negatively correlates with body mass in mammalian species ranging from the mouse to the horse (51, 52). This phenomenon has primarily been attributed to smaller animals having liver mitochondria with greater inner mitochondrial membrane surface area (per vol matrix and per mass protein) (52). Morphologically, a greater inner mitochondrial membrane surface area is reflected in a greater number of invaginations of the inner mitochondrial membrane into cristae. This is an efficient manner of increasing surface area with minimal changes in volume, yet it imposes physical limitations on the surface area that can be achieved. Mouse mitochondria may be less responsive to induction of inner membrane surface area by T3 because of this physical limitation.
Thyroidectomy for 5-8 wk tended to reduce the specific and total proton leak activities of isolated liver and skeletal muscle mitochondria compared with mitochondria from sham-operated mice. Thus loss of physiological levels of thyroid hormone over a 5- to 8-wk period is sufficient to induce in vitro changes in mouse liver and skeletal muscle mitochondria. These changes were not accompanied by a reduction in UCP-2 and UCP-3 expression in the tissues studied, indicating that if UCP-2 and UCP-3 are involved in the leak, their depressed activities may be the result of changes in inner mitochondrial membrane phospholipid composition (an effect known to alter the activity of some integral membrane proteins). Furthermore, the data imply that chronic treatment of Tx mice with T3 at 2.5 µg/100 g body mass would cause in vitro changes to liver and skeletal muscle mitochondria. Therefore, long-term regulation of RMR by thyroid hormone-induced changes in proton leak may involve both changes in protein-mediated leak (via the amount and activities of UCP-2 and UCP-3) and changes in nonspecific leak (via the total area of the inner mitochondrial membrane).
Surprisingly, RMR was not depressed in Tx mice compared with sham-operated mice. This observation has been verified with a separate group of Tx and sham-operated mice whose RMR was measured at 26°C (n = 4/group, unpublished observations). The absence of an affect of thyroidectomy on RMR may be due to the possibility that 26-28°C is more of a cold stress to Tx mice than to sham-operated euthyroid mice (i.e., the thermoneutral zone for Tx mice is higher than that for sham-operated mice). We know of no studies addressing this specific issue in mice, but it is known that hypothyroid rats at 20-23°C exhibit enhanced sympathetic nervous system activity, as evidenced by studies reporting increased urinary catecholamines and tissue norepinephrine turnover (16, 59). Furthermore, Mory et al. (45) and Ikemoto et al. (33) found that brown adipose tissue of hypothyroid rats and mice at 20-25°C share some characteristics with brown adipose tissue of cold-exposed euthyroid animals (e.g., increased brown adipose tissue weight, protein content, and oxygen consumption). Thus, for a given temperature, hypothyroid rodents appear to be under a greater cold stress than are euthyroid rodents. Nonetheless, the thermogenic mechanisms activated (e.g., brown adipose tissue) are not sufficient to raise the hypothyroid rat RMR to the level of its euthyroid control. Mice may differ from rats in their ability to recruit thyroid hormone-independent thermogenic mechanisms for maintenance of core temperature. If so, the RMR values we report for Tx mice are overestimates of their true RMR.
In addition to the postulated effects on in vivo proton leak, T3 regulation of RMR can partly be ascribed to activation of ATP-consuming pathways (24, 35). Thyroid hormone stimulation of protein synthesis and active ion transport is well established (13, 48). In our study, mice treated with T3 gained more weight than those treated with vehicle. Although we did not determine body composition, this suggests that protein synthesis is an important component contributing to the elevated RMR of T3-treated mice. The anabolic effect of T3 indicates that our dose regimen did not induce pronounced hyperthyroidism, as this is typically associated with a reduction in body mass (e.g., Ref. 42).
In summary, administration of T3 to Tx- and sham-operated mice at a concentration six times lower than that previously shown to stimulate in vitro rat mitochondrial proton leak had no such effect on isolated liver and skeletal muscle mitochondria despite significant effects on RMR. This indicates that T3 did not alter the inner mitochondrial membrane surface area-to-protein ratio or the inherent lipid bilayer leakiness. Although correlations do not establish causality, the fact that gastrocnemius, heart, and liver UCP-2 and gastrocnemius and heart UCP-3 mRNA levels were correlated with RMR suggests that these proteins may contribute to RMR. If UCP-2 and UCP-3 function as leak pathways, the leak in vitro may have been unaffected because their in vivo activity requires the presence of an allosteric factor that is lost on isolation of mitochondria. The effect of T3 on ATP-consuming reactions is also likely to be an important contributor to the increase in RMR.
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NOTE ADDED IN PROOF |
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A recent study reported a discordance between UCP-3-expressing yeast respiration and respiration of the isolated mitochondria. This provides further support for the hypothesis that the activity of UCP-3 is subject to posttranslational regulation. (Zhang, C.-Y., T. Hagen, U. K. Mootha, L. J. Slieker, and B. Lowell. Assessment of uncoupling protein 3 using a yeast heterologous expression system. FEBS Lett. 449: 129-134, 1999).
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ACKNOWLEDGEMENTS |
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We thank Jock Hamilton for technical support and insightful comments.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-32907 (to B. A. Horwitz), DK-52581 (to C. H. Warden), and DK-35747 (to C. Halsted).
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 reprint requests and other correspondence: B. A. Horwitz, NPB, Univ. California, One Shields Ave., Davis, CA 95616-8519 (E-mail: bahorwitz{at}ucdavis.edu).
Received 15 December 1998; accepted in final form 22 April 1999.
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REFERENCES |
---|
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---|
1.
Boss, O.,
S. Samec,
F. Kuhne,
P. Bijlenga,
F. Assimacopoulos-Jeannet,
J. Seydoux,
J. P. Giacobino,
and
P. Muzzin.
Uncoupling protein-3 expression in rodent skeletal muscle is modulated by food intake but not by changes in environmental temperature.
J. Biol. Chem.
273:
5-8,
1998
2.
Brand, M. D.
The proton leak across the mitochondrial inner membrane.
Biochim. Biophys. Acta
1018:
128-133,
1990[Medline].
3.
Brand, M. D.,
L. F. Chien,
E. K. Ainscow,
D. F. S. Rolfe,
and
R. K. Porter.
The causes and functions of mitochondrial proton leak.
Biochim. Biophys. Acta
1187:
132-139,
1994[Medline].
4.
Brand, M. D.,
P. Couture,
and
A. J. Hulbert.
Liposomes from mammalian liver mitochondria are more polyunsaturated and leakier to protons than those from reptiles.
Comp. Biochem. Physiol. Biochem. Mol. Biol.
108:
181-188,
1994[Medline].
5.
Brand, M. D.,
D. Steverding,
B. Kadenbach,
P. M. Stevenson,
and
R. P. Hafner.
The mechanism of the increase in mitochondrial proton permeability induced by thyroid hormones.
Eur. J. Biochem.
206:
775-781,
1992[Abstract].
6.
Brookes, P. S.,
A. J. Hulbert,
and
M. D. Brand.
The proton permeability of liposomes made from mitochondrial inner membrane phospholipids: no effect of fatty acid composition.
Biochim. Biophys. Acta
1330:
157-164,
1997[Medline].
7.
Brookes, P. S.,
D. F. S. Rolfe,
and
M. D. Brand.
The proton permeability of liposomes made from mitochondrial inner membrane phospholipids: comparison with isolated mitochondria.
J. Membr. Biol.
155:
167-174,
1997[Medline].
8.
Brown, G. C.
The leaks and slips of bioenergetic membranes.
FASEB J.
6:
2961-2965,
1992
9.
Brown, G. C.,
and
M. D. Brand.
On the nature of the mitochondrial proton leak.
Biochim. Biophys. Acta
1059:
55-62,
1991[Medline].
10.
Cadenas, S., R. B. Jones, and M. D. Brand.
Proton conductance is insensitive to nucleotides in skeletal
muscle mitochondria (Hot Topic Abstract HTP10). Proc.
8th Int. Congr. Obesity, Paris August 1998.
11.
Chavin, K. D.,
S. Yang,
H. Z. Lin,
J. Chatham,
V. P. Chacko,
J. B. Hoek,
E. Walajtys-Rode,
A. Rashid,
C.-H. Chen,
C.-C. Huang,
T.-C. Wu,
M. D. Lane,
and
A. M. Diehl.
Obesity induces expression of uncoupling protein-2 in hepatocytes and promotes liver ATP depletion.
J. Biol. Chem.
274:
5692-5700,
1999
12.
Chen, Y. D. I.,
and
F. L. Hoch.
Thyroid control over biomembranes.
Arch. Biochem. Biophys.
181:
470-483,
1977[Medline].
13.
Clausen, T.,
C. V. Hardeveld,
and
M. E. Everts.
Significance of cation transport in control of energy metabolism and thermogenesis.
Physiol. Rev.
71:
733-774,
1991
14.
Desautels, M.,
and
J. Himms-Hagen.
Roles of noradrenaline and protein synthesis in the cold-induced increase in purine nucleotide binding by rat brown adipose tissue mitochondria.
Can. J. Biochem.
57:
968-976,
1979[Medline].
15.
Desautels, M.,
G. Zaror-Behrens,
and
J. Himms-Hagen.
Increased purine nucleotide binding, altered phospholipid composition, and thermogenesis in brown adipose tissue mitochondria of cold-acclimated rats.
Can. J. Biochem.
56:
378-383,
1978[Medline].
16.
Dicker, A.,
A. Raasmaja,
B. Cannon,
and
J. Nedergaard.
Increased 1-adrenoceptor density in brown adipose tissue indicates recruitment drive in hypothyroid rats.
Am. J. Physiol.
263 (Endocrinol. Metab. 26):
E654-E662,
1992
17.
Fleury, C.,
M. Neverova,
S. Collins,
S. Raimbault,
O. Champigny,
C. Levi-Meyrueis,
F. Bouillaud,
M. F. Seldin,
R. S. Surwit,
D. Ricquier,
and
C. H. Warden.
Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia.
Nat. Genet.
15:
269-272,
1997[Medline].
18.
Garlid, K. D.,
A. D. Beavis,
and
S. K. Ratkje.
On the nature of ion leaks in energy-transducing membranes.
Biochim. Biophys. Acta
976:
109-120,
1989[Medline].
19.
Gimeno, R. E.,
M. Dembski,
X. Weng,
N. Deng,
A. W. Shyjan,
C. J. Gimeno,
F. Iris,
S. J. Ellis,
E. A. Woolf,
and
L. A. Tartaglia.
Cloning and characterization of an uncoupling protein homolog.
Diabetes
46:
900-906,
1997[Abstract].
20.
Gong, D. W.,
Y. He,
M. Karas,
and
M. Reitman.
Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, 3-adrenergic agonists, and leptin.
J. Biol. Chem.
272:
24129-24132,
1997
21.
Hafner, R. P.,
C. D. Nobes,
A. D. McGown,
and
M. D. Brand.
Altered relationship between protonmotive force and respiration rate in non-phosporylating liver mitochondria isolated from rats of different thyroid hormone status.
Eur. J. Biochem.
178:
511-518,
1988[Abstract].
22.
Harper, M.-E.,
and
M. D. Brand.
The quantitative contributions of mitochondrial proton leak and ATP turnover reactions to the changed respiration rates of hepatocytes from rats of different thyroid hormone status.
J. Biol. Chem.
268:
14850-14860,
1993
23.
Harper, M.-E.,
and
M. D. Brand.
Use of top-down elasticity analysis to identify sites of thyroid hormone-induced thermogenesis.
Proc. Soc. Exp. Biol. Med.
208:
228-237,
1995[Abstract].
24.
Harper, M.-E.,
and
M. D. Brand.
Hyperthyroidism stimulates mitochondrial proton leak and ATP turnover in rat hepatocytes but does not change the overall kinetics of substrate oxidation reactions.
Can. J. Physiol. Pharmacol.
72:
899-908,
1994[Medline].
25.
Heusner, A. A.
Biological similitude: statistical and functional relationships in comparative physiology.
Am. J. Physiol.
246 (Regulatory Integrative Comp. Physiol. 15):
R839-R845,
1984[Medline].
26.
Heusner, A. A.
Body size and energy metabolism.
Annu. Rev. Nutr.
5:
267-293,
1985[Medline].
27.
Hill, R. W.
Determination of oxygen consumption by use of the paramagnetic oxygen analyzer.
J. Appl. Physiol.
33:
261-263,
1972
28.
Hoch, F. L.
Lipids and thyroid hormones.
Prog. Lipid Res.
27:
199-270,
1988[Medline].
29.
Hodny, Z.,
P. Kolarova,
M. Rossmeisl,
M. Horakova,
M. Nibbelink,
L. Penicaud,
L. Casteilla,
and
J. Kopecky.
High expression of uncoupling protein-2 in foetal liver.
FEBS Lett.
425:
185-190,
1998[Medline].
30.
Horwitz, B. A.,
J. S. Hamilton,
and
K. S. Kott.
GDP binding to hamster brown fat mitochondria is reduced during hibernation.
Am. J. Physiol.
249 (Regulatory Integrative Comp. Physiol. 18):
R689-R693,
1985[Medline].
31.
Hulbert, A. J.
The thyroid hormones: a thesis concerning their action.
J. Theor. Biol.
73:
81-100,
1978[Medline].
32.
Hulbert, A. J.,
M. L. Augee,
and
J. K. Raison.
The influence of thyroid hormones on the structure and function of mitochondrial membranes.
Biochim. Biophys. Acta
455:
597-601,
1976[Medline].
33.
Ikemoto, H.,
T. Hiroshige,
and
S. Ito.
Oxygen consumption of brown adipose tissue in normal and hypothyroid mice.
Jpn. J. Physiol.
17:
516-522,
1967[Medline].
34.
Jekabsons, M.,
and
B. A. Horwitz.
Effect of nucleotides on rat liver and skeletal muscle mitochondrial non-phosphorylating respiration and membrane potential (Abstract).
FASEB J.
12:
A813,
1998.
35.
Kelly, J. M.,
and
B. W. McBride.
The sodium pump and other mechanisms of thermogenesis in selected tissues.
Proc. Nutr. Soc.
49:
185-202,
1990[Medline].
36.
Kopecky, J.,
Z. Hodny,
P. Kolarova,
M. Horakova,
and
R. Kolenovska.
Ontogenic changes of uncoupling protein 1 in white fat of the aP2-UCP transgenic and nontransgenic mice: posttranscriptional control of gene expression (Abstract).
Obes. Res.
5, Suppl. 1:
65S,
1997.
37.
Lanni, A.,
L. Beneduce,
A. Lombardi,
M. Moreno,
O. Boss,
P. Muzzin,
J. P. Giacobino,
and
F. Goglia.
Expression of uncoupling protein-3 and mitochondrial activity in the transition from hypothyroid to hyperthyroid state in rat skeletal muscle.
FEBS Lett.
444:
250-254,
1999[Medline].
38.
Lanni, A.,
M. D. Felice,
A. Lombardi,
M. Moreno,
C. Fleury,
D. Ricquier,
and
F. Goglia.
Induction of UCP2 mRNA by thyroid hormones in rat heart.
FEBS Lett.
418:
171-174,
1997[Medline].
39.
Larkin, S.,
E. Mull,
W. Miao,
R. Pittner,
K. Albrandt,
C. Moore,
A. Young,
M. Denaro,
and
K. Beaumont.
Regulation of the third member of the uncoupling protein family, UCP3, by cold and thyroid hormone.
Biochem. Biophys. Res. Commun.
240:
222-227,
1997[Medline].
40.
Larrouy, D.,
P. Laharrague,
G. Carrera,
N. Viguerie-Bascands,
C. Levi-Meyrueis,
C. Fleury,
C. Pecqueur,
M. Nibbelink,
M. Andre,
L. Casteilla,
and
D. Ricquier.
Kuppfer cells are a dominant site of uncoupling protein 2 expresssion in rat liver.
Biochem. Biophys. Res. Commun.
235:
760-764,
1997[Medline].
41.
Liu, Q.,
C. Bai,
F. Chen,
R. Wang,
T. MacDonald,
M. Gu,
Q. Zhang,
M. A. Morsy,
and
C. T. Caskey.
Uncoupling protein-3: a muscle-specific gene upregulated by leptin in ob/ob mice.
Gene
207:
1-7,
1998[Medline].
42.
Masaki, T.,
H. Yoshimatsu,
T. Kakuma,
S. Hidaka,
M. Kurokawa,
and
T. Sakata.
Enhanced expression of uncoupling protein 2 gene in rat white adipose tissue and skeletal muscle following chronic treatment with thyroid hormone.
FEBS Lett.
418:
323-326,
1997[Medline].
43.
Millet, L.,
H. Vidal,
F. Andreelli,
D. Larrouy,
J. P. Riou,
D. Ricquier,
M. Laville,
and
D. Langin.
Increased uncoupling protein-2 and -3 mRNA expression during fasting in obese and lean humans.
J. Clin. Invest.
100:
2665-2670,
1997
44.
Monemdjou, S.,
L. P. Kozak,
and
M.-E. Harper.
Mitochondrial proton leak in brown adipose tissue mitochondria of UCP1 knockout mice is GDP-insensitive.
Am. J. Physiol.
276 (Endocrinol. Metab. 39):
E1073-E1082,
1999
45.
Mory, G.,
D. Ricquier,
P. Pesquies,
and
P. Hemon.
Effects of hypothyroidism on the brown adipose tissue of adult rats: comparison with the effects of adaptation to cold.
J. Endocrinol.
91:
515-524,
1981[Abstract].
46.
Negre-Salvayre, A.,
C. Hirtz,
G. Carrera,
R. Cazenave,
M. Troly,
R. Salvayre,
L. Penicaud,
and
L. Casteilla.
A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation.
FASEB J.
11:
809-815,
1997
47.
Nicholls, D. G.,
and
R. M. Locke.
Thermogenic mechanisms in brown fat.
Physiol. Rev.
64:
1-64,
1984
48.
Nobes, C. D.,
P. L. Lakin-Thomas,
and
M. D. Brand.
The contribution of ATP turnover by the Na+/K+-ATPase to the rate of respiration of hepatocytes. Effects of thyroid status and fatty acids.
Biochim. Biophys. Acta
976:
241-245,
1989[Medline].
49.
Oh, S. S.,
and
M. L. Kaplan.
Early treatment of obese (ob/ob) mice with triiodothyronine increases oxygen consumption and temperature and decreases body fat content.
Proc. Soc. Exp. Biol. Med.
207:
260-267,
1994[Abstract].
50.
Porter, R. K.,
and
M. D. Brand.
Causes of differences in respiration rate of hepatocytes from mammals of different body mass.
Am. J. Physiol.
269 (Regulatory Integrative Comp. Physiol. 38):
R1213-R1224,
1995
51.
Porter, R. K.,
and
M. D. Brand.
Body mass dependence of H+ leak in mitochondria and its relevance to metabolic rate.
Nature
362:
628-630,
1993[Medline].
52.
Porter, R. K.,
A. J. Hulbert,
and
M. D. Brand.
Allometry of mitochondrial proton leak: influence of membrane surface area and fatty acid composition.
Am. J. Physiol.
271 (Regulatory Integrative Comp. Physiol. 40):
R1550-R1560,
1996
53.
Rolfe, D. F. S.,
and
M. D. Brand.
Contribution of proton leak to skeletal muscle respiration and to standard metabolic rate.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1380-C1389,
1996
54.
Rolfe, D. F. S.,
and
G. C. Brown.
Cellular energy utilization and molecular origin of standard metabolic rate in mammals.
Physiol. Rev.
77:
731-758,
1997
55.
Rolfe, D. F. S.,
A. J. Hulbert,
and
M. D. Brand.
Characteristics of mitochondrial proton leak and control of oxidative phosphorylation in the major oxygen-consuming tissues of the rat.
Biochim. Biophys. Acta
1118:
405-416,
1994.
56.
Ruggiero, F. M.,
G. V. Gnoni,
and
E. Quagliariello.
Lipid composition of brown adipose tissue mitochondria and microsomes in hyperthyroid rats.
Int. J. Biochem.
21:
327-332,
1989[Medline].
57.
Sanchis, D.,
C. Fleury,
N. Chomiky,
M. Goubern,
Q. Huang,
M. Neverova,
F. Gregoire,
J. Easlick,
S. Raimbault,
C. Levi-Meyrueis,
B. Miroux,
S. Collins,
M. Seldin,
D. Richard,
C. Warden,
F. Bouillaud,
and
D. Ricquier.
BMCP1, a novel mitochondrial carrier with high expression in the central nervous system of humans and rodents, and respiration uncoupling activity in recombinant yeast.
J. Biol. Chem.
273:
34611-34615,
1998
58.
Schrauwen, P.,
J. Xia,
C. Bogardus,
R. E. Pratley,
and
E. Ravussin.
Skeletal muscle uncoupling protein 3 expression is a determinant of energy expenditure in Pima Indians.
Diabetes
48:
146-149,
1999[Abstract].
59.
Sellers, E. A.,
K. V. Flattery,
and
G. Steiner.
Cold acclimation of hypothyroid rats.
Am. J. Physiol.
226:
290-294,
1974[Medline].
60.
Tata, J. R.,
L. Ernster,
O. Lindberg,
E. Arrhenius,
S. Pedersen,
and
R. Hedman.
The action of thyroid hormones at the cell level.
Biochem. J.
86:
408-428,
1963.