From the Pennington Biomedical Research Center, Baton
Rouge, Louisiana 70808 and the § Department of Biochemistry,
Faculty of Medicine, University of Ottawa, Ottawa,
Ontario, K 1 H 8M5 Canada
Received for publication, January 17, 2001
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ABSTRACT |
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An interaction between free fatty acids and UCP1
(uncoupling protein-1)
leading to de-energization of mitochondria was assumed to be a key
event for triggering heat production in brown fat. Recently, Matthias
et al., finding indistinguishable de-energization of
isolated brown fat mitochondria by fatty acids in UCP1-deficient mice
and control mice, challenged this assumption (Matthias, A., Jacobsson,
A., Cannon, B., and Nedergaard, J. (1999) J. Biol. Chem. 274, 28150-28160). Since their results were obtained using UCP1-deficient and control mice on an undefined genetic background, we
wanted to determine unambiguously the phenotype of UCP1 deficiency with
the targeted Ucp1 allele on congenic C57BL/6J and 129/SvImJ backgrounds. UCP1-deficient congenic mice have a very pronounced cold-sensitive phenotype; however, deficient mice on the F1 hybrid background were resistant to cold. We propose that heterosis provides a
mechanism to compensate for UCP1 deficiency. Contrary to the results of
Matthias et al., we found a significant loss of fatty acid-induced de-energization, as reflected by membrane potential and
oxygen consumption, in brown fat mitochondria from UCP1-deficient mice.
Unlike cold sensitivity, fatty acid-induced uncoupling of mitochondria
was independent of the genetic background of UCP1-deficient mice. We
propose that intracellular free fatty acids directly regulate
uncoupling activity of UCP1 in a manner consistent with models
described in the literature.
Brown adipose tissue
(BAT)1 plays an important
role in heat production and is considered to contribute to energy
balance (5, 6). Stimulation by the sympathetic nervous system causes an up-regulation in the metabolic rate of BAT that is reflected in an
increase of heat production (7, 8). The ability to generate heat is
attributed to the high number of mitochondria containing UCP1
(uncoupling protein-1)
(9). This transmembrane protein is thought to be an ion carrier that
uncouples respiration from ATP synthesis, allowing mitochondria to
produce heat. The importance of UCP1 for thermogenesis was proven by
the observation that UCP1-deficient mice are cold-sensitive (10).
However, the fact that some UCP1-deficient mice are resistant to cold
and that adiposity is not increased led us to postulate that additional
thermogenic mechanisms can compensate for UCP1 deficiency. In this
respect, UCP1-deficient mice can help not only to understand the
mechanisms that control BAT thermogenesis, but can also be used to
identify alternative pathways for heat production.
Although it is known that the sympathetic nervous system controls heat
production of brown adipocytes (11, 12), the intracellular signaling
pathway remains unclear. It has been proposed that free fatty acids
(FFAs), released by the action of hormone-sensitive lipase, serve both
as an energy substrate and as an activator of the proton carrier
function of UCP1, thereby triggering heat production. It has also been
shown that FFAs can increase respiration of isolated brown adipocytes
(13) and that isolated BAT mitochondria are more prone to
de-energization by FFAs compared with liver mitochondria (14). Finally,
experiments with reconstituted UCP1 in proteoliposomes (4) and with
ectopic expression of UCP1 in yeast (15) demonstrate that FFAs are
necessary for UCP1 to act as a proton carrier.
The model for the activation of UCP1 by FFAs has recently been
challenged by Matthias et al. (1) by the analysis of the bioenergetics of isolated BAT mitochondria from UCP1-deficient mice. In
agreement with the findings of Monemdjou et al. (16), Matthias et al. found a high mitochondrial membrane
potential in UCP1-deficient mice that was independent of GDP addition.
This confirmed that GDP inhibited UCP1-dependent proton
flux. Matthias et al. also presented provocative findings
that the ability of FFAs to uncouple BAT does not depend on UCP1. Their
data suggested that wild-type and UCP1-deficient BAT mitochondria had
the same ratio of de-energization after the addition of the same
amounts of fatty acid. In a second study, Matthias et al.
(17) showed that the stimulation of oxygen consumption by noradrenaline
and fatty acid in isolated brown adipocytes depended on Ucp1
expression. Matthias et al. concluded that the uncoupling
effect of FFA is not directly mediated by UCP1; rather an alternative
activator such as a metabolite of FFAs enhances the proton transport
activity of UCP1.
Considering the long history of biochemical studies pointing toward an
interaction between UCP1 and FFAs in the activation of proton flux, we
reevaluated UCP1-independent FFA uncoupling in UCP1-deficient mice that
have defined genetic backgrounds. Our results show that FFA-induced
uncoupling of respiration in isolated BAT mitochondria is strongly
reduced in UCP1-deficient mice. The basis of the difference between our
results and those of the Matthias et al. (1, 17) is
not clear since FFA-induced uncoupling is reduced in UCP1-deficient
mice irrespective of the genetic background of the targeted mice. On
the other hand, differences in genetic background are extremely
important for thermoregulation in UCP1-deficient mice since targeted
mice on congenic backgrounds are extremely sensitive to cold, whereas
mice on a F1 hybrid background are resistant to cold. Clearly some, as
yet to be identified, thermogenic mechanisms allow the latter mice to
resist cold.
Animals--
Ucptm1 knockout mice on a mixed
129/SvPas and C57BL/6J background were backcrossed to 129/SvImJ and
C57BL/6J for 10 generations. At the 10th backcross generation, each
line was typed with 135 microsatellite markers to establish its genetic
purity. These mice were intercrossed to obtain mice homozygous for the
Ucp1tm1 allele. The absence of Ucp1
mRNA was confirmed by real-time reverse transcription-polymerase
chain reaction using a Taqman® probe. Littermates with two
wild-type Ucp1 alleles were used as control mice.
129/SvImJ × C57BL/6J hybrid mice were obtained by crossing
homozygous Ucp1tm1 knockout mice from the two
congenic lines. All mice were kept in a 12-h light cycle in 27 °C
and fed a 4.5% fat chow diet. Animals of both genders between the ages
of 8 and 12 weeks were used.
Cold Sensitivity--
Cold sensitivity was estimated using the
Vital View Data Acquisition System® (Mini Mitter Co.,
Inc., Sunriver, OR). Temperature probes were implanted in the
peritoneal cavity 1 week prior to the experiment. Mice were
single-caged and exposed to 4 °C on ER-4000 receiver units. The body
temperatures were monitored online with a sampling rate of 12/h. Cold
exposure was maintained for 18 h, and all hypothermic mice
reaching a body core temperature of <30 °C were removed from the
cold environment.
Mitochondrial Preparations--
Mitochondria were isolated in a
manner similar to that described by Cannon and Lindberg (18).
Each step was performed on ice or at 4 °C. Mice were killed by
cervical dislocation, and interscapular BAT was removed. The fat pads
were homogenized immediately in homogenization medium (250 mM sucrose, 1 mM HEPES (pH 7.2), and 0.2 mM EGTA) in the presence of 0.5% BSA in a Potter-Elvehjem homogenizer. The suspension was centrifuged at 900 × g for 10 min and filtered through a 100-µm nylon mesh
filter. The mitochondrial pellet was obtained by centrifugation at
6000 × g for 16 min and resuspended in homogenization
medium with 0.1% albumin. The mitochondria were then centrifuged at
6000 × g and resuspended in high potassium medium (100 mM KCl, 2 mM HEPES (pH 7.2), and 1 mM EGTA). Resuspension directly in respiration buffer (125 mM sucrose, 4 mM HEPES (pH 7.2), 1 mM EGTA, 2 mM MgCl2, 4 mM Tris phosphate, 20 mM KCl, 0.1% BSA, 5 µM rotenone, and 6 µg/ml oligomycin) caused a higher
fluorescent quench, but did not alter the final results. Mitochondrial
protein content was determined by the Lowry method (35), and the
suspensions were kept on ice for no longer than 4 h for the
experiments. Biochemical properties of mitochondria were characterized
by determination of activities for mitochondrial glycerol-3-phosphate
dehydrogenase (19) and cytochrome c oxidase (20).
Oxygen Consumption--
All measurements were performed at
37 °C with constant stirring. A Clark-type oxygen electrode from
Hansatech was used for monitoring oxygen consumption. Mitochondria were
incubated in respiration buffer at a concentration of 0.05 mg/ml
protein. Glycerol phosphate was added to a final concentration of 5 mM. The UCP1-mediated proton leak across the inner
mitochondrial membrane was inhibited by the addition of GDP (1 mM final concentration). De-energization of mitochondria
was achieved by the addition of FFA. A concentration of 1 nM FCCP was sufficient to disrupt the electrochemical
gradient and to observe maximum mitochondrial respiration.
Protonmotive Force--
Fluorescent quench of the cationic dye
rhodamine 123 was used for estimation of protonmotive force (21, 22).
The measurements were performed under the same conditions as the oxygen
measurements, except that 0.13 µM fluorescent dye was
added. Fluorescence was monitored in a PerkinElmer Life Sciences LS 50B
spectrofluorometer. According to our spectral data, we chose
wavelengths of 500 and 525 nm for excitation and emission,
respectively. The fluorescence quench was converted to membrane
potential using the Nernst equation. The conversion was calibrated
using different potassium concentrations of the incubation medium and
generation of diffusion potential by the addition of 29 ng/ml
valinomycin, a potassium ionophore. Liver mitochondria were treated
identically to BAT mitochondria, except that succinate at 10 mM was used as a substrate.
Expression Analysis--
RNA was isolated using
TRI®-Reagent and subjected to quantitative real-time
reverse transcription-polymerase chain reaction using the ABI Prism
7700 sequence detection system (23). Polymerase chain reaction primers
and Taqman® probes were designed corresponding to cDNA
sequences of Ucp1, Ucp2, and Ucp3
(GenBankTM/EBI accession numbers U63419, AB012159, and
AF032902, respectively).
Chemicals--
All chemicals were purchased from Sigma. GDP and
DL-glycerol 3-phosphate were ordered as Tris and
di(monocyclohexylammonium) salts, respectively. Only charcoal-treated
fatty acid-free BSA was used. Lauric acid was used as a Tris salt in
water; all other fatty acids were dissolved in ethanol.
Phenotype of UCP1-deficient Mice on Congenic
Backgrounds--
UCP1-deficient mice are sensitive to cold, showing
the pivotal function of UCP1 for thermogenesis (10). However, in our initial description of the mice, we reported that ~15% of the individuals were resistant to cold, suggesting that variation in
genetic background could alter cold sensitivity. To address this issue,
we established UCP1-deficient mice on inbred 129/SvImJ and C57BL/6J
backgrounds. Incipient mice developed normally with regard to growth
and development. As listed in Table I for
the 129/SvImJ background, UCP1-deficient mice showed an
increased BAT depot weight, a comparable content of mitochondrial
protein, decreased cytochrome c oxidase activity, and
increased mitochondrial glycerol-3-phosphate dehydrogenase activity
compared with wild-type mice.
Profound differences in sensitivity to cold were observed as the
offspring approached the 10th backcross generation. To keep homozygous
postnatal mice alive, the breeding room temperature was increased to
27 °C. In adult mice, the ability to produce heat under cold stress
was characterized with cold sensitivity tests. Fig.
1 shows the traces of body temperature of
mice during cold exposure. 129/SvImJ and C57BL/6J wild-type mice could
maintain their body temperature in an environment at 4 °C for an
indefinite time (Fig. 1A). UCP1-deficient mice congenic on
129/SvImJ and C57BL/6J backgrounds lost body core temperature rapidly
(Fig. 1, B and C). The average rate of
temperature loss of 4.9° and 3.9°/h was similar for both
backgrounds. We also analyzed UCP1-deficient mice on a (129/SvImJ × C57BL/6J) F1 hybrid background (Fig. 1D). These hybrid
mice were generated by out-crossing parental lines of
Ucp1 Uncoupling of Mitochondrial Oxidative Phosphorylation--
FFAs,
released by the action of hormone-sensitive lipase, play a key role in
thermogenesis as an energy substrate for mitochondrial
The quantitative effects of fatty acids on membrane potential were
determined using mitochondria from mice on a 129/SvImJ background. The
experiments were carried out as described above, and de-energization
was expressed in millivolts. Fig. 3 shows dose-response curves for linoleic acid in control BAT mitochondria and
UCP1-deficient mitochondria from BAT and liver. The concentration of
unbound linoleic acid in the presence of 0.1% BSA was calculated according to Richieri et al. (24). Values were fitted by
graphs similar to Michaelis-Menten kinetics, and initial
de-energization rates were estimated. The de-energization in
UCP1-deficient BAT mitochondria was 35% of that in wild-type BAT
mitochondria. For comparison, liver mitochondria with 0.6 mV/nM are still less prone to de-energization compared with
UCP1-deficient BAT mitochondria with 1.6 mV/nM.
The abilities of lauric, linoleic, and stearic acids to de-energize
mitochondria are summarized in Table II.
All measurements were carried out in the presence of 0.1% BSA;
therefore, the concentrations of unbound fatty acids are much lower
than the nominal concentrations. Because correction coefficients for
calculating the concentration of unbound molecules are not known for
some fatty acids, the nominal concentrations are presented. As already
shown by Matthias et al. (1), the maximum membrane potential
was slightly increased in UCP1-deficient mitochondria. Contradictory to
the findings of this group, we observed that with a nominal
concentration of 66 µM lauric acid, a 36% reduction in
membrane potential occurred in wild-type mice, compared with a 16%
reduction in UCP1-deficient mice. The ability of linoleic and stearic
acids to de-energize was also reduced in UCP1-deficient BAT
mitochondria. We measured reductions from 30 to 14% and from 31 to
15% for the 27 µM nominal concentration of linoleic and
stearic acids, respectively.
An alternative strategy for showing the importance of UCP1 for fatty
acid-induced de-energization of isolated BAT mitochondria is based on
the fact that long-term cold exposure increases UCP1 in BAT
mitochondria (25, 26). Accordingly, we exposed normal mice to 4 °C
for 1 week and performed the same de-energization experiments as
described above. BAT mitochondria from cold-exposed mice showed 39 ± 3% de-energization after the addition of 66 µM lauric
acid versus 25 ± 6% in control mice at 27 °C,
indicating a positive correlation between higher content of UCP1 and
fatty acid-induced de-energization.
Affect of Oxygen Consumption--
The key step in heat production
of BAT is the activation of energy expenditure by uncoupling the
respiration from ATP synthesis by UCP1. Respiratory studies were
carried out to determine whether impaired de-energization in
UCP1-deficient mitochondria leads to impaired activation of
respiration. Fig. 4 (A and
B) shows representative traces for oxygen consumption in BAT
mitochondria. The BAT mitochondria from control mice reduced oxygen
consumption after the addition of 1 mM GDP, whereas
respiration of UCP1-deficient mitochondria was not affected by GDP.
After the addition of 20 µM linoleic acid, the coupled
mitochondria from control mice showed a 20% increase in oxygen
consumption (Fig. 4A) compared with UCP1-deficient mitochondria, whose respiration remained unaffected upon the addition of the same amount of fatty acid (Fig. 4B). Maximum
respiration was achieved by the addition of 2 µM FCCP. To
show quantitative effects on fatty acid-stimulated respiration, we
performed dose-response experiments. We coupled BAT mitochondria with 1 mM GDP and monitored oxygen consumption after the addition
of varying amounts of lauric acid. These results are summarized in Fig.
4C. The nominal concentration of fatty acid that caused an
increase in respiration was between 25 and 50 µM for
wild-type mitochondria and between 75 and 100 µM for
UCP1-deficient mitochondria.
Expression of Uncoupling Proteins in UCP1-deficient Mice on a
Hybrid Background--
Our finding that fatty acid-induced
de-energization of isolated BAT mitochondria is affected by UCP1
deficiency is contrary to the findings of Matthias et al.
(1). The only known variable that might account for the differences
between our results and those of Matthias et al. is the
genetic background of these mice. The cold resistance of UCP1-deficient
mice on a hybrid background compared with cold sensitivity on inbred
backgrounds suggests the existence of an alternative thermogenic
mechanism induced in hybrid mice. To determine if this thermogenic
pathway is mediated by the UCP1 homologs UCP2 and UCP3, we performed
expression analysis at the mRNA level. Consistent with previous
reports (1, 10), the Ucp2 mRNA levels in BAT from
UCP1-deficient mice were elevated over the controls (Fig.
5A). However, the degree of
Ucp2 overexpression varied between 250% for the C57BL/6J
genetic background and 600% for the 129/SvImJ genetic background.
Ucp2 mRNA levels in cold-resistant F1 progeny were
intermediate with respect to the parental strains, suggesting that
variation in Ucp2 mRNA levels may be co-dominantly inherited. There were no differences in Ucp3 expression
levels compared with control mice. Knowing that Ucp3 is
mainly expressed in skeletal muscle and has a thermogenic potential
(27), we also analyzed skeletal muscle. Differences in Ucp2
and Ucp3 mRNA levels in skeletal muscle cannot account
for cold resistance of F1 hybrid Ucp1 knockout mice
(Fig. 5B).
Determining the interrelationships between thermogenesis, energy
expenditure, and body weight regulation, particularly with respect to
energy reserves, has both fundamental and practical importance in
mammalian biology. However, progress in understanding how thermogenesis
can be manipulated to affect energy expenditure and body weight has
been slow since the number of potential mechanisms that can produce
heat is very large. UCP1-dependent non-shivering thermogenesis in brown adipose tissue is a well defined mechanism that
can be up-regulated pharmacologically, and several genetically altered
mouse models provide experimental systems to evaluate its role in the
regulation of body weight. Transgenic mice in which Ucp1 is
overexpressed have consistently provided strong evidence that elevated
UCP1 can reduce excess adiposity. However, paradoxically, there is no
evidence to suggest that the absence of UCP1 will produce the opposite
phenotype, i.e. increased adiposity. Other phenotypes of
mice carrying the targeted inactivation of Ucp1 need
explanation. In our original publication (10), we also reported
that 15% of the homozygous targeted mice were resistant to cold
exposure. How is it that occasionally mice homozygous for the inactive
allele are resistant to cold? Subsequently, Matthias et al.
(1) obtained data showing that mitochondria isolated from
targeted Ucp1 mice are uncoupled by FFAs to the same degree as mitochondria from wild-type mice, suggesting that FFAs do not directly activate UCP1-dependent proton flux. Since these
effects of the mutation on body weight gain and thermosensitivity and the effects of FFAs were contrary to our expectations, we established the Ucp1 mutation on congenic backgrounds to eliminate
uncontrolled genetic variability as a confounding factor. Experiments
on thermosensitivity and FFA activation of UCP1 have been described
here; the effects of the targeted Ucp1 mutation on body
weight gain is under investigation.
Cold sensitivity is a complex phenotype that was linked to metabolic
disorders some 40 years ago with the measurement of body temperature in
genetically obese (ob/ob) mice during cold exposure (28, 29). Since
then, mouse models lacking key proteins for non-shivering thermogenesis
have been established, but not all of them proved to be cold-sensitive.
Mice lacking the We have shown that UCP1-deficient mice are cold-sensitive on both
129/SvImJ and C57BL/6J backgrounds, but not on the (129/SvImJ × C57BL/6J) F1 hybrid background. The resistance to cold in the hybrid
animal cannot be explained by the characteristics of its mitochondrial
physiology, at least within the limits of the experiments on membrane
potentials and respiration and activation by FFAs. The results with
inbred and hybrid UCP1-deficient mice are indistinguishable. In
addition, there are no differences in expression levels of Ucp2 and Ucp3 in BAT or skeletal muscle
that would suggest that hybrid mice are resistant to cold due to
elevated expression of these genes. In addition to the resistance to
cold found in the hybrid mice, UCP1-deficient mice on an inbred
background can be adapted to tolerate cold by gradually reducing the
ambient temperature; however, the acquisition of this property is
variable.2 We interpret these
results to suggest that the cold sensitivity phenotype is very
sensitive to the environment and that alternative thermogenic
mechanisms can be induced to compensate for UCP1-based thermogenesis.
The adaptation of C57BL/6J and 129/SvImJ mice to cold exposure or the
constitutive resistance in hybrid mice can provide a biological system
to identify alternative mechanisms of thermogenesis.
The cold resistance of UCP1-deficient mice on a hybrid background is a
dramatic example of heterosis or hybrid vigor. Heterosis has been
extensively studied in plants with respect to growth; it has also been
studied in mammals for complex phenotypes like behavioral traits (32).
This might also account for cold resistance of UCP1-deficient mice on a
F1 hybrid background as well. One of the more relevant examples of
heterosis comes from a study of hybrids of four parental lines of
chicken in which positive heterosis was observed for norepinephrine
levels in the hypothalamus in a line-specific manner (33). In these
same hybrids, heterosis was found for dihydroxyphenylacetic acid in the
optic tectum only. These results suggest that heterosis can
affect the level of hormones that are relevant to thermogenesis. At the
gene level, mice heterozygous for the inactive allele for
Using UCP1-deficient mice on 129/SvImJ, C57BL/6J, and F1 hybrid
backgrounds, we observed a significant reduction of fatty acid-induced
de-energization of BAT mitochondria compared with control mice. This
indicates that FFAs directly activate the uncoupling activity of UCP1
in BAT mitochondria. However, UCP1-deficient BAT mitochondria are still
more prone to fatty acid-induced de-energization compared with liver
mitochondria. This indicates that despite similar protonmotive force,
UCP1-deficient BAT mitochondria and liver mitochondria are not similar,
as already shown by the lack of ATP synthase activity in the former
(1). It also suggests that UCP1-deficient mitochondria retain a
significant capacity to generate a membrane potential that can be
uncoupled by fatty acids. It is uncertain whether this residual
uncoupling contributes to heat production.
Our finding that fatty acids have a reduced ability to de-energize BAT
mitochondria in the absence of UCP1 irrespective of the genetic
background of the mice does not agree with the findings of Matthias
et al. (1). They showed no impaired fatty acid-induced de-energization of UCP1-deficient BAT mitochondria compared with control mice. However, inspection of their data generated from UCP1-deficient mice on an undefined genetic background shows a trend
toward UCP1 dependence. The uncoupling activity for seven fatty acids
(as shown in Table III of Ref. 1) indicates that the uncoupling
activity for each fatty acid is slightly decreased in UCP1-deficient
BAT mitochondria. Considering all seven fatty acids together, an
average value of 1.03 mV/µM can be calculated for
wild-type mice versus 0.67 mV/µM FFA in
UCP1-deficient mice. It should be emphasized that their data were
expressed as millivolts/nominal FFA concentration in the presence of
0.1% BSA. Due to the exponential relationship between nominal and free
(unbound) concentrations of fatty acids in the presence of BSA, the
differences, expressed as millivolts/unbound FFA, would be more
remarkable. However, using mice on an undefined background, their
analysis did not show statistically significant differences. The reason
why the impairment of fatty acid-induced de-energization by UCP1
deficiency is more significant in a mouse on a congenic background (our
mice) is presently unknown. We conclude that, as previously postulated from biochemical studies, FFAs are vital physiological activators of
UCP1-mediated uncoupling of respiration by the stimulation of proton flux.
There are two models describing how FFAs interact with UCP1 to uncouple
respiration. According to Winkler and Klingenberg (4), FFAs provide
carboxyl groups for transporting protons together with amino acid
carboxyl groups in the transmembrane channel of UCP1. In the second
model, the function of UCP1 is indirect (2, 3). This model proposes
that neutral FFAs can pass the inner mitochondrial membrane
transporting protons. After deprotonation in the matrix, UCP1
transports the carboxylate anions back across the membrane, which
completes the fatty acid cycle of proton shuttling. Although our
experiments do not provide any insight as to which model is correct,
they do show that BAT mitochondria from UCP1-deficient mice have an
impaired fatty acid-induced de-energization. We suggest that the
interaction of FFAs and UCP1 enables changes in the intercellular
concentration of FFA to regulate the uncoupling activity of UCP1. It is
also evident from our results on heterosis that the absence of a
UCP1-based thermogenesis does not preclude the emergence of alternative
mechanisms of thermogenesis to protect mice from cold exposure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Parameters of interscapular brown fat pad and isolated mitochondria in
congenic 129/SvImJ mice
14, animals of
both genders and age of 65 ± 5 days. Values are expressed as
means ± S.D.
/
mice. Remarkably, these mice were resistant
to cold, despite the absence of UCP1. We observed also that ~1 out of
10 mice of the cold-resistant groups lost body temperature (Fig. 1,
A and D). It is not unexpected to occasionally
find a cold-sensitive mouse in a cold-resistant group since anything
that affects the health of a mouse impairs cold resistance. It is quite
possible that the surgery to implant temperature probes affected the
health of the mice.
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Fig. 1.
Body temperature of mice during cold
exposure. After moving mice to a 4 °C environment, temperatures
were monitored using implanted probes and receiver units. A,
129/SvImJ and C57BL/6J wild-type mice; B and C,
UCP1-deficient mice congenic on 129/SvImJ and C57BL/6J backgrounds,
respectively; D, UCP1-deficient mice on a (129/SvImJ × C57BL/6J)/F1 hybrid background.
-oxidation.
Additionally, they are also thought to stimulate respiration by direct
activation of UCP1. To establish the validity of this interaction, we
investigated the ability of fatty acids to de-energize BAT
mitochondria, as evidenced by mitochondrial membrane potential.
UCP1-deficient mice and control mice on different genetic backgrounds
were kept at 27 °C prior to dissection, and the protonmotive force
of isolated BAT mitochondria was measured. Fig.
2 shows representative traces for
wild-type (A, C, and E) and
UCP1-deficient (B, D, and F)
mitochondria on 129/SvImJ (A and B), C57BL/6J
(C and D), and F1 hybrid (E and
F) backgrounds. As expected, BAT mitochondria in the control
mice showed very little increase in protonmotive force after the
addition of substrate, but a large increase after purine nucleotide
addition (Fig. 2A, C, and E). As
previously shown (1, 16), mitochondria in the mutant mice became
maximally charged following the addition of substrate only (Fig. 2,
B, D, and F) and showed no further
increase upon the addition of 1 mM GDP. After achievement
of maximum protonmotive force, BAT mitochondria were subjected to
de-energization by stepwise addition of FFA. The final concentrations
of lauric acid were 33, 67, and 100 µM. In this phase,
UCP1-deficient mitochondria (Fig. 2, B, D, and
F) were substantially less responsive to the addition of the
same amount of FFA compared with BAT mitochondria in control mice
(A, C, and E). The last step of each
experiment was always the total de-energization with FCCP, a
nonspecific uncoupler. Overall, the traces in Fig. 2 show an impaired
fatty acid-induced de-energization in UCP1-deficient BAT mitochondria that is independent of the genetic background.
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Fig. 2.
Protonmotive force in BAT mitochondria from
UCP1-deficient mice and control mice on different genetic
backgrounds. Fluorescent quench of rhodamine 123 was converted
into protonmotive force as described under "Experimental
Procedures." Additions were substrate glycerol phosphate
(sub) and GDP at final concentrations of 5 and 1 mM, respectively. Stepwise de-energization was achieved by
the addition of lauric acid with nominal FFA concentrations (33, 67, and 100 µM) in the presence of 0.1% BSA. FCCP was added
to a final concentration of 1 nM. Representative traces are
shown for wild-type and UCP1-deficient mice on different genetic
backgrounds: 129/SvImJ (A and B), B57BL/6J
(C and D), and (129/SvImJ × C57BL/6J)/F1
hybrid (E and F).
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Fig. 3.
Dose-response curves for fatty acid-induced
de-energization of mitochondria. Mitochondria were charged
by the addition of substrate in the presence of 10 mM GDP
and de-energized by the addition of linoleic acid. The concentration of
free acid in the presence of 0.1% BSA was calculated. , wild-type
BAT mitochondria;
, UCP1-deficient mitochondria;
, liver
mitochondria. Values are expressed as means ± S.D. Graphs were
fitted with the least-square method, and initial de-energization was
calculated.
Proton-motive force and de-energization by fatty acids in BAT
mitochondria from Ucp1 knockout mice and control mice
p and the
maximum of
p. Concentrations of FFA are given as nominal
concentrations in the presence of 0.1% BSA. n
9, animals of both genders and age of 65 ± 5 days. Values are
expressed as means ± S.D.
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Fig. 4.
Effect of UCP1 deficiency on stimulation of
respiration of BAT mitochondria. A and B,
representative traces for wild-type and UCP1-deficient mitochondria,
respectively. Oxygen consumption was calculated by numeric
differentiation of oxygen content, which was measured using a
Clark-type electrode. Additions were 5 mM glycerol
phosphate as substrate, 1 mM GDP/Tris salt, and 20 µM linoleic acid in the presence of 0.1% albumin and 4 µM FCCP. C, respiration in the presence of 1 mM GDP and different concentrations of lauric acid. ,
wild-type mitochondria;
, UCP1-deficient mitochondria. Six male mice
were used for each group. Values are expressed means ± S.E. Age
was 65 ± 5 days.
View larger version (14K):
[in a new window]
Fig. 5.
Expression levels of UCP2 and UCP3 in
UCP1 knockout mice of different genetic backgrounds.
RNAs from BAT (A) and skeletal muscle (B) were
isolated using TRI®-Reagent. mRNA levels for UCP2
(open bars) and UCP3 (closed bars) were estimated
by real-time reverse transcription-polymerase chain reaction. All
expression levels are shown as percentage of control mice. At least
four mice were used in each group. Values are expressed as
means ± S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3-adrenergic receptor, which has been
postulated to be essential for the differentiation of the brown
adipocyte (30), can be exposed to 4 °C without symptoms of
hypothermia, apparently through compensation by
1-adrenergic receptors (31). More relevant with respect
to the UCP1-deficient mice are the dopamine
-hydroxylase-deficient
mice that are unable to synthesize norepinephrine (12). Even though
these mice are on an undefined genetic background, they are even more
sensitive to cold compared with UCP1-deficient mice, and there is no
evidence that any of the mutant animals are resistant to cold. The fact that these mice have low levels of Ucp1 mRNA suggests
that the extreme sensitivity to cold is based on the impairment of
mechanisms of thermogenesis in brown adipocytes besides UCP1 levels.
Certainly the ability of these dopamine
-hydroxylase-deficient mice
to generate fatty acids for
-oxidation from the triglyceride pools has been strongly reduced, as evidenced by the accumulation of lipid in
the cytoplasm of the brown adipocytes. Other processes, including the
stimulation of blood flow through the tissue, must be reduced in the
absence of norepinephrine.
-calcium/calmodulin kinase II gene show much more defensive
aggression than either the homozygous wild-type or mutant mice (34). An
explanation of the mechanisms for these effects is not known, but it is
clear that heterosis is not an uncommon phenomenon. Despite the fact
that heterosis is poorly understood, hybrid effects may explain the
cold resistance of some UCP1-deficient mice on a mixed undefined
background in our original study (10).
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ACKNOWLEDGEMENTS |
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We thank Robert Koza and Martin Rossmeisl for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Pfizer and the National Institutes of Health.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.
¶ To whom correspondence should be addressed: Pennington Biomedical Research Center, 6400 Perkins Rd., Baton Rouge, LA 70808. Tel.: 225-763-2771; Fax: 225-763-3030; E-mail: KozakLP@pbrc.edu.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M100466200
2 W. E. Hofmann, X. Liu, C. M. Bearden, M.-E. Harper, and L. P. Kozak, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: BAT, brown adipose tissue; FFAs, free fatty acids; BSA bovine serum albumin, FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.
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REFERENCES |
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