1 Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5; and 3 Food Research Program, Agriculture and Agri-Food Canada, Guelph, Ontario N1G 5C9, Canada; and 2 Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808
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ABSTRACT |
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Uncoupling protein-3 (UCP3) is a mitochondrial carrier protein
of as yet undefined physiological function. To elucidate
characteristics of its function, we studied the effects of fasting on
resting metabolic rate, respiratory quotient, muscle Ucp3
expression, and mitochondrial proton leak in wild-type and
Ucp3(/
) mice. Also analyzed were the fatty acid
compositions of skeletal muscle mitochondria in fed and fasted
Ucp3(
/
) and wild-type mice. In wild-type mice, fasting
caused significant increases in Ucp3 (4-fold) and
Ucp2 (2-fold) mRNA but did not significantly affect
mitochondrial proton leak. State 4 oxygen consumption was not affected
by fasting in either of the two groups. However, protonmotive force was
consistently higher in mitochondria of Ucp3(
/
) animals
(P = 0.03), and fasting further augmented protonmotive
force in Ucp3(
/
) mice; there was no effect in wild-type
mitochondria. Resting metabolic rates decreased with fasting in both
groups. Ucp3(
/
) mice had higher respiratory quotients
than wild-type mice in fed resting states, indicating impaired fatty
acid oxidation. Altogether, results show that the fasting-induced
increases in Ucp2 and Ucp3 do not correlate with
increased mitochondrial proton leak but support a role for UCP3 in
fatty acid metabolism.
uncoupling proteins; uncoupling protein-2; uncoupling protein-3; proton leak; mitochondria; skeletal muscle; thermogenesis; energy expenditure
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INTRODUCTION |
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TO DATE, FIVE PUTATIVE uncoupling proteins (UCPs) have been cloned on the basis of their homology to the original uncoupling protein, UCP1, found exclusively in brown adipose tissue. Although a role for UCP1 in thermogenesis is unequivocal (15, 20), the physiological functions of the newer uncoupling proteins are as yet unclear. UCP2 and UCP3 share an ~58% homology at the amino acid level with UCP1, and in vitro studies demonstrate their ability to uncouple respiration in yeast and in proteoliposomes (9, 14, 16, 17). UCP2 is expressed in most tissues, whereas UCP3 is expressed selectively in skeletal muscle of humans and also in brown adipose tissue of rodents (9). Because of its homology to UCP1, and the significant proportion of resting muscle oxygen consumption that is due to a mitochondrial proton leak, the mechanism of which is poorly understood (29), it was hypothesized that UCP3 plays an important role in energy expenditure (9, 17).
The hypothesis that UCP3 plays an important role in the regulation of
energy expenditure is, however, controversial. Although the
Ucp3(/
) mouse is not obese and has a normal thermogenic response to cold and thyroid hormones (18, 33), the
overexpression of human Ucp3 in muscle of mice has a thermogenic
effect, and mice are resistant to the development of obesity and
insulin insensitivity (12). Recent in vivo studies of
skeletal muscle ATP synthesis have also revealed in fasting
Ucp3(
/
) mice that the rate of ATP synthesis is over
fourfold higher and the ratio of ATP to ADP is 31% higher than in
control mouse tissue (13). Interestingly, however, there
were no changes in the rate of TCA cycle flux or in whole body energy
expenditure (calculated from the turnover of doubly labeled water).
Given that overall energy expenditure and substrate oxidation reactions
(i.e., TCA cycle activity) are unchanged, there must be either a
decrease in proton leak reactions or an increase in a futile cycle of
ATP turnover that does not require increased substrate oxidation.
Ucp3, similar to Ucp1 and Ucp2, is highly regulated at the transcriptional level. In brown adipose tissue, the downregulation of expression induced by food restriction and fasting (8, 17, 30, 31) is indeed consistent with a thermogenic role in that tissue. However, the upregulation of Ucp3 expression in muscle during food restriction and fasting runs counter to the idea that the primary function of UCP3 is to uncouple respiration (8). Many groups have shown that increased circulating levels of fatty acids increase the expression of Ucp3 in muscle (17, 21, 32, 34). The latter, in conjunction with findings showing that mutations in human Ucp3 gene affect fat oxidation in humans (1), suggests that UCP3 may play a more important role in fatty acid metabolism than in energy expenditure per se.
To further understand the physiological role(s) of UCP3, we have
studied in Ucp3(/
) and wild-type mice characteristics of total body energy expenditure, respiratory quotients, muscle
mitochondrial proton leak, and mitochondrial fatty acid content in both
fed and fasting states. In wild-type mice, we determined whether
fasting-induced increases in the expression of Ucp3 in
muscle correlated with changes in mitochondrial proton leak. On the
basis of the upregulation of Ucp3 during fasting, we
assessed whether fasting caused perturbations in mitochondrial
metabolism in the absence of Ucp3 expression.
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EXPERIMENTAL PROCEDURES |
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Treatment of animals.
Male Ucp3(/
) and wild-type C57BL/6J mice
(18) were obtained at 3-6 mo of age from the research
colonies of Dr. Marc Reitman (National Institute of Diabetes and
Digestive and Kidney Diseases). Mice were group housed (3/cage), given
free access to Charles River-5075 rodent chow (4.5% fat by weight) and
water, and kept at 23°C with light at 0700-1900. Mice were cared
for in accordance with the principles and guidelines of the Canadian
Council on Animal Care and the Institute of Laboratory Animal Resources
(National Research Council).
Indirect calorimetry. Oxygen consumption and carbon dioxide production were measured using a four-chamber Oxymax system with automatic temperature and light controls (Columbus Instruments, Columbus, OH). Temperature was maintained at 23°C, and lights were on from 0700 to 1900. System settings included a flow rate of 1 l/min, a purge time of 2 min, and a measurement period of 60 s. Twenty-four hours before the collection of tissues and starting at 0900, mice were placed in separate calorimetry chambers (each with a volume of 2.5 liters). One mouse had ad libitum access to chow and water, and the other had access only to water. Resting metabolic rates were calculated as the mean of the three lowest points over each of the specified time periods. The respiratory quotient (RQ) is a ratio of carbon dioxide produced (in liters) divided by oxygen produced (in liters). The oxidation of carbohydrate results in an RQ of 1.00, whereas the oxidation of fatty acids results in a value of ~0.70 (2). For each mouse, RQ values were recorded at the time points used for resting metabolic rate determinations.
Muscle mRNA levels of Ucp2 and Ucp3. Real-time RT-PCR was carried out with the use of TAQMAN probes and primers complementary to cDNAs of Ucp2 and Ucp3 (accession nos. AB012159 and AF032902).
Isolation of mitochondria from skeletal muscle.
Mitochondria were isolated from hindlimb and forelimb skeletal muscles
of 16 Ucp3(/
) and 16 wild-type mice. Muscle was placed immediately in ice-cold medium (in mM: 100 sucrose, 10 EDTA, 100 Tris · HCl, 46 KCl, pH 7.4 with KOH) and minced in cold medium containing defatted bovine serum albumin (0.5% wt/vol) to bind fatty
acids liberated during tissue homogenization. The protease Nagarse
XXVII (Sigma, St. Louis, MO) was added at a final concentration of 0.8 U/ml, and the suspension was incubated for 2 min at room temperature.
Tissue was disrupted in a Potter Elvjehm homogenizer set at low speed
and spun at 750 g for 10 min at 4°C. The supernatant was
then spun at 10,000 g for 10 min at 4°C; the mitochondrial pellet was resuspended in isolation medium (without albumin) and spun
again as above. Mitochondria were resuspended in 0.175 ml of
albumin-free suspension medium. Protein concentration was assayed by
the Biuret method, using bovine serum albumin as the reference standard.
Measurement of mitochondrial oxygen consumption.
Mitochondrial respiration was measured using a Clark-type oxygen
electrode (Hansatech, Norfolk, UK), the incubation chamber of which was
kept at 37°C and magnetically stirred. Mitochondria were incubated at
0.5 mg of mitochondrial protein in 1.0 ml of suspension medium (120 mM
KCl, 20 mM sucrose, 20 mM glucose, 10 mM
KH2PO4, 5 mM HEPES, 2 mM MgCl2, and
1 mM EGTA, pH 7.2 with KOH). Respiration rates were determined
simultaneously and in parallel with measurements of protonmotive force
(p). Titrations were carried out in the presence of 10 mM succinate,
80 ng nigericin/ml (to convert
pH to voltage units), and 5.0 µM
of rotenone (to prevent oxidation of any endogenous NAD-linked
substrates). State 4 oxygen consumption was determined in the presence
of maximal amounts of the ATP synthase-specific inhibitor oligomycin (8 µg/mg protein). Further additions of oligomycin caused no additional inhibition of oxygen consumption and no further increase in
p, confirming that ATP synthase was completely inhibited.
Measurement of mitochondrial p.
p was determined by use of a methyltriphenylphosphonium
(TPMP+)-sensitive electrode as previously described
(22).
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Overall kinetics of mitochondrial proton leak.
The kinetic response of the proton leak to p was assessed in the
presence of maximal amounts of the ATP synthase inhibitor oligomycin
and by titrating the activity of the respiratory chain with incremental
concentrations of malonate (0.20-2.0 mM), as previously described
(5, 23).
Extraction of total mitochondrial fatty acids.
Mitochondria remaining from the metabolic studies were pooled into
their four respective groups for fasting and fed Ucp3(/
) and wild-type mice. Each of the isolated composite mitochondrial samples (~10-18 mg protein) in aqueous buffer was transferred into 10-ml Nalgene fluorinated ethylene propylene (FEP) centrifuge tubes adapted with screw caps. The volume was brought up to 1.2 ml with
distilled water. Methanol (3.0 ml) and chloroform (1.5 ml) were added
to the tube to form a single Bligh and Dyer phase (3). The
content was sonicated at one-half power for 1 min with the tube
immersed in an ice bath. To this solution, 1.5 ml of chloroform and 1.5 ml of distilled water were added in this order, mixed thoroughly after
each addition, and centrifuged. The lower chloroform layer was removed,
and the upper phase was reextracted with chloroform (3.0 ml). The
chloroform layers containing total lipids were combined and reduced to
dryness under a stream of nitrogen.
Preparation and analysis of fatty acid methyl esters. The total lipids were methylated by addition of two drops of benzene and 2 ml of 5% HCl in anhydrous methanol (wt/wt) and heating of the solution at 80°C for 1 h. After methylation, 0.1 ml of water and 2 ml of hexane were added, and the content was well mixed and centrifuged. The hexane layer was removed, reduced in volume, and analyzed by gas chromatography (GC; Hewlett-Packard model no. 5890). The GC was equipped with an autosampler (model no. 7673), a split injector, an ionization detector, an HP ChemStation, and a fused silica papillary column (CP Sil 88; 100 m × 0.25 mm inner diameter × 0.2 µm film thickness; Varian, Mississauga, ON). The following conditions were used: H2 as carrier gas at 170 kPa; split ratio of 1:15; injector and detector temperature at 250°C; and temperature program for 2 min at 150°C, 1°C/min to 200°C, followed by 5°C/min to 215°C and then maintainence at 215°C for 20 min. The fatty acid methyl esters (FAME) were identified by comparison with authentic FAME standards obtained from Nu-Chek Prep (Elysian, MN).
Statistical analysis. Two sample comparisons were conducted with the use of two-tailed t-tests. Effects of mouse genotype and fasting were examined using two-way ANOVA. P value < 0.05 was considered significant. Unless otherwise stated, results are presented as means ± SE.
Materials. Oligomycin, malonate, valinomycin, bovine serum albumin (fraction V), TPMP+, succinate, nigericin, and rotenone were from Sigma. 3H2O, 86Rb-labeled Cl, [14C]sucrose, and [3H]TPMP bromide were from Mandel Dupont NEN (Guelph, ON, Canada).
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RESULTS |
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In vivo studies of food intake, metabolic rate, and respiratory
quotient.
Food intake (in
g · day1 · mouse
1) was
monitored over a 5-day period in which the 16 Ucp3(
/
)
and 16 wild-type mice had free access to water and food. There were no
significant differences between the Ucp3(
/
) and
wild-type mice (results not shown), confirming earlier findings
(18).
|
|
Body and tissue weights.
Consistent with some impairment in fatty acid oxidation is the
observation that weight loss during fasting was greater in wild-type
mice than in Ucp3(/
) mice (P = 0.001;
Table 3). As expected, fasting resulted
in significant decreases in fat stores in both the wild-type and
Ucp3(
/
) mice: interscapular brown adipose tissue (IBAT;
P < 0.001), inguinal white adipose tissue (INWAT;
P < 0.05), and epididymal white adipose tissue (EWAT; P < 0.001). Interestingly, the fat pad weights for the
fasted wild-type mice were significantly smaller than the weights for the fasted Ucp3(
/
) mice, when expressed as a percentage
of the corresponding fed values. Note, however, that these percentages are necessarily based on post mortem values from eight mice that were
randomly assigned at the outset to the fed or the fasting groups.
|
mRNA expression of Ucp2 and Ucp3.
In wild-type mice, fasting resulted in a fourfold increased
expression of Ucp3 (Fig.
1A) and a twofold increase in
Ucp2 mRNA expression (Fig. 1B). In
Ucp3(/
) mice, fasting resulted in a 1.5-fold increase in
Ucp2 mRNA (Fig. 1B).
|
Overall kinetics of muscle mitochondrial proton leak in Ucp3(/
)
mice and wild-type mice.
The overall kinetics of mitochondrial proton leak for wild-type and
Ucp3(
/
) mice are shown in Fig.
2, A and B. In
mitochondria from wild-type mice (Fig. 2A), maximal
leak-dependant oxygen consumption rates (state 4 respiration rates)
were similar between the fed and fasted mice. Values were 160.9 ± 9.1 (n = 6) and 173.5 ± 15.8 (n = 6) nmol O · mg
protein
1 · min
1, respectively. Thus
the increases in Ucp2 and Ucp3 expression induced
by fasting (Fig. 1) did not correspond to any increase in maximal
leak-dependent oxygen consumption. Moreover,
p was not significantly
affected by the increased expression of Ucp2 and
Ucp3 mRNA. Values at state 4 were 165.9 ± 5.8 (n = 7) and 167.0 ± 5.2 (n = 7)
mV, respectively, for wild-type fed and fasting states. The kinetics of
muscle mitochondrial proton leak, i.e., the overall response of
leak-dependent oxygen consumption to
p (represented by the overall
shape of the curve in Fig. 2A), was not affected by fasting
in the wild-type mice.
|
Fatty acid analyses.
We then analyzed the fatty acid composition of the mitochondria
remaining from our metabolic studies. The rationale for the analyses
was based on the consistency with which Ucp3 mRNA expression is correlated with metabolic situations of increased fatty acid oxidation (17, 21, 32, 34), on the documented interactions of fatty acids with UCP1 (25), and on the very recent
hypothesis suggesting that UCP3 may export fatty acids from the
mitochondrial matrix (19). The mitochondria remaining from
the studies of mitochondrial proton leak were pooled into their four
respective groups of fasting and fed Ucp3(/
) and
wild-type mice. After pooling, there was 10-18 mg of mitochondrial
protein for each of the four groups, allowing for the measurements
reported in Table 4. In this study, the
fatty acid composition of the total mitochondrial lipids, rather than
their individual lipid classes, is presented because of the limited
amount of lipid involved.
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DISCUSSION |
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Many studies in the literature have reported on altered patterns of mRNA expression for the novel uncoupling proteins and have related the altered expression patterns to possible physiological functions. Interpretation of the data has led to a significant amount of controversy surrounding the hypothesized mechanisms and physiological functions of the novel uncoupling proteins. However, the increases in Ucp3 expression with fasting have remained paradoxical on the basis of the well-known decrease in energy expenditure in muscle with fasting.
To clarify some of the many questions surrounding the expression and
purported function of UCP3, we studied the effects of fasting on muscle
mitochondrial metabolism and proton leak in wild-type mice and in
Ucp3(/
) mice. In this report, we demonstrate that
fasting results in significantly upregulated expression of both
Ucp3 (4-fold) and Ucp2 (2-fold) mRNA levels in
wild-type skeletal muscle and increased Ucp2 expression
(1.5-fold) in Ucp3(
/
) muscle but no significant increase
in proton leak compared with the fed state. Thus increased expression
of Ucp3 and Ucp2 in response to fasting does not
correlate with an increase in mitochondrial proton leak, the function
originally proposed for these proteins. These results support the idea
that the primary function of UCP3 is not mitochondrial proton leak.
Our findings on UCP3-deficient mice significantly extend earlier
findings. Two groups originally reported on metabolic characteristics of Ucp3(/
) mice. Both groups independently reported that
Ucp3(
/
) mice are healthy and are not more predisposed to
obesity than their wild-type counterparts. Vidal-Puig et al.
(33) showed that skeletal muscle mitochondria lacking UCP3
are better coupled and produce a significantly higher amount of
reactive oxygen species than control mitochondria. Gong et al.
(18) reported an increased mitochondrial protonmotive
force consistent with, but not conclusive of, decreased proton leak.
The fact that Gong et al. (18) report no
significant change in state 4 respiration is significant, as state 4 respiration represents the maximum leak-dependent respiration.
Increased expression of Ucp3 mRNA in muscle with fasting has been observed in a number of studies (6, 8, 28). Importantly, Cadenas et al. (10) showed increased expression of Ucp2 and Ucp3 in muscle of starved rats but that mitochondrial proton conductance was unchanged. Others have shown that Ucp2 expression, like that of Ucp3, is increased with fasting (7). These findings suggest that UCP2 and UCP3 do not catalyze the basal proton conductance in skeletal muscle mitochondria. Our results support and extend these findings.
As previously observed (18, 33), Ucp2 levels were not affected by the absence of Ucp3 (Fig. 1), thus eliminating the possibility of compensation by Ucp2 in the absence of Ucp3.
In muscle mitochondria from fed Ucp3(/
) mice, we
observed higher protonmotive force values than in wild-type mice, just as was observed by Gong et al. (18). However, the
increased protonmotive force was not accompanied by a decrease in state 4 respiration, as would be expected in the absence of an uncoupling protein. It can, however, be correctly stated that mitochondrial oxygen
consumption at any given protonmotive force value is lower in the
Ucp3(
/
) mice than in the wild-type mice. Vidal-Puig et al. (33) did not measure mitochondrial protonmotive force
but did show greater coupling in the Ucp3(
/
) mice. It is
not known why we are again unable to detect differences in
coupling efficiency between mitochondria from Ucp3(
/
)
mice and controls. The higher mitochondrial protonmotive force supports
some electrogenic role for UCP3. However, the differences could be
related to differences in contaminating fatty acids in the
mitochondrial preparations from Ucp3(
/
) mice and
controls, rather than underlying mechanistic differences, despite the
presence of defatted bovine serum albumin during the isolation
procedures. This possibility warrants further investigation.
In the Ucp3(/
) mice, fasting had no effect on state 4 oxygen consumption rates, suggesting no significant increase in
uncoupling of oxidative phosphorylation despite the increased
expression of Ucp2. In addition, we observed consistently
higher protonmotive force values rather than lower, as would be
expected if uncoupling were occurring. In the wild-type mice, fasting
caused significant increases in Ucp3 (4-fold) and
Ucp2 (2-fold) mRNA but did not significantly affect state 4 oxygen consumption or the overall kinetics of mitochondrial proton leak
reactions. Altogether, our findings support the idea that UCP2 and UCP3
have a physiological function other than mitochondrial proton leak.
This study is the first in Ucp3(/
) mice to report
significant differences in whole body fatty acid metabolism. We show
that respiratory quotients are significantly higher in
Ucp3(
/
) mice than in wild-type controls under resting
metabolic rate conditions when mice have ad libitum access to food.
Although the trend was also for higher respiratory quotients in
Ucp3(
/
) mice under fasting conditions, this was
not statistically significant. Our findings therefore support the idea
that UCP3 plays an important role in fatty acid oxidation. This would
not be surprising, given the tight correlation between the expression
of Ucp3 and Ucp2 and fasting. Our results
therefore support those of Argyropoulos et al.
(1), who found in studies of a Gullah-speaking
African American population that basal fat oxidation rates were reduced by 50% and that respiratory quotients were markedly increased in
persons heterozygous for an exon 6-splice donor mutation in the
Ucp3 gene. Chung et al. (11) detected the
same splice site mutation in African Americans in a Maywood, IL,
population but were unable to detect phenotypic effects. The latter may
be related to the higher degree of gene admixture in the Maywood
population (26, 27) and the small sample size
(n = 2, n = 1) used to study
characteristics of fatty acid oxidation.
It is generally well acknowledged that UCP2 and UCP3 play some role in energy metabolism during situations in which fatty acid oxidation is high; however, there are very few reports relating to possible mechanisms and functions. While at this point it is only a hypothesis, we have suggested that UCP3 may function in conjunction with mitochondrial thioesterase to export fatty acid anions from the mitochondrial matrix, to liberate CoA-SH and facilitate rapid rates of fatty acid oxidation (19). The findings herein, including our observations of mitochondrial fatty acid changes with fasting and our recent findings that mitochondrial thioesterase-1 is significantly upregulated in muscle of mice that overexpress the human form of Ucp3 in muscle (24), lend significant support for this hypothesis.
Further studies need be directed at mechanisms through which UCP3 may play a key role in mitochondrial fatty acid metabolism. Focus should be placed on the hypothesis that UCP3 may function to translocate fatty acid anion out of the mitochondrial matrix for the physiological purpose of continued high rates of fatty acid oxidation in the face of large supplies of fatty acyl-CoA.
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ACKNOWLEDGEMENTS |
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This research was supported by funding from Natural Sciences and Engineering Research Council of Canada (to M.-E. Harper) and National Heart, Lung, and Blood Institute Grant HD-08431 (to L. P. Kozak).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M.-E. Harper, Dept. of Biochemistry, Microbiology, and Immunology, Univ. of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada (E-mail: mharper{at}uottawa.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 23 May 2001; accepted in final form 20 June 2001.
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