Effects of fasting on muscle mitochondrial energetics and fatty acid metabolism in Ucp3(-/-) and wild-type mice

Véronic Bézaire1, Wolfgang Hofmann2, John K. G. Kramer3, Leslie P. Kozak2, and Mary-Ellen Harper1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Delta p). Titrations were carried out in the presence of 10 mM succinate, 80 ng nigericin/ml (to convert -Delta 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 Delta p, confirming that ATP synthase was completely inhibited.

Measurement of mitochondrial Delta p. Delta p was determined by use of a methyltriphenylphosphonium (TPMP+)-sensitive electrode as previously described (22).

The calibration of the TPMP+-sensitive electrode, the determination of mitochondrial matrix volumes, and the calculation of Delta p from TPMP+ electrode data were carried out as earlier described (4). Delta p is calculated by use of the Nernst equation as
&Dgr;p<IT>=</IT>61.5<IT>·</IT>log (a<SUB>m</SUB><IT>·</IT>TPMP<SUP>+</SUP><SUB>m</SUB>/TPMP<SUP>+</SUP><SUB>e</SUB>)
where TPMP<UP><SUB>m</SUB><SUP>+</SUP></UP>/TPMP<UP><SUB>e</SUB><SUP>+</SUP></UP> represents the ratio of the accumulation of the cation inside and external to the mitochondria. The nonspecific binding of TPMP+ in mitochondria is reflected in am. The latter indicates the proportion of probe that is free (i.e., not bound). am was determined using the method that adjusts the TPMP+ accumulation ratio to the accumulation ratio for 86Rb, a K+ congener that does not bind, over a range of membrane potentials and ignores any effect of matrix volume on the relative binding of TPMP+ (4). Correction factors were 0.31 and 0.36, respectively, for Ucp3(-/-) and wild-type mice, and average matrix volumes were 0.52 ± 0.16 µl/mg protein (n = 2) in Ucp3(-/-) mice and 0.48 ± 0.18 µl/mg protein (n = 2) in controls (18).

Overall kinetics of mitochondrial proton leak. The kinetic response of the proton leak to Delta 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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In vivo studies of food intake, metabolic rate, and respiratory quotient. Food intake (in g · day-1 · 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).

Resting metabolic rate (RMR) was studied in an indirect calorimeter over the 24-h period of fasting or feeding that preceded tissue collection. There were no significant differences in RMR between Ucp3(-/-) and wild-type mice over the 24-h period, again confirming earlier findings (18). However, RMR in both types of mice decreased significantly with fasting (Table 1). In Ucp3(-/-) mice, RMR decreased by 40% compared with measurements taken within the first 2 h of fasting; in wild-type mice, the decrease was 49%.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Resting metabolic rates of wild-type and Ucp3(-/-) mice during the 24-h fast

RQs decreased significantly with fasting at each time point in both Ucp3(-/-) and wild-type mice (Table 2), as expected during a shift from the oxidation of carbohydrate to fat. More importantly, the respiratory quotients were found to be lower in wild-type than Ucp3(-/-) mice at the 0000-0200 (P < 0.01) and 2200-2400 (P < 0.01) time points, indicating impaired fatty acid oxidation in Ucp3(-/-) mice under the ad libitum feeding conditions. During fasting, RQ values also tended to be lower for the eight wild-type than for the eight Ucp3(-/-) mice; the differences were, however, not statistically significant.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   RQs of wild-type and Ucp3(-/-) mice during the 24-h fast

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Body weight, adipose tissue weights, and the effects of fasting in wild-type and Ucp3(-/-) mice

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).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   Increases in Ucp2 and Ucp3 expression in skeletal muscle of wild-type and Ucp3(-/-) mice after a 24-h fast. A: Ucp3 expression in wild-type mice. B: Ucp2 expression in wild-type and Ucp3(-/-) mice. RT-PCR was carried out by use of TAQMAN probes and primers (see EXPERIMENTAL PROCEDURES). ** P < 0.01 and *** P < 0.001.

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, Delta 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 Delta p (represented by the overall shape of the curve in Fig. 2A), was not affected by fasting in the wild-type mice.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Mitochondrial proton leak in skeletal muscle mitochondria from wild-type and Ucp3(-/-) mice. A: wild-type fed (black-triangle) vs. fasted (). B: Ucp3(-/-) fed (black-triangle) vs. fasted (). The kinetic response of proton leak-dependent O2 consumption to protonmotive force (Delta p) was assessed in the presence of maximal amounts of the ATP synthase inhibitor, oligomycin, and by titration of the activity of the respiratory chain with incremental concentrations of malonate (0.20-2.0 mM). State 4 respiration rates are indicated by the values to the far right of each curve.

Similar to the results from wild-type mice, in mitochondria from Ucp3(-/-) mice, fasting did not affect state 4 respiration rates (Fig. 2B). State 4 oxygen consumption rates were 168.7 ± 12.8 (n = 6) and 169.5 ± 17.9 (n = 6) nmol O · mg protein-1 · min-1 for fed and fasted mice, respectively. Delta p values under state 4 conditions were similar in the fed and fasted mice; values were 176.9 ± 4.3 (n = 7) and 178.3 ± 4.0 (n = 7) mV, respectively. The proton leak kinetics are represented in Fig. 2B and show that, overall, Delta p values over the range of incubation conditions were higher in the mitochondria from fasted mice. This is the opposite direction of change that would be expected on the basis of the increased expression of Ucp2 with fasting.

Finally, a comparison of results between wild-type and Ucp3(-/-) mice (Fig. 2, A and B) shows no significant difference in state 4 respiration rates but significantly higher Delta p values (P = 0.03) at state 4 in Ucp3(-/-) mice, corroborating earlier findings (18).

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Effect of fasting on fatty acid composition of muscle mitochondria in wild-type and Ucp3(-/-) mice

The mitochondrial lipids had a characteristically high content of docosahexaenoic acid (22:6n-3) at ~29%, with smaller amounts of linoleic (18:2n-6) and arachidonic acid (20:4n-6) at ~15 and 7%, respectively. Saturated fatty acids accounted for ~30%, with palmitic acid (16:0) at ~17% and stearic acid (18:0) at ~13%. There appeared to be slight differences in the mitochondrial fatty acid composition between the two strains of mice, but that could not be tested statistically because the samples were pooled. In general, the polyunsaturated fatty acids (PUFA) from both the linoleic (n-6) and linolenic (n-3) family of acids were lower in the wild-type compared with the Ucp3(-/-) mice, at the expense of saturated fatty acids, specifically 16:0. Fasting consistently depleted 22:6n-3 in both strains of mice, but in the wild-type mouse, it was at the expense of 16:0, whereas in the Ucp3(-/-) mouse, this was at the expense of 18:2n-6. Fasting did not appear to change the relative concentration of 18:0 and 20:4n-6 and many of the metabolites of the n-3 and n-6 PUFA, although 16:0 and 18:2n-6 responded in opposite directions. Future studies must attempt to perform individual analyses to provide a statistical evaluation, and, if possible, to provide compositional analyses of the individual major lipid classes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Argyropoulos, G, Brown AM, Willi SM, Zhu J, He Y, Reitman M, Gevao SM, Spruill I, and Garvey WT. Effects of mutations in the human uncoupling protein 3 gene on the respiratory quotient and fat oxidation in severe obesity and type 2 diabetes. J Clin Invest 102: 1345-1351, 1998[Abstract/Free Full Text].

2.   Blaxter, K. Energy Metabolism in Animals and Man. Cambridge, MA: Cambridge Univ. Press, 1989, p. 16-17.

3.   Bligh, EG, and Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911-917, 1959[ISI].

4.   Brand, MD. Measurement of protonmotive force. In: Bioenergetics: a Practical Approach, edited by Brown GC, and Cooper CE.. Oxford: IRL, 1995, p. 39-62.

5.   Brand, MD. Top-down elasticity analysis and its application to energy metabolism in isolated mitochondria and intact cells. Mol Cell Biochem 184: 13-20, 1998[ISI][Medline].

6.   Boss, O, Muzzin P, and Giacobino JP. The uncoupling proteins, a review. Eur J Endocrinol 139: 1-9, 1998[ISI][Medline].

7.   Boss, O, Samec S, Dulloo A, Seydoux J, Muzzin P, and Giacobino JP. Tissue-dependent upregulation of rat uncoupling protein-2 expression in response to fasting or cold. FEBS Lett 412: 111-114, 1997[ISI][Medline].

8.   Boss, O, Samec S, Kuhne F, Bijlenga P, Assimacopoulos-Jeannet F, Seydoux J, Giacobino JP, and Muzzin P. 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[Abstract/Free Full Text].

9.   Boss, O, Samec S, Paoloni-Giacobino A, Rossier C, Dulloo A, Seydoux J, Muzzin P, and Giacobino JP. Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression. FEBS Lett 408: 39-42, 1997[ISI][Medline].

10.   Cadenas, S, Buckingham JA, Samec S, Seydoux J, Din N, Dulloo AG, and Brand MD. UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged. FEBS Lett 462: 257-260, 1999[ISI][Medline].

11.   Chung, WK, Luke A, Cooper RS, Rotini C, Vidal-Puig A, Rosenbaum M, Chua M, Solanes G, Zheng M, Zhao L, LeDuc C, Eisberg A, Chu F, Murphy E, Schreier M, Aronne L, Caprio S, Kahle B, Gordon D, Leal SM, Goldsmith R, Andreu AL, Bruno C, DiMauro S, and Leibel RL. Genetic and physiologic analysis of the role of uncoupling protein 3 in human energy homeostasis. Diabetes 48: 1890-1895, 1999[Abstract].

12.   Clapham, JC, Arch JR, Chapman H, Haynes A, Lister C, Moore GB, Piercy V, Carter SA, Lehner I, Smith SA, Beeley LJ, Godden RJ, Herrity N, Skehel M, Changani KK, Hockings PD, Reid DG, Squires SM, Hatcher J, Trail B, Latcham J, Rastan S, Harper AJ, Cadenas S, Buckingham JA, Brand MD, and Abuin A. Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean. Nature 406: 415-418, 2000[ISI][Medline].

13.   Cline, GW, Vidal-Puig AJ, Dufour S, Cadman KS, Lowell BB, and Shulman GI. In vivo effects of uncoupling protein-3 gene disruption on mitochondrial energy metabolism. J Biol Chem. 276: 20240-20244, 2001[Abstract/Free Full Text].

14.   Echtay, KS, Winkler E, Frischmuth K, and Klingenberg M. Uncoupling proteins 2 and 3 are highly active H(+) transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone). Proc Natl Acad Sci USA 98: 1416-1421, 2001[Abstract/Free Full Text].

15.   Enerback, S, Jacobsson A, Simpson EM, Guerra C, Yamashita H, Harper ME, and Kozak LP. Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese. Nature 387: 90-94, 1997[ISI][Medline].

16.   Fleury, C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, and Warden CH. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet 15: 223-224, 1997[ISI][Medline].

17.   Gong, DW, He Y, Karas M, and Reitman M. Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, beta3-adrenergic agonists, and leptin. J Biol Chem 272: 24129-24132, 1997[Abstract/Free Full Text].

18.   Gong, DW, Monemdjou S, Gavrilova O, Leon LR, Marcus-Samuels B, Chou CJ, Everett C, Kozak LP, Li C, Deng C, Harper ME, and Reitman ML. Lack of obesity and normal response to fasting and thyroid hormone in mice lacking uncoupling protein-3. J Biol Chem 275: 16251-16257, 2000[Abstract/Free Full Text].

19.   Himms-Hagen, J, and Harper ME. Physiological role of UCP3 may be export of fatty acids from mitochondria when fatty acid oxidation predominates. Exp Biol Med (Maywood) 226: 78-84, 2001[Abstract/Free Full Text].

20.   Hofmann, WE, Liu X, Bearden CM, Harper ME, and Kozak LP. Effects of genetic background on thermoregulation and fatty acid-induced uncoupling of mitochondria in UCP1-deficient mice. J Biol Chem 276: 12460-12465, 2001[Abstract/Free Full Text].

21.   Lin, B, Coughlin S, and Pilch PF. Bidirectional regulation of uncoupling protein-3 and GLUT-4 mRNA in skeletal muscle by cold. Am J Physiol Endocrinol Metab 275: E386-E391, 1998[Abstract/Free Full Text].

22.   Monemdjou, S, Hofmann WE, Kozak LP, and Harper ME. Increased mitochondrial proton leak in skeletal muscle mitochondria of UCP1-deficient mice. Am J Physiol Endocrinol Metab 279: E941-E946, 2000[Abstract/Free Full Text].

23.   Monemdjou, S, Kozak LP, and Harper ME. Mitochondrial proton leak in brown adipose tissue mitochondria of Ucp1-deficient mice is GDP insensitive. Am J Physiol Endocrinol Metab 276: E1073-E1082, 1999[Abstract/Free Full Text].

24.   Moore, GBT, Himms-Hagen J, Harper ME, and Clapham JC. Overexpression of UCP-3 in skeletal muscle of mice results in increased expression of mitochondrial thioesterase mRNA. Biochem Biophys Res Commun 283: 785-790, 2001[ISI][Medline].

25.   Nicholls, DG, and Rial E. A history of the first uncoupling protein, UCP1. J Bioenerg Biomembr 31: 399-406, 1999[ISI][Medline].

26.   Parra, EJ, Kittles RA, Argyropoulos G, Pfaff CL, Hiester K, Bonilla C, Sylvester N, Parrish-Gause D, Garvey WT, Jin L, McKeigue PM, Kamboh MI, Ferrell RE, Pollitzer WS, and Shriver MD. Ancestral proportions and admixture dynamics in geographically defined African Americans living in South Carolina. Am J Phys Anthropol 114: 18-29, 2001[ISI][Medline].

27.   Parra, EJ, Marcini A, Akey J, Martinson J, Batzer MA, Cooper R, Forrester T, Allison DB, Deka R, Ferrell RE, and Shriver MD. Estimating African American admixture proportions by use of population-specific alleles. Am J Hum Genet 63: 1839-1851, 1998[ISI][Medline].

28.   Ricquier, D, and Bouillaud F. The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP. Biochem J 345: 161-179, 2000[ISI][Medline].

29.   Rolfe, DF, and Brand MD. Proton leak and control of oxidative phosphorylation in perfused, resting rat skeletal muscle. Biochim Biophys Acta 1276: 45-50, 1996[ISI][Medline].

30.   Samec, S, Seydoux J, and Dulloo AG. Role of UCP homologues in skeletal muscles and brown adipose tissue: mediators of thermogenesis or regulators of lipids as fuel substrate? FASEB J 12: 715-724, 1998[Abstract/Free Full Text].

31.   Sivitz, WI, Fink BD, and Donohoue PA. Fasting and leptin modulate adipose and muscle uncoupling protein: divergent effects between messenger ribonucleic acid and protein expression. Endocrinology 140: 1511-1519, 1999[Abstract/Free Full Text].

32.   Tsuboyama-Kasaoka, N, Tsunoda N, Maruyama K, Takahashi M, Kim H, Ikemoto S, and Ezaki O. Up-regulation of uncoupling protein 3 (UCP3) mRNA by exercise training and down-regulation of UCP3 by denervation in skeletal muscles. Biochem Biophys Res Commun 247: 498-503, 1998[ISI][Medline].

33.   Vidal-Puig, AJ, Grujic D, Zhang CY, Hagen T, Boss O, Ido Y, Szczepanik A, Wade J, Mootha V, Cortright R, Muoio DM, and Lowell BB. Energy metabolism in uncoupling protein 3 gene knockout mice. J Biol Chem 275: 16258-16266, 2000[Abstract/Free Full Text].

34.   Weigle, DS, Selfridge LE, Schwartz MW, Seeley RJ, Cummings DE, Havel PJ, Kuijper JL, and BeltrandelRio H. Elevated free fatty acids induce uncoupling protein 3 expression in muscle: a potential explanation for the effect of fasting. Diabetes 47: 298-302, 1998[Abstract].


Am J Physiol Endocrinol Metab 281(5):E975-E982
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society