1

Exercise induces an increase in muscle UCP3 as a component of the increase in mitochondrial

biogenesis

Terry E. Jones, Keith Baar, Edward Ojuka, May Chen, John O. Holloszy

Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

Running Title: Exercise, UCP3 and mitochondrial biogenesis

Please send correspondence to:

John O. Holloszy, MD Washington University School of Medicine Department of Medicine Campus Box 8113 4566 Scott Avenue St. Louis, MO 63110

Phone: 314-362-3506 Fax: 314-362-7657 E-Mail: jhollosz@im.wustl.edu

Copyright 2002 by the American Physiological Society.

AJP-Endo Articles in PresS. Published on September 17, 2002 as DOI 10.1152/ajpendo.00316.2002

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ABSTRACT

Previous studies have indicated that exercise acutely induces large increases in UCP3 in

skeletal muscle, while endurance training results in marked decreases in muscle UCP3. Because

UCP3 expression appears to be regulated by the same mechanism as other mitochondrial

constituents, it seemed unlikely that exercise would result in such large and divergent changes in

mitochondrial composition. The purpose of this study was to test the hypothesis that major

changes in UCP3 protein concentration do not occur independently of mitochondrial biogenesis

and that UCP3 increases as a component of the exercise-induced increase in mitochondria. We

found a large increase in UCP3 mRNA immediately and 3h after a bout of swimming. UCP3

protein concentration was increased ~35% 18h after a single exercise bout, ~63% after 3d and

~84% after 10d of exercise. These increases in UCP3 roughly paralleled those of other

mitochondrial marker proteins. Our results are consistent with the interpretation that endurance

exercise induces an adaptive increase in mitochondria that have a normal content of UCP3.

gene expression; cytochrome c; cytochrome oxidase; skeletal muscle

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INTRODUCTION

The mitchondrial protein UCP3 was identified on the basis of its homology with the

uncoupling protein UCP1 present in brown adipose tissue (14). It has been proposed on the basis

of its homology with UCP1 that UCP3 also functions as an uncoupling protein (5, 14).

Furthermore, some investigators have interpreted the results of studies of the heterologous

expression of UCP3 in yeast (36) and of overexpression or knockout of UCP3 in transgenic mice

(8, 9, 34) as providing evidence for uncoupling.

UCP3 is expressed primarily in skeletal muscle (14). Because of its possible role in

energy metabolism, a number of investigators have studied the adaptive response of UCP3 in

skeletal muscle to exercise (11, 23, 29, 33, 37). Tsuboyama-Kasaoka et al. (33) reported that

UCP3 mRNA was increased 7-fold in skeletal muscle of mice three hours after a single bout of

treadmill running, and 16-fold 3 h after exercise in mice that had been trained for 3 wk by

swimming. They speculated that “upregulation of UCP3 mRNA may be a defense mechanism

against extra energy supply to consume extra energy in skeletal muscle.” Similarly, Zhou et al.

(37) reported that UCP3 mRNA was increased ~7-fold immediately after a 200 min long bout of

swimming. A similar response was seen following a bout of treadmill running. They also

reported that UCP3 protein concentration was increased 3.5-fold immediately after 100 min of

treadmill running and 5.6-fold immediately after 200 min of running (37). In studies in humans

Pilegaard et al. (23) found a ~2.5-fold increase in UCP3 mRNA 4 h after a 4 h bout of cycling,

and Schrauwen et al. (29) reported a ~2-fold increase in UCP3 mRNA 4 h after exercise in men

who performed cycle exercise for 2 h in the fasting state.

Although there were differences in the time course and magnitudes of the increases, all of

the studies on the acute effect of exercise have shown that UCP3 mRNA is increased after a bout

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of exercise. However, Boss et al. (6) reported that in contrast to the acute effects of exercise,

endurance exercise training by means of an 8 week long program of treadmill running resulted in

76% and 59% decreases in UCP3 mRNA in tibialis anterior and soleus muscles of rats. They

speculated that “a need for high metabolic efficiency is associated with decreased mRNA

expression of UCPs in skeletal muscle which would decrease energy dissipation” in the trained

state. Similarly, Schrauwen et al. (29) reported that endurance trained men had significantly

reduced UCP3 mRNA levels in quadriceps muscles compared to untrained men, and that UCP3

mRNA was negatively correlated with maximal oxygen uptake capacity in these subjects.

Taken together the results of these studies have lead to the hypotheses that the “acute

regulation of UCP3 gene expression has immediate and functionally important consequences”

(37), while the decrease in UCP3 mRNA with long term training results in an increase in the

metabolic efficiency of muscle (6, 30). These hypotheses imply that exercise results in major

changes in mitochondrial composition with a great short-term increase in UCP3, and a large

long-term decrease in the amount of UCP3 relative to the other mitochondiral constituents.

Because UCP expression appears to be regulated by the same mechanisms as other

mitochondrial constituents (25, 35), it seemed improbable to us that major changes in UCP3

protein concentration would occur independently of mitochondrial biogenesis. Therefore, the

purpose of the present study was to test the hypothesis that UCP3 protein concentration increases

as a component of the increase in skeletal muscle mitochondria induced by exercise (4, 18).

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MATERIALS AND METHODS

Materials. Reagents for ECL were obtained from Amersham Pharmacia Biotech.

Reagents for SDS-polyacrylamide gel electrophoresis and Zeta-Probe membranes were from

Bio-Rad. TRIzol Reagent, for isolation of RNA was purchased from Invitrogen. [ 32P]dATP

was purchased from NEN Life Science Products. ULTRAhyb and the Strip EZ DNA labeling kit

were obtained from Ambion. A rabbit polyclonal antibody directed against the 20 carboxy-

terminal amino acids of citrate synthases was generated by Alpha Diagnostic International. A

mouse anti-human cytochrome oxidase (COX) subunit I monoclonal antibody was purchased

from Molecular Probes. A mouse anti-cytochrome c monoclonal antibody was purchased from

Pharmingen International. Horseradish peroxidase-conjugated secondary antibodies were from

The Jackson Laboratory. All other reagents were purchased from Sigma.

Animals. Male wistar rats (~100 g) were purchased from Charles River. Purina chow

and water were provided ad libitum. This study was approved by the Animal Studies Committee

of Washington University School of Medicine.

Exercise. The rats were accustomed to swimming for 2 days, 10 min/day before

performing the exercise protocol of swimming for two-3 h long periods separated by a 45 min

long rest period, as described previously (24, 26). One group of rats performed the exercise

protocol for one day, one group of rats performed three daily bouts of exercise, and a third group

performed 10 daily bouts of exercise. Animals were anesthetized with pentobarbital sodium (5

mg/100 g body wt) immediately, 3h or 18h after the last bout of swimming and triceps and

epitrochlearis muscles were collected. In a separate experiment, soleus, and white and red

portions of quadriceps muscles were harvested from sedentary rats. All muscles were clamp-

frozen and stored at -80°C. The anesthetized rats were killed by exsanguination.

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Northern blot analysis. Triceps muscle was homogenized in TRIzol. Total RNA was

precipitated using chloroform and isoproopanol. 25 µg of total RNA was size-fractionated in a

1% formaldehyde agarose gel and transferred to Zeta-Probe membrane. Hybridization was

carried out overnight in ULTRAhyb containing a cDNA probe with specific radioactivity of 106

cpm/ml. The cDNA utilized in this study was rat UCP3 (a generous gift from G. Lynis Dohm)

labeled by using a Strip-EZ DNA kit and [ 32P]dATP. Autoradiographs of the membranes were

made using Kodak Biomax MR film.

Western immunoblotting. Epitrochlearis, triceps, soleus, white quadriceps, or red

quadriceps muscle was homogenized in a buffer containing 20 mM HEPES, 1 mM EDTA, 250

mM sucrose, pH 7.4. Protein content was measured using BCA (Pierce). Aliquots of

homogenate were solubilized in Laemmli sample buffer, and subjected to SDS-polyacrylamide

gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes.

Membranes were blocked in PBS or TBS containing 5% nonfat dry milk. Blots were probed

with antibodies directed against UCP3, cytochrome c, citrate synthase and COX subunit I. Blots

were then incubated with secondary antibody conjugated to horseradish peroxidase. Antibody

bound protein was detected by ECL. Films were scanned and analyzed using Sigma Scan

(Jandel Scientific).

Statistical analyses. Results are expressed as mean ± SE. Statistical comparisons were

made using unpaired t-tests with the level of statistical significance set at p<0.05.

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RESULTS

Response of UCP3 to a single bout of exercise. We were unable to quantify the

magnitude of the increase of UCP3 mRNA, as a UCP3 mRNA signal was not detected in non-

exercised muscles. However, Fig. 1A clearly shows that UCP3 mRNA expression was increased

in triceps muscles of rats immediately after, and three hours after, a bout of swimming. This

finding confirms the results of Zhou et al. (37). However, in contrast to the finding of Zhou et al.

(37) that UCP3 protein was increased ~ 5 fold immediately after 200 min of exercise, there was

no increase in UCP3 protein in muscles of our rats ~10 min after 6h of exercise. (Fig. 1B)

Adaptive response of UCP3 and mitochondrial marker enzymes to a program of daily

swimming. Eighteen hours after a bout of swimming UCP3 protein concentration was increased

~35% in epitrochlearis muscle (Fig. 1B, Fig. 2). There was a further progressive increase in

UCP3 protein concentration in the epitrochlearis muscle in response to 3 and 10 days of daily

swimming (Fig. 2). This pattern of increase was similar to that of cytochrome oxidase subunit I.

Except for a more rapid initial increase, the increments in UCP3 also paralleled those of two

other mitochondrial markers, cytochrome c and citrate synthase. A similar pattern of increase of

mitochondrial marker enzymes was seen in triceps muscle (data not shown). Thus, it appears

that the adaptive increase in UCP3 in response to exercise reflects the overall increase in

mitochondrial biogenesis.

Relationship between UCP3 and other mitochondrial constituents in the three muscle

fiber types. It has been reported that expression of UCP3 protein is most abundant in type IIb

fibers, less in type IIa fibers, and lowest in type I fibers (17). This report appears to be in

conflict with our hypothesis that the UCP3 content of skeletal muscles is proportional to their

content of mitochondria, because type IIb fibers have a lower content of mitochondria than type

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IIa or type I fibers. We therefore compared the pattern of UCP3 expression with that of two

mitochondrial marker proteins, cytochrome oxidase subunit I and cytochrome c in the

superficial, white and deep, red portions of the quadriceps muscle, and in the soleus muscle. The

superficial, white portion of the quadriceps contains a high proportion of type IIb fibers, the deep

red portion of the quadriceps is made up predominantly of type IIa fibers, while the soleus has a

high proportion of type I fibers (1). As shown in Fig. 3, the concentration of UCP3 protein

roughly parallels those of two other mitochondrial marker proteins.

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DISCUSSION

The results of this study support our hypothesis that the increase in UCP3 protein that

occurs in response to exercise does not represent an increase in UCP3 protein per se. Instead it

appears that UCP3, a mitochondrial constituent, increases as a component of the exercise-

induced increase in muscle mitochondria. Previous studies have shown that mitochondria from

endurance-trained muscle are tightly coupled (2, 18, 22). An increase in tightly coupled muscle

mitochondria of normal composition, such as occurs with endurance training, does not result in

an increase in resting metabolic rate (31), because there is no increase in the energy requirement

of trained resting muscle, or, therefore, in the availability of ADP and inorganic phosphate (10,

12)

Previous studies of the acute effects of exercise showed that UCP3 mRNA is increased

immediately or shortly after a bout of exercise (23, 33, 37). This finding led to speculation

regarding the functional significance of increased UCP3 expression. Tsuboyama-Kasaoka, et al.

(33) hypothesized that the large up-regulation of UCP3 mRNA may play a role in “fine

adjustments in energy expenditure that may be a defense mechanism against extra supply to

consume extra energy in skeletal muscles”. They also speculated that this increase in UCP3

expression might play a role in the increase in glucose uptake seen three hours after exercise.

While increases in mRNA are frequently referred to as increases in gene expression,

mRNA only provides the information that can lead to increased gene expression. Gene

expression can be controlled at various points beyond transcription in the sequence of events

leading to an increase in the concentration of a protein and modification of the phenotype.

Increased gene expression and its physiological consequences do not occur until there is an

increase in the concentration of the protein encoded by the gene. As illustrated by the response of

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UCP3 to exercise, the magnitude of an adaptive increase in the concentration of a protein can be

very different from that of the increase in its mRNA, i.e., 7-16 fold increases in mRNA after a

bout of exercise (33, 37) versus a ~35 % increase in UCP3 protein concentration 18 hours after

one bout and a ~ 84 % increase after 10 daily exercise bouts (Fig. 2).

Zhou et al. (37) hypothesized that the rapid increase in UPC3 expression induced by

exercise functions to decrease production of reactive oxygen species in contracting muscles. This

hypothesis seemed reasonable in the context of their finding that the amount of UCP3 protein

was increased 5.6-fold in leg muscles of rats immediately after a single, 200 min long bout of

treadmill running. This large and very rapid increase in protein concentration, which paralleled

the increase in UCP3 mRNA, is, to the best of our knowledge, unprecedented. The largest and

most rapid increases in mitochondrial proteins in skeletal muscle in response to exercise reported

previously are probably those of GLUT4 and ALA synthase both of which can increase as much

as 2-fold during an 18 hour post-exercise period (19, 26). In any case, we were unable to detect

an increase in UCP3 protein immediately post-exercise and found only a 35% increase 18 h after

one bout of exercise. We have no explanation for this discrepancy.

Boss et al. (6) reported that a strenuous 13 wk long program of treadmill exercise training

resulted in decreased expression of UCP3 (an average decrease of ~65 %) in rat skeletal muscles.

They concluded that, in keeping with their hypothesis, this “decreased expression of uncoupling

protein in skeletal muscle would allow for a higher level of metabolic efficiency . . . which

would . . . favor energy storage . . . recovery.” Similarly Schrauwen et al. (29) reported that

UCP3 expression was significantly lower in muscles of trained, as compared to untrained, men,

and that maximal oxygen uptake capacity was negatively correlated with UCP3. In these studies

UCP3 mRNA, rather than actual UCP3 expression, was measured. Nevertheless the finding of a

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decrease in UCP3 mRNA concentration is puzzling and seems incompatible with our finding of a

progressive increase in UCP3 protein concentration during 10d of exercise-training (Fig. 2).

This finding of Boss et al. (6) is also in disagreement with the results of Tsuboyama-Kasaoka et

al. (33), who found a large increase in UCP3 mRNA after 3 wk of exercise training. Our results

provide evidence that exercise does not result in major changes in the ratio of UCP3 to other

mitochondrial constituents. This in contrast to the picture provided by previous studies of

marked alterations in mitochondrial composition, with an initial large disproportional increase in

UCP3 with one exercise bout (33, 37) and a large decrease in UCP3 with training (6, 29).

There is considerable controversy regarding the functional role of UCP3. UCP1 mediates

adaptive thermogenesis in brown adipose tissue by uncoupling mitochondrial respiration from

ATP synthesis (27). Because UCP3 is a homologue of UCP1, it has been proposed that UCP3

also functions as an uncoupling protein that plays a role in skeletal muscle thermogenesis and

energy expenditure (6, 9, 14, 20).

The results of two studies on UCP3 knockout mice were interpreted to indicate that

mitochondria were more tightly coupled, with an increased state 3/state 4 ratio in one study (34),

and, in another study (9), a 4-fold increase in the rate of ATP synthesis with no change in citrate

cycle flux, CO2 production or energy expenditure. The latter finding is surprising, since the

theoretical maximum P/O ratio (i.e. number of ATP’s generated per oxygen atom) is 3; while

more recently values of ~2.5 have been obtained (3). These findings also do not seem to fit with

the apparent absence of an altered phenotype in UCP3 knockout mice. They do not have a

decreased metabolic rate and are not obese (7, 9, 15, 34), and respond normally to thyroid

hormone (15). A subsequent study on UCP3 knockout mice by Cadenas et al. (7) failed to

confirm the earlier findings of more tightly coupled mitochondria (9, 34); instead they found that

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respiratory control and proton conductance were unchanged in mitochondria lacking UCP3.

There is evidence that UCP3 can uncouple mitochondria when expressed in yeast (36) or

overexpressed in mouse muscle (7, 8). However, as discussed by Brand and co-workers (7, 16)

this uncoupling appears to be an expression artifact due to disruption of mitochondrial function

by the enormous, 20 to 66-fold, unphysiological increases in UCP3. Although studies in which

UCP3 expression was altered by physiological interventions also do not support an uncoupling

role for UCP3 (21, 28, 32), further research is needed to firmly establish whether or not UCP3

functions as an uncoupling protein muscle under some physiologic conditions such as, for

example, cold exposure.

Another hypothesis regarding the biological role of UCP3 in skeletal muscle is that it has

no significant uncoupling function under physiological conditions, but serves to transport

superoxide anions as a component of the defense mechanism against reactive oxygen species

(ROS) (5, 13). If this concept is correct, our results would indicate that endurance exercise-

training results in an increase in skeletal muscle mitochondria that have a normal capacity to

protect against ROS by the UCP3 mediated transport of superoxide anions.

In conclusion, our results provide evidence that endurance exercise results in an increase

in UCP3 protein in skeletal muscle as a component of the exercise-induced increase in

mitochondrial biogenesis. In contrast to the results of previous studies, they indicate that this

adaptive response results in mitochondria with a normal content of UCP3, rather than in

mitochondria with a markedly increased or decreased compliment of UCP3.

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ACKNOWLEDGEMENTS

We are grateful to Mrs. Victoria Reckamp for expert technical assistance with

preparation of this manuscript, and to Dr. Lorraine Nolte for technical assistance. We thank Dr.

Lynis Dohm for kindly providing the UCP3 cDNA.

This research was supported by National Institutes of Health Grant AG00425. Terry E.

Jones, Keith Baar and Edward Ojuka were supported by National Institute on Aging Institutional

National Research Service Award AG00078.

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FIGURE LEGENDS

Fig. 1. Effects of one bout of exercise on triceps muscle UCP3 mRNA and protein content. A.

Northern blots were performed on RNA from triceps muscle of sedentary rats, and on muscles of

exercised rats obtained ~10 min, 3h or 18h after exercise, as described in Materials and Methods.

B. Western blots were performed on homogenates of epitrochlearis muscles from sedentary rats,

and on muscles of exercised rats obtained ~10 min and 18h after exercise, as described in

Materials and Methods. Each bar represents the mean ± SE for muscles from 6-11 rats.

Fig. 2. Time course of the increases in UCP3 protein and a number of other mitochondrial

proteins in response to exercise. Epitrochlearis muscles were obtained from sedentary rats and

from rats exercised by swimming either once, for 3d or for 10d. Muscles were taken ~18-20h

after the last exercise bout. Enzyme protein levels were determined by Western blot analysis as

described under Materials and Methods. Each point represents the mean obtained on muscles

from 3-12 rats.

Fig. 3. Comparison of UCP3, cytochrome c and cytochrome oxidase subunit I (COXI) levels in

the soleus and the deep, red and superficial, white portions of the quadriceps muscle. Muscle

homogenates were used to determine UCP3, cytochrome c and cytochrome oxidase subunit I

protein levels as described under Materials and Methods. Bars are means ± SE for muscles from

3-4 rats.

21

Control 10 min 18 h

UC

P3 (n

orm

aliz

ed to

tal i

nten

sitie

s)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

A

Control 10 min 3 h 18 h Post-exercise

B

Control 10 min 18 h Post-exercise

Figure 1

UCP3 mRNA

UCP3 Protein

Post-exercise

*

22

Exercise (days)0 2 4 6 8 10

Perc

ent i

ncre

ase

0

20

40

60

80

100

120 UCP3Cytochrome cCOX subunit ICitrate synthase

Figure 2

23

Nor

mal

ized

per

cent

age

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6UCP3Cytochrome cCOX subunit I

soleus white red quadriceps quadriceps

Figure 3