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Uncoupling protein 3 transcription is regulated by
peroxisome proliferator-activated receptor ␣ in the
adult rodent heart
MARTIN E. YOUNG, SARITA PATIL, JUN YING, CHRISTOPHE DEPRE,
HARLEEN SINGH AHUJA,* GREGORY L. SHIPLEY,* STANISLAW M. STEPKOWSKI,
†
PETER J. A. DAVIES,* AND HEINRICH TAEGTMEYER
1
Division of Cardiology, *Department of Integrative Biology, and the
†
Division of Organ
Transplantation, University of Texas Houston Medical Center, Houston, Texas 77030, USA
ABSTRACT Relatively little is known concerning the
regulation of uncoupling proteins (UCPs) in the heart.
We investigated in the adult rodent heart 1) whether
changes in workload, substrate supply, or cytokine
(TNF-␣) administration affect UCP-2 and UCP-3 ex-
pression, and 2) whether peroxisome proliferator-acti-
vated receptor ␣ (PPAR␣) regulates the expression of
either UCP-2 or UCP-3. Direct comparisons were made
between cardiac and skeletal muscle. UCP-2, UCP-3,
and PPAR␣ expression were reduced when cardiac
workload was either increased (pressure overload by
aortic constriction) or decreased (mechanical unload-
ing by heterotopic transplantation). Similar results were
observed during cytokine administration. Reduced di-
etary fatty acid availability resulted in decreased expres-
sion of both cardiac UCP-2 and UCP-3. However, when
fatty acid (the natural ligand for PPAR␣) supply was
increased (high-fat feeding, fasting, and STZ-induced
diabetes), cardiac UCP-3 but not UCP-2 expression
increased. Comparable results were observed in rats
treated with the specific PPAR␣ agonist WY-14,643.
The level of cardiac UCP-3 but not UCP-2 expression
was severely reduced (20-fold) in PPAR␣
ⴚ/ⴚ
mice
compared to wild-type mice. These results suggest that
in the adult rodent heart, UCP-3 expression is regu-
lated by PPAR␣. In contrast, cardiac UCP-2 expression
is regulated in part by a fatty acid-dependent, PPAR␣-
independent mechanism.—Young, M. E., Patil, S.,
Ying, J., Depre, C., Ahuja, H. S., Shipley, G. L.,
Stepkowski, S. M., Davies, P. J. A., Taegtmeyer H.
Uncoupling protein 3 transcription is regulated by
peroxisome proliferator-activated receptor ␣ in the
adult rodent heart. FASEB J. 15, 833–845 (2001)
Key Words: diabetes 䡠 fasting 䡠 fatty acids 䡠 hypertrophy 䡠 un-
loading
Very little is known about either the function or the
regulation of uncoupling proteins (UCPs) in the adult
heart. There are three known members of the family of
UCPs. Thermogenin, also known as UCP-1, was first
discovered in brown adipose tissue as the principal
mediator of nonshivering thermogenesis (1). UCP-1 is
a transmembrane protein found in the inner mitochon-
drial membrane, where it uncouples ATP synthesis and
mitochondrial electron transport, dissipating the en-
ergy as heat (2). More recently, two genes have been
described with high sequence homology to UCP-1—
UCP-2 and UCP-3 (3–5)—both of which have been
shown to possess uncoupling activity in cells (6). UCP-2
is ubiquitously expressed whereas UCP-3 is highly ex-
pressed in skeletal muscle, adipose tissue, and, to a
lesser extent, the heart. Although much research has
focussed on UCPs in skeletal muscle, the mechanism(s)
by which cardiac UCP expression alter(s) require elu-
cidation.
There are four primary hypotheses concerning the
physiological roles of UCP-2 and UCP-3 (7): thermo-
genesis, regulation of fatty acid oxidation, regulation of
ATP synthesis, and reduction of mitochondrial reactive
oxygen species (ROS) generation. Whether UCPs in
the heart play a role in one or several of the above
mentioned functions is unknown. The roles of UCPs in
tissues, such as skeletal muscle, as well as white and
brown adipocytes have been investigated by determin-
ing the changes in gene expression of the UCPs in
response to various stimuli. For example, increases in
plasma free fatty acid levels result in increased skeletal
muscle UCP expression, suggesting a role in fatty acid
utilization and/or prevention of lipotoxicity and insu-
lin resistance (7–9). Thus, determining UCP expres-
sion in the heart in response to altered workload (in
which ATP demand, substrate utilization, and ROS
generation are altered) and/or substrate availability
might aid an understanding of the role of cardiac
UCPs. In addition, tumor necrosis factor ␣ (TNF-␣) has
been shown to increase skeletal muscle UCP expres-
sion, suggesting a role in cytokine-induced thermogen-
esis (10). TNF-␣ levels are known to increase in the
failing heart(11), which has also been described as
‘energy starved’ (12). Whether increased TNF-␣ levels
result in increased UCP expression and whether the
1
Correspondence: Division of Cardiology,, University of
Texas Houston Medical School, 6431 Fannin, MSB 1.246,
Houston, TX 77030, USA. E-mail: ht@heart.med.uth.tmc.edu
8330892-6638/01/0015-0833 © FASEB
latter affects the efficiency of the failing heart are
unknown.
The purpose of the present study was twofold. First,
we wanted to investigate both physiological and patho-
physiologic situations under which cardiac UCP-2 and
UCP-3 expression might be altered. These included
increased and decreased workload and altered sub-
strate availability (e.g., fasting/refeeding, altered di-
etary fatty acid composition, streptozotocin-induced
diabetes), as well as cytokine (TNF-␣) challenge. Sec-
ond, we were curious about the potential role of
peroxisome proliferator-activated receptor ␣ (PPAR␣,a
nuclear transcription factor that has been suggested to
play a role in skeletal muscle and adipocyte UCP
expression) in the changes observed in UCP expres-
sion. We 1) measured PPAR␣ expression under condi-
tions in which UCP expression altered; 2) determined
the effects of specific activation of PPAR␣ in vivo, with
WY-14,643, on UCP expression; and 3) measured the
level of UCP expression in PPAR␣
⫺/⫺
mice. Where
possible, we made direct comparisons between cardiac
and skeletal muscle to determine the similarities and
differences of UCP control in these two tissues. The
results show that during increased availability of fatty
acids, the heart increases the expression of UCP-3 only,
with no effect on cardiac UCP-2 expression. In contrast,
in skeletal muscle, the expression of both UCP-2 and
UCP-3 is increased with PPAR␣ activation. UCP-3, but
not UCP-2, expression was severely reduced in the
heart of PPAR␣
⫺/⫺
mice compared to wild-type mice.
Together, these findings strongly implicate PPAR␣ as
the major regulator of UCP-3, but not UCP-2, expres-
sion in the adult rodent heart. During mechanical
unloading and pressure overloading of the heart,
UCP-2, UCP-3, and PPAR␣ expression all decrease. The
results provide new hypotheses for the roles of UCPs in
the adult heart.
MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats (200–225 g initial weight) were
kept in the Animal Care Center of the University of Texas
Houston Medical School under controlled conditions
(23⫾1°C; 12 h light/12 h dark cycle), and received standard
laboratory chow and water ad libitum unless otherwise stated.
Changes in workload
In the first series of experiments, cardiac unloading was
induced by heterotopic transplantation of a rat heart into the
abdomen of a recipient rat, as described earlier (13, 14). After
2 wk, the animals were anesthetized and both donor (unload-
ed) and recipient (control) hearts were removed, freeze
clamped, and stored at ⫺80°C prior to RNA extraction.
In the second series of experiments, cardiac pressure
overload was induced by banding the ascending aorta, with a
20-gauge needle, as described previously (14, 15). In control
animals, sham operations were performed without banding of
the aorta. Either 7 or 9 days after aortic constriction (to be
specified), the animals were anesthetized, hearts were re-
moved, freeze-clamped, and stored at ⫺80°C.
Changes in substrate availability
In the first set of experiments, rats were fed either a high-
carbohydrate/low-fat (HC/LF) diet or a low-carbohydrate/
high-fat (LC/HF) diet (Purina Mills). These isocaloric diets
varied only in the proportion of energy obtained from
carbohydrate and fat. The contribution of carbohydrate, fat
and protein to total energy available were 71%, 6% and 23%
for the HC/LF diet, and 24%, 53% and 23% for the LC/HF
diet, respectively. The source of carbohydrate was a combina-
tion of sucrose and dextrin, while the source of fat was a
combination of corn oil and lard. Nonnutritive fiber was also
increased in the LC/HF diet. The length of time in which the
rats were fed the special diets is specified for individual
experiments. In selected experiments, soleus (skeletal) mus-
cle was removed in addition to the heart.
The effects of fasting and refeeding on cardiac gene
expression were investigated. Rats were fasted for either 1 or
2 days, after which the heart and soleus muscles were isolated.
A subset of fasted rats were refed with the HC/LF diet for an
additional 4 days.
Diabetes was induced through a single injection of strep-
tozotocin [STZ; 55 mg/kg intravenous (i.v.)]. Control ani-
mals were administered with buffer (Hank’s buffer; Life
Technologies, Inc., Grand Island, N.Y.) only. Five, 7, 14, or
182 days (6 months) after the initial injection, the animals
were anesthetized, hearts were removed, freeze-clamped, and
stored at
⫺80°C. Animals were considered diabetic if their
blood glucose level was greater than 300 mg per deciliter.
Pharmacological interventions
To test the effects of specific PPAR␣ activation, WY-14,643 was
added to standard powdered Purina rodent chow at a con-
centration of 0.01% (w/w). Rats were fed a WY-14,643-
containing diet for 4 days. Control animals received powered
rodent chow only.
In a separate set of experiments, rats received a single i.v.
(tail vein) injection with TNF-␣ (human recombinant, 30
g/kg body weight in 1 ml 0.9% NaCl); controls were
injected with NaCl only. Twelve hours later, heart and soleus
muscles were removed. As cytokine administration decreases
rodent food intake (which can potentially affect gene expres-
sion), rats were injected at 07.00 h. During the following 12 h
period in the light, rodent ingestion is minimal. To ensure
standardized experimental conditions, food was withdrawn
from all animals at the time of injection.
PPAR␣
ⴚ/ⴚ
mice
Isolated RNA from hearts of age matched wild-type and
PPAR␣
⫺/⫺
mice was a kind gift from Dr L. Nagy (The Salk
Institute for Biological Studies, Gene Expression Laboratory,
La Jolla, Calif.).
RNA extraction and quantitative reverse transcription-
polymerase chain reaction (RT-PCR)
RNA extraction and quantitative RT-PCR of samples was
performed using previously described methods well estab-
lished in our laboratory (14, 16–18). Specific quantitative
assays were designed from the rat sequences available in
GenBank (Table 1). Primers and probes were designed from
unconserved sequences of the genes (allowing for isoform
834 Vol. 15 March 2001 YOUNG ET AL.The FASEB Journal
specificity). The correlation between C
t
(the number of PCR
cycles required for the fluorescent signal to reach a detection
threshold) and the amount of standard was linear overa5log
range of RNA for all assays (Fig. 1 illustrates the values for
rat/mouse UCP-2 and rat UCP-3). The level of transcripts for
the constitutive housekeeping gene product cyclophilin was
quantitatively measured in each sample to control for sample-
to-sample differences in RNA concentration. PCR data are
reported as the number of transcripts per number of cyclo-
philin molecules.
Statistical analysis
Data are presented as the mean ⫾ se. Statistically significant
differences between groups were calculated by the Student’s
t test. A value of P⬍0.05 was considered significant.
RESULTS
Mechanical unloading and pressure overload both
decrease cardiac UCP expression
Decreased workload results in significant atrophy over
the course of 2 wk, at which time there is a 55%
decrease in the heart weight-to-body weight ratio (14).
The expression of UCP-2 and UCP-3 were decreased in
unloaded hearts (Fig. 2A, B). Pressure overload re-
sulted in cardiac hypertrophy (after 1 wk the heart
weight to body weight ratios were 3.42⫾0.06 vs.
2.98⫾0.05 in the experimental and control groups;
P⬍0.05). Hypertrophy also decreased the expression of
UCP-2 and UCP-3 (Fig. 2C, D). In all hearts, levels of
UCP-2 expression were 100-fold higher compared to
UCP-3 (Fig. 2).
Mechanical unloading decreases cardiac PPAR␣
expression
A possible mechanism by which reduced cardiac work-
load results in decreased UCP expression is a decrease
in the level of PPAR␣ expression. Unloading reduced
cardiac PPAR␣ expression by half (0.137⫾0.027 vs.
0.282⫾0.018 in experimental and control groups;
P⬍0.05).
TABLE 1. Primer and probe sequences used in the quantitative PCR for UCP-2, UCP-3, PPAR␣, PDK-2, PDK-4, muscle CPT-I, iNOS,
and cyclophilin
Gene Primer/probe Sequence
r/m UCP-2
a
Forward 5⬘-TCATCAAAGATACTCTCCTGAAAGC-3⬘
Reverse 5⬘-TGACGGTGGTGCAGAAGC-3⬘
Probe 5⬘-FAM-TGACAGACGACCTCCCTTGCCACT-TAMRA-3⬘
r UCP-3 Forward 5⬘-GTGACCTATGACATCATCAAGGA-3⬘
Reverse 5⬘-GCTCCAAAGGCAGAGACAAAG-3⬘
Probe 5⬘-FAM-CTGGACTCTCACCTGTTCACTGACAACTTCC-TAMRA-3⬘
m UCP-3 Forward 5⬘-TGCTGAGATGGTGACCTACGA-3⬘
Reverse 5⬘-CCAAAGGCAGAGACAAAGTGA-3⬘
Probe 5⬘-FAM-AAGTTGTCAGTAAACAGGTGAGACTCCAGCAA-TAMRA-3⬘
r PPAR␣ Forward 5⬘-ACTACGGAGTTCACGCATGTG-3⬘
Reverse 5⬘-TTGTCGTACACCAGCTTCAGC-3⬘
Probe 5⬘-FAM-AGGCTGTAAGGGCTTCTTTCGGCG-TAMRA-3⬘
r PDK-2 Forward 5⬘-TCAGCAAGTTCTCCCCGTC-3⬘
Reverse 5⬘-ATGAAGTTTTCTCGCAGGCA-3⬘
Probe 5⬘-FAM-TGCTGGATCCGAAGTCTAGAAACTGCTTCAT-TAMRA-3⬘
r PDK-4 Forward 5⬘-TTCACACCTTCACCACATGC-3⬘
Reverse 5⬘-AAAGGGCGGTTTTCTTGATG-3⬘
Probe 5⬘-FAM-CGTGGCCCTCATGGCATTCTTG-TAMRA-3⬘
r muscle CPT-I Forward 5⬘-ATCATGTATCGCCGCAAACT-3⬘
Reverse 5⬘-ACCCATGTGCTCCTACCAGAT-3⬘
Probe 5⬘-FAM-TCAAGCCGGTAATGGCACTGGG-TAMRA-3⬘
r iNOS Forward 5⬘-GAGAAGCTGAGGCCCAGG-3⬘
Reverse 5⬘-ACCTTCCGCATTAGCACAGA-3⬘
Probe 5⬘-FAM-CAGTCTTGGTGAAAGCGGTGTTCTTTG-TAMRA-3⬘
r/m Cyclophilin Forward 5⬘-CTGATGGCGAGCCCTTG-3⬘
Reverse 5⬘-TCTGCTGTCTTTGGAACTTTGTC-3⬘
Probe 5⬘-FAM-CGCGTCTGCTTCGAGCTGTTTGCA-TAMRA-3⬘
a
r denotes a rat-specific assay; m denotes a mouse-specific assay; r/m denotes a rat- and mouse-compatible assay.
Figure 1. Sensitivity of the quantitative assays for UCP-2 and
UCP-3. Each graph shows the C
t
obtained with various
amounts of standard RNA molecules. The relation between
the number of standard molecules and the C
t
was linear over
the range investigated (from ⬃10
3
to ⬃10
7
).
835CARDIAC UCP GENE EXPRESSION
Pressure overload induced changes in UCP
expression are dependent on dietary fatty acids
The involvement of PPAR␣ in decreased UCP expres-
sion during pressure overload was investigated in two
ways. First, we measured the expression of PPAR␣
during overloading. Second, we determined whether
the availability of the natural ligand for PPAR␣ (fatty
acids) affected pressure overload induced alterations in
UCP expression. Therefore, rats were fed one of two
diets: either a high-carbohydrate/low-fat (HC/LF) diet
or a low-carbohydrate/high-fat (LC/HF) diet. On day 7
after the initiation of the special diets, half of the rats
underwent aortic banding, whereas the other half were
operated without banding. Rats were maintained on
their specific diets for 9 days after surgery, then the
hearts were isolated. Aortic banding resulted in similar
degrees of cardiac hypertrophy in both groups (Table
2).
Hearts isolated from sham-operated rats fed the
LC/HF diet compared to sham-operated rats fed the
HC/LF diet possessed significantly higher levels of both
UCP-2 and UCP-3 expression (Table 2). However, the
fold induction of UCP-3 was far greater than that of
UCP-2. As observed during standard laboratory chow
feeding, banding significantly reduced the expression
of both UCP-2 and UCP-3 when rats were fed the
LC/HF diet (Table 2). In contrast, pressure overload
did not decrease the cardiac expression of either UCP-2
or UCP-3 for rats fed the HC/LF diet (Table 2).
Banding resulted in a significant decrease in PPAR␣
expression for rats fed either diet (Table 2). Further-
more, rats fed the HC/LF diet showed significantly
higher cardiac PPAR␣ expression compared to rats fed
the LC/HF diet (Table 2).
Increased dietary fat increases cardiac UCP-3, but not
UCP-2, expression; heart vs. skeletal muscle
To investigate the time course over which cardiac UCP
expression changes, rats were fed the LC/HF diet for
various lengths of time (1, 2, 4, or 8 days), after which
the expression of cardiac UCP-2 and –3 as well as
PPAR␣ were measured (Fig. 3). At all time points,
feeding with the LC/HF diet had no effect on cardiac
UCP-2 expression (Fig. 3A). In contrast, UCP-3 expres-
sion was rapidly induced by the LC/HF diet (within
24 h), and this induction was maintained (Fig. 3B). In
the case of PPAR␣, the LC/HF diet repressed expres-
sion, an effect that was partially normalized after 8 days
of continuous feeding (Fig. 3C).
We also measured UCP and PPAR␣ expression in
soleus muscle (Fig. 3D–F). Feeding of rats with the
LC/HF diet resulted in a rapid increase in soleus
muscle UCP-2 and UCP-3 expression (Fig. 3D, E). This
increase was maintained for the full duration of the
feeding study. PPAR␣ expression was not affected by
the LC/HF diet (Fig. 3F).
Figure 2. UCP-2 and UCP-3 expression during cardiac un-
loading and overloading. Altered expression of UCP-2 and
UCP-3 in unloaded (A, B) and overloaded (C, D) hearts.
Values are shown as the mean ⫾ se for 9 or 10 separate
observations. All values are normalized against the expression
of the housekeeping gene cyclophilin. *P ⬍ 0.05 vs. control.
TABLE 2. Effects of banding and altered dietary fatty acid content on body weight (BW), heart weight (HW), hypertrophy (HW/BW
ratio), and expression of UCP-2, UCP-3, and PPAR␣
HC/LF sham HC/LF banded LC/HF sham LC/HF banded
Body weight (g) 255 ⫾ 7 257 ⫾ 6 267 ⫾ 6 262 ⫾ 6
Heart weight (g) 0.81 ⫾ 0.03 0.99 ⫾ 0.09* 0.85 ⫾ 0.02 1.02 ⫾ 0.07
$
HW/BW
a
3.16 ⫾ 0.08 3.91 ⫾ 0.24* 3.17 ⫾ 0.07 3.90 ⫾ 0.13
$
UCP-2/cyclo 0.278 ⫾ 0.024 0.306 ⫾ 0.021 0.360 ⫾ 0.018* 0.285 ⫾ 0.019
$
UCP-3/cyclo
b
0.214 ⫾ 0.041 0.345 ⫾ 0.061 1.147 ⫾ 0.215*** 0.719 ⫾ 0.115
$
PPAR␣/cyclo 0.146 ⫾ 0.007 0.106 ⫾ 0.014* 0.092 ⫾ 0.006** 0.074 ⫾ 0.004***
$
a
HW/BW ratio is multiplied by a factor of 1000.
b
UCP-3/cyclo ratio is multiplied by a factor of 100. The table shows the body weight (BW), heart weight (BW), and heart weight to body
weight ratio (HW/BW; a marker of cardiac hypertrophy) for sham operated and banded animals fed either a high-carbohydrate/low-fat
(HC/LF) diet or a low-carbohydrate/high-fat (LC/HF) diet, as well as the expression of UCP-2, UCP-3, and PPAR␣. Duration of diet feeding
was a total of 16 days (7 days before and 9 days after surgery). Values are shown as the mean ⫾ se for 5–10 observations. All gene expression
values are normalized against the expression of the housekeeping gene cyclophilin (cyclo).
* P ⬍ 0.05, ** P ⬍ 0.01 and *** P ⬍ 0.001 vs. HC/LF control.
$
P ⬍ 0.05 vs. LC/HF control.
836 Vol. 15 March 2001 YOUNG ET AL.The FASEB Journal
Fasting and refeeding modulates cardiac UCP-3, but
not UCP-2, expression; heart vs. skeletal muscle
Rats were fasted for either 1 or 2 days, after which a
subset was refed for an additional 4 days on the HC/LF
diet. Fasting had no effect on cardiac UCP-2 expres-
sion, although refeeding with the HC/LF diet resulted
in a significant decrease in expression (Fig. 4A.In
contrast, fasting significantly increased UCP-3 expres-
sion by threefold within 24 h (Fig. 4B). This increased
UCP-3 expression was severely blunted when fasting
continued for 2 days (Fig. 4B). Refeeding returned
cardiac UCP-3 expression to normal levels (Fig. 4B).
Fasting for either 1 or 2 days significantly lowered the
expression of cardiac PPAR␣, an effect that was re-
versed on refeeding (Fig. 4C).
To investigate further differences in UCP regulation
between cardiac and skeletal muscle, we also deter-
mined the effects of fasting and refeeding on soleus
muscle UCP expression. Fasting resulted in significant
increases in UCP-2 and -3 expression (Fig. 4D, E,
respectively). In the case of UCP-3, this increase ob-
served after 1 day was severely reduced at the second
day of fasting, a result similar to that observed in the
heart (Fig. 4E, B). Refeeding normalized soleus muscle
UCP (both -2 and -3) expression (Fig. 4D, E, respective-
ly). Soleus muscle PPAR␣ expression was not affected
by either fasting or refeeding (Fig. 4F).
STZ-induced diabetes increases cardiac UCP-3
expression
STZ-induction of diabetes through pancreatic -cell
destruction resulted in significant elevations in plasma
glucose levels at 5 (3.12-fold; P⬍0.001), 7 (4.45-fold;
P⬍0.001), 14 (3.18-fold; P⬍0.001), and 182 (5.00-fold;
P⬍0.001) days after STZ injection (compared to age-
matched controls). UCP-2 expression in the heart was
not affected by STZ-induced diabetes (Fig. 5A). How-
ever, cardiac UCP-3 expression increased rapidly in
STZ-induced diabetes and remained elevated at all time
points investigated (Fig. 5B). The highest fold induc-
tion in cardiac UCP-3 expression occurred 14 days after
the initial STZ injection (6.4-fold). Despite a mainte-
nance in the induction of UCP-3 at day 182 (4.6-fold),
there was a substantial decrease in the absolute level of
cardiac UCP-3 expression in both control and STZ
diabetic animals (Fig. 5B). Although there was no
significant difference in cardiac UCP-2 expression with
respect to time, there was a trend for older animals to
show reduced expression (Fig. 5A). Thus, cardiac UCP
expression declined with age.
We also investigated the effects of STZ-induced dia-
betes on cardiac PPAR␣ gene expression. After either 5
or 7 days of diabetes, there is no effect on PPAR␣
expression (Fig. 5C). After 14 days of diabetes there is
a decrease in PPAR␣ expression, although this effect is
Figure 3. Time course for altered cardiac and soleus muscle UCP-2, UCP-3, and PPAR␣ expression during increased dietary fat
content. The time course for altered UCP-2, UCP-3, and PPAR␣ expression in cardiac (A—C) and soleus (D—F) muscle in
response to feeding on the low-carbohydrate/high-fat (LC/HF) diet. Values are shown as the mean ⫾ se for five separate
observations. All values are normalized against the expression of the housekeeping gene cyclophilin. *P⬍0.05, **P⬍0.01, and
***P⬍0.001 vs. control (day zero).
837CARDIAC UCP GENE EXPRESSION
not significant (Fig. 5C). However, there is a significant
decrease in PPAR␣ expression at 182 days after the
induction of diabetes (Fig. 5C). Age alone had no effect
on cardiac PPAR␣ expression (Fig. 5C).
The PPAR␣ agonist WY-14,643 alters heart and soleus
muscle gene expression
Rats were fed either control diet or diet containing the
PPAR␣ agonist WY-14,643 (0.01% w/w) for 4 days, after
which cardiac and soleus muscle UCP-2, UCP-3,
PPAR␣, PDK-2, PDK-4, and muscle CPT-I expression
were measured. WY-14,643 caused an increase in soleus
muscle UCP-2 expression, with no effect on cardiac
UCP-2 expression (Fig. 6A). Both cardiac and soleus
muscle UCP-3 expression was increased by WY-14,643
(3.5- and 13.4-fold, respectively; Fig. 6B). PPAR␣ ex-
pression was not affected by WY-14,643 in either heart
or soleus muscle (Fig. 6C). To determine the effective-
ness of WY-14,643 feeding, we measured pyruvate de-
hydrogenase kinase-4 (PDK-4) and muscle-specific car-
nitine palmitoyltransferase-I (CPT-I) expression, genes
known to be induced by PPAR␣ activation (19, 20).
PDK-4 and muscle CPT-I expression increased in re-
sponse to WY-14,643 feeding in both heart and soleus
muscle (Fig. 6C, F). PDK-2, whose expression is not
believed to be regulated by PPAR␣ (19), was not
affected by WY-14,643 (Fig. 6D).
TNF-␣ decreases cardiac UCP-2, UCP-3, and PPAR␣
expression
We investigated whether cytokine exposure alters car-
diac gene expression by injecting rats with TNF-␣.
TNF-␣ administration (30 g/kg; i.v.) resulted in a
significant decrease in cardiac PPAR␣ expression after
12h(Fig. 7C). Reduced PPAR␣ expression in response
to TNF-␣ was accompanied by decreased cardiac UCP-2
and UCP-3 expression (Figs. 7A, B). Similarly, TNF-␣
administration resulted in decreased soleus muscle
UCP-3 and PPAR␣ expression (Fig. 7B, C). In contrast,
soleus muscle UCP-2 expression increased (Fig. 7A). To
determine the effectiveness of TNF-␣ administration,
cardiac and soleus muscle inducible nitric oxide syn-
Figure 4. Effects of fasting and refeeding on cardiac and soleus muscle UCP-2, UCP-3, and PPAR␣ expression. The effects of
fasting (either 1 or 2 days) and refeeding on UCP-2, UCP-3, and PPAR␣ expression in cardiac (A—C) and soleus (D—F) muscle.
Animals were refed the high-carbohydrate/low-fat (HC/LF) diet. Values are shown as the mean ⫾ se for four or five separate
observations. All values are normalized against the expression of the housekeeping gene cyclophilin. *P⬍0.05, **P⬍0.01, and
***P⬍0.001 vs. control (day zero).
838 Vol. 15 March 2001 YOUNG ET AL.The FASEB Journal
thase (iNOS) expression was also measured. TNF-␣
increased both cardiac and soleus muscle iNOS expres-
sion to similar extents (Fig. 7D).
Genetic mutation of the PPAR␣ gene decreases
expression of cardiac UCP-3, but not UCP-2
We investigated whether PPAR␣ was essential for the
expression of cardiac UCP-2 and UCP-3 by comparing
wild-type and PPAR␣
⫺/⫺
mouse hearts. The level of
cardiac UCP-2 and UCP-3 in wild-type mice were rela-
tively similar (Table 3). There was a 20-fold lower level
of cardiac UCP-3 expression in PPAR␣
⫺/⫺
mice com
-
pared to wild-type hearts (Table 3). No differences were
observed in the level of UCP-2 expression in hearts
isolated from PPAR␣
⫺/⫺
mice compared to those iso
-
lated from wild-type mice (Table 3).
DISCUSSION
We show that cardiac UCP-2 and UCP-3 expression
changes in response to altered physiological and patho-
physiologic states; in the case of UCP-3, these changes
are regulated by PPAR␣. In contrast to soleus muscle,
cardiac UCP-2 expression does not significantly change
in response to high-fat feeding, fasting, diabetes, or
WY-14,643 treatment, all of which lead to PPAR␣ acti-
vation. However, UCP-3 expression increases in cardiac
and soleus muscle under the same conditions. Expres-
sion of both UCP-3 and PPAR␣ decrease during cardiac
mechanical unloading and pressure overloading. Mu-
tation of the PPAR␣ gene results in a near complete
inhibition of cardiac UCP-3 expression, with no effect
on UCP-2 expression. These results suggest that in the
adult rodent heart, UCP-3 expression is regulated by
PPAR␣ whereas UCP-2 expression is not.
Cardiac UCP-2 expression decreased during mechan-
ical unloading and pressure overloading. Furthermore,
decreased UCP-2 expression during pressure overload
was dependent on the presence of dietary fatty acids.
Decreased dietary fatty acid intake alone reduced car-
diac UCP-2 and UCP-3 expression. These results sug-
gest that UCP-2 expression in the heart is regulated, at
least in part, by a fatty acid-dependent, PPAR␣-indepen-
Figure 5. Time course for altered cardiac UCP-2, UCP-3, and
PPAR␣ expression by streptozotocin-induced diabetes. The
effects of streptozotocin (STZ; 55 mg/kg i.v.) -induced dia-
betes on cardiac UCP-2 (A), UCP-3 (B), and PPAR␣ (C)
expression either 5, 7, 14, or 182 days after the initial
injection. Values are shown as the mean ⫾ se for five separate
observations. All values are normalized against the expression
of the housekeeping gene cyclophilin. *P ⬍ 0.05, **P ⬍ 0.01,
and ***P ⬍ 0.001 vs. age-matched controls.
839CARDIAC UCP GENE EXPRESSION
dent mechanism. Last, we show that TNF-␣ reduces the
expression of both UCP-2 and UCP-3 in the heart,
suggesting that TNF-␣ induced UCP expression in the
failing heart is not involved in energetic dysfunction.
The results of the present study must be interpreted
in a wider context. The uncoupling of mitochondrial
electron transport from ADP phosphorylation causes a
collapse of the proton gradient across the inner mito-
chondrial membrane and thereby limits ATP genera-
tion through oxidative phosphorylation. Instead, the
potential energy ‘stored’ by the gradient is liberated as
heat, an essential process in nonshivering thermogen-
esis (1, 2). Such a proton leak is catalyzed by UCPs.
Potential roles for UCP-2 and UCP-3 have been sug-
gested for skeletal muscle and adipose tissue, primarily
from studies investigating regulation of their expres-
sion in response to different stimuli. For example, cold
exposure, thyroid hormone, TNF-␣, elevated dietary fat
composition, insulin-dependent diabetes mellitus, and
specific PPAR agonists have all been shown to increase
skeletal muscle UCP expression (8–10, 21–23). In
contrast, exercise training lowers skeletal muscle UCP
expression (23). These results, plus consideration of
the sheer mass of skeletal muscle in the body, have
implicated skeletal muscle UCPs in the processes of
heat generation, obesity, and perhaps maintenance of
insulin sensitivity (9, 24, 25). However, the role(s) of
UCPs in the adult heart have not been addressed and
are more difficult to rationalize, which gives rise to the
following considerations.
Altered cardiac workload affects both UCP-2 and
UCP-3 expression
In response to increased or decreased workload, the heart
increases glucose utilization and decreases fatty acid utili-
zation (26–28). This change is mirrored by changes in the
expression of several genes encoding metabolic enzymes,
including decreases in the expression of PDK-4 (M. E.
Young et al., unpublished observation), muscle-specific
CPT-I, and medium chain acyl CoA dehydrogenase
(MCAD) in the heart (14, 29). All three genes are
regulated by PPAR␣ in the heart (19, 20, 30, 31). The
present study has found that PPAR␣ expression decreases
during both unloading and overloading (see Results
section and Table 2), which is responsible for the ob-
served changes in PDK-4, CPT-I, and MCAD. Likewise,
cardiac expression of both UCP-2 and UCP-3 decreased
during unloading and overloading (Fig. 2). This decrease
in UCP expression depended on the presence of fatty
acids, as pressure overloaded hearts isolated from animals
Figure 6. Effects of the specific PPAR␣ agonist WY-14,643, on cardiac and soleus muscle UCP-2, UCP-3, PPAR␣, PDK-2, PDK-4,
and muscle CPT-I expression. The effects of feeding animals with a diet containing WY-14,643 (0.01% w/w) on cardiac and
soleus muscle UCP-2 (A), UCP-3 (B), PPAR␣ (C), PDK-2 (D), PDK-4 (E), and muscle CPT-I (F) expression. Values are shown
as the mean ⫾ se for six separate observations. All values are normalized against the expression of the housekeeping gene
cyclophilin. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. tissue-matched control.
840 Vol. 15 March 2001 YOUNG ET AL.The FASEB Journal
fed a low-fat diet (HC/LF) did not possess decreased UCP
expression compared to diet-matched controls, despite a
decrease in PPAR␣ expression (Table 2). These results
initially suggested to us that PPAR␣ acts in the decreased
UCP expression during hypertrophy only in the presence
of its ligand (fatty acids). Similarly, the decreased PPAR␣
expression during unloading could potentially result in
decreased UCP expression. Consistent with the decrease
observed in UCP expression during pressure overload,
exercise training, which is associated with cardiac hyper-
trophy, reduces UCP expression in the heart (as well as in
skeletal muscle) (23).
Fatty acid availability influences both cardiac and
skeletal muscle UCP expression
High-fat feeding, fasting, and insulin-dependent diabe-
tes mellitus would be expected to activate PPAR␣ (30,
32, 33). In certain experiments, a direct comparison
was made between expression of UCPs in heart and a
slow-twitch skeletal muscle (soleus) that is relatively
similar to the heart (34, 35). Still, there were differ-
ences between the muscles. High-fat feeding, fasting,
and diabetes all increased the expression of cardiac
UCP-3 expression but had no effect on UCP-2 expres-
sion (Figs. 3–5). These results agree with a recent study
that investigated cardiac UCP expression during diabe-
tes and fasting (36). In contrast to the heart, high-fat
feeding and fasting caused induction of both UCP-2
and UCP-3 in soleus muscle (Figs. 3 and 4); previously
reported studies have shown both UCP-2 and UCP-3
expression to increase in skeletal muscle during STZ-
induced diabetes (21). The fold induction of UCP-3 in
soleus muscle was greater than that of the heart during
high-fat feeding and fasting. Specific activation of
PPAR␣ through feeding with WY-14,643 resulted in a
similar pattern of UCP expression observed with eleva-
tion of plasma fatty acid levels: increased UCP-3 expres-
sion in both heart and soleus muscles, with the largest
induction in the latter, whereas UCP-2 was induced
only in soleus, not heart, muscle (Fig. 6).
Cardiac UCP-3, but not UCP-2, expression is
dependent on PPAR␣ signaling in the adult rodent
heart
Table 4 summarizes the relationship between PPAR␣
and the expression of cardiac UCP-2 and UCP-3, as
observed in the present study. Decreased PPAR␣ ex-
pression during cardiac unloading and overloading
occurs in concert with decreased expression of UCP-2
and UCP-3. Furthermore, the decreased UCP expres-
sion during hypertrophy is dependent on the presence
of fatty acids in the diet, providing evidence for the
hypothesis that this decrease is related to the reduced
PPAR␣ expression. Similarly, on low-fat feeding (either
postfasting or comparing the HC/LF and LC/HF feed-
ing effects), when PPAR␣ activation should be reduced,
UCP-2 and UCP-3 expression both decrease (Fig. 4 and
Table 2). However, situations in which PPAR␣ is stim-
ulated (high-fat feeding, fasting, diabetes, and WY-
14,643 feeding) increase the expression of cardiac
UCP-3, with no effect on cardiac UCP-2 expression.
These observations are consistent with the hypothesis
that UCP-3 expression is regulated by PPAR␣. One
possible explanation for these observations was that in
the adult rat heart, UCP-2, but not UCP-3, expression is
maximal through PPAR␣ signaling. Thus, a further
stimulation of PPAR␣ would have no effect on UCP-2
expression, yet reduced PPAR␣ signaling would reduce
UCP-2 expression. To investigate this hypothesis fur-
ther, we determined the level of expression of cardiac
UCP-2 and UCP-3 in PPAR␣
⫺/⫺
mice. In this model,
expression of various PPAR␣-regulated genes is de-
creased (37). Because we find reduced expression of
TABLE 3. Expression of cardiac UCP-2 and UCP-3 in
PPAR␣
⫺/⫺
and wild-type mice
Wild-type PPAR␣
⫺/⫺
UCP-2/cyclo 0.248 ⫾ 0.034 0.259 ⫾ 0.076
UCP-3/cyclo 0.193 ⫾ 0.074 0.010 ⫾ 0.002*
a
Expression of UCP-2 and UCP-3 in wild-type and PPAR␣
⫺/⫺
hearts. Values are shown as the mean ⫾ se for four (wild-type) or five
(PPAR␣
⫺/⫺
) separate observations. All values are normalized against
the expression of the housekeeping gene cyclophilin (cyclo).
* P ⬍ 0.05 vs wild-type.
Figure 7. Effects of TNF-␣ administration on cardiac and
soleus muscle UCP-2, UCP-3, PPAR␣ and iNOS expression.
The effects of TNF-␣ injection (30 g/kg; i.v.) on cardiac and
soleus muscle UCP-2 (A), UCP-3 (B), PPAR␣ (C), and iNOS
(D) expression. Values are shown as the mean ⫾ se for six
separate observations. All values are normalized against the
expression of the housekeeping gene cyclophilin. *P ⬍ 0.05
and **P ⬍ 0.01 vs. tissue-matched control.
841CARDIAC UCP GENE EXPRESSION
UCP-3, but not UCP-2, in PPAR
⫺/⫺
mice, these results
provide evidence that UCP-2 expression in the adult
rodent heart is not regulated by PPAR␣. Instead, there
must be a fatty acid-dependent, PPAR␣-independent
mechanism modulating cardiac UCP-2 expression,
which is maximally activated under normal physiologi-
cal circumstances. PPAR␥, another member of the
family of PPAR transcription factors, does not appear to
regulate UCP-3 expression in the same way as PPAR␣ in
the heart. Treatment of adult rats with the PPAR␥
agonist troglitazone (0.1% w/w in the diet for 4 days),
has no effect on UCP-3 (nor UCP-2) expression in the
heart (M. E. Young et al., unpublished observation).
This lack of an effect by troglitazone treatment (which
did induce known PPAR␥-regulated genes in the skel-
etal muscle) on cardiac UCP gene expression is most
likely due to the very low abundance of PPAR␥ in the
heart, as compared with both adipose tissue and skele-
tal muscle.
TNF-␣ reduces cardiac UCP expression
TNF-␣ is known to cause contractile dysfunction (38),
and the expression of TNF-␣ increases in the failing
heart (11). Furthermore, TNF-␣ antagonism appears to
have beneficial effects in subjects with heart failure
(39). TNF-␣ has been shown to induce UCP expression
in skeletal muscle (10). As overexpression of UCPs
might limit mitochondrial ATP production, we hypoth-
esized that TNF-␣-induced cardiac UCP expression
could result in the contractile dysfunction observed
previously. However, administration of TNF-␣ to adult
rats caused a significant reduction in cardiac UCP
expression (Fig. 7). Whether reduced cardiac UCP
expression in response to TNF-␣ administration was
due to a direct effect of TNF-␣ on the cardiomyocyte or
to a systemic effect cannot be determined in the
present study. It is also still possible that local TNF-␣
generation by the cardiomyocyte, as observed in failing
myocardium, induces UCP expression.
Complexity of cardiac and skeletal muscle UCP gene
expression regulation
There are obvious differences in the regulation of
UCP-2 and UCP-3 expression, some of which are sum-
marized in Table 4. Under physiological conditions,
cardiac UCP-2 expression is ⬃100-fold greater than that
of UCP-3. Expression of UCP-3 is regulated by PPAR␣
in the adult rodent heart, whereas UCP-2 is not. On the
second day of exposure to elevated fatty acid levels (for
both the high-fat feeding and fasting experiments),
both cardiac and soleus muscle UCP-3 expression fall
transiently (Figs. 3 and 4). As fatty acid exposure
continues, UCP-3 expression increases again (Fig. 3).
Increased age was associated with decreased cardiac
UCP expression. In addition, TNF-␣ increases soleus
muscle UCP-2 expression while decreasing cardiac
UCP-2 expression (Fig. 7). Thus, multiple mechanisms
for the regulation of UCP expression must operate in
heart and skeletal muscle. These might include altered
expression and/or activity in PPAR␣ (as observed in
the heart for the present study), PPAR␣ dimerization
partners (e.g., RXR) or coactivators (e.g., PGC-1),
which confer specificity between UCP-2 and UCP-3
promoter regions, or even as yet unidentified PPAR␣-
independent mechanisms (40–42). A study published
during the preparation of this manuscript suggests that
cardiac UCP-2 expression is regulated by PPAR␣ (43).
In this study, neonatal cardiomyocytes were cultured in
the presence of triiodothyronine, fatty acids or WY-
14,643, resulting in increased UCP-2 expression. From
these observations, it was concluded that increased
cardiac UCP-2 expression on birth was due to com-
bined stimulation of the thyroid hormone receptor and
PPAR␣. However, at birth there is a substantial increase
in mitochondrial biogenesis, which might explain the
increased expression of a mitochondrial protein such
as UCP-2 (44). It should be noted that the results
observed in the present study are not consistent with
altered mitochondrial biosynthesis (e.g., acute pressure
overload results in increased mitochondrial biogenesis
whereas UCP expression decreases; 45). It is possible
that neonatal cardiomyocytes in culture possess a spe-
cific factor (e.g., a PPAR␣ dimerization partner or
coactivator) that is not present in the adult heart.
Increased fatty acid availability reduces cardiac
PPAR␣ expression
The present results suggest that conditions associated
with increased cardiac fatty acid utilization (high-fat
feeding, fasting, and diabetes) result in decreased
PPAR␣ expression, a mechanism not acutely observed
in skeletal muscle (Figs. 3–5). Two hypotheses can be
drawn regarding the potential mechanism by which this
phenomenon occurs. First, increased signaling through
PPAR␣ might cause the altered expression of a protein
that affects the expression of PPAR␣. This autoregula-
tion mechanism would therefore be PPAR␣-depen-
dent. A second, PPAR␣-independent mechanism can
TABLE 4. Relationship between PPAR␣ and the expression of
UCP-2 and UCP-3 in the heart
a
Model
PPAR␣
(expression/ligand
availability)
UCP-2
expression
UCP-3
expression
Unloading 2Expression 22
Pressure overload 2 Expression 22
TNF␣ administration 2 Expression 22
PPAR␣
⫺/⫺
mouse
2 Expression ⫽ 2
High-fat feeding 1 Ligand ⫽ 1
Fasting 1 Ligand ⫽ 1
STZ-induced
diabetes 1 Ligand ⫽ 1
WY-14,643 treatment 1 Ligand ⫽ 1
a
1 represents an increase, 2 represents a decrease, and ⫽
represents no change.
842 Vol. 15 March 2001 YOUNG ET AL.The FASEB Journal
be postulated wherein fatty acids activate a specific
pathway that regulates the expression of PPAR␣. Thus,
the first mechanism is fatty acid independent and
PPAR␣ dependent whereas the latter mechanism is
fatty acid dependent and PPAR␣ independent. To
investigate which mechanism was responsible for the
observed results, we used a specific PPAR␣ agonist
(WY-14,643) that would lead to PPAR␣ activation in the
absence of elevated fatty acid levels. WY-14,643 in-
creased cardiac and skeletal muscle UCP-3, PDK-4, and
muscle CPT-I expression, suggesting PPAR␣ was acti-
vated. In contrast to fatty acids, WY-14,643 feeding had
no effect on cardiac PPAR␣ expression (or soleus
muscle PPAR␣ expression). The results are consistent
with the hypothesis that a fatty acid-dependent mecha-
nism is responsible for the observed down-regulation of
cardiac PPAR␣. Further evidence toward this hypothe-
sis is the observation that hearts isolated from the obese
Zucker rat, a model of insulin resistance in which
plasma fatty acid levels are elevated, have severely
reduced PPAR␣ expression (46). It could be hypothe-
sized that repression of PPAR␣ expression by fatty acids
serves as a mechanism to prevent excessive fluctuations
in PPAR␣ signaling in this tissue during altered fatty
acid availability.
Potential roles of UCPs in the adult rodent heart
The present study has focused on uncovering the
mechanisms by which gene expression of the uncou-
pling proteins is regulated in the adult heart during
both physiological and pathophysiological conditions.
Determination of UCP protein levels (requiring anti-
body generation) and altered mitochondrial function is
beyond the scope of the present study. It is believed
that elucidating the mechanisms by which the heart
alters the expression of the UCPs might aid in the
understanding of their function. Possible major func-
tions for UCPs in the heart include thermogenesis,
regulation of fatty acid oxidation, regulation of ATP
synthesis, and reduction of ROS formation. Due to its
size (compared with the body), the heart is unlikely to
play a role in global thermogenesis. UCP-2 expression
is relatively insensitive to substrate availability, suggest-
ing it plays little function in cardiac fatty acid utilization
(which increases, for example, in diabetes). However,
UCP-3 is highly responsive to fatty acids. Whether
UCP-3 induction helps prevent lipotoxicity when fatty
acid levels are high or acts in an antioxidant capacity
when oxidative metabolism increases (fatty acids can-
not be metabolized anaerobically) are distinct possibil-
ities. When the heart requires increased efficiency, for
example, during increased or decreased workload or
substrate limitation (as observed during low dietary
fatty acid availability, as fatty acids are the primary fuel
for the heart under physiological conditions), cardiac
UCP expression decreases. If cardiac UCPs affect mito-
chondrial ATP synthesis, decreased UCP expression
would be expected to increase mitochondrial effi-
ciency. Last, evidence suggests that uncoupling pro-
teins can act as antioxidants (47, 48). The heart is a
continuously contracting organ, with high oxidative
metabolism. The observation that cardiac UCP-2 ex-
pression is relatively high (even higher than skeletal
muscle) and relatively constant during various condi-
tions suggests that it has an essential, constitutive role
in the heart, such as prevention of ROS formation.
Indirect evidence for this hypothesis comes from the
observations that TNF-␣ administration, which causes a
rapid decrease in cardiac UCP-2 and UCP-3 expression,
is known to increase oxidative stress (49).
Limitations of the study
Whether changes in UCP gene expression result in
changes in either UCP protein or activity or ultimately
lead to altered cardiac function has not been deter-
mined. Future studies are required to address these
issues. For example, the expression of UCP-2 in the
heart is ⬃100-fold greater than that of UCP-3. There-
fore, if both UCP-2 and UCP-3 possess uncoupling
activity, what is the physiological significance of UCP-3
induction during increased fatty acid availability? It is
possible that UCP-2 and UCP-3 possess different intrin-
sic activities, are differentially regulated post-transcrip-
tionally, or are located within different regions of the
inner mitochondrial membrane, akin to the light har-
vesting complexes and photosystems of chloroplasts.
Whether allosteric factors other than guanosine nucle-
otides and fatty acids differentially affect the activity of
UCP-2 and UCP-3 is unknown. Another concern is that
each intervention investigated in the present study
could potentially affect multiple factors in these com-
plex in vivo models. For example, nervous activity,
workload, and multiple growth factor and cytokine
signaling cascades are all altered in both the unloaded
and pressure overloaded heart. In addition, various
hormonal alterations occur during nutritional manip-
ulation and diabetes. Even in knockout mice, compen-
satory mechanisms become activated, allowing for ad-
aptation in the absence of a specific gene. All these
factors could potentially affect cardiac UCP gene ex-
pression. However, one common factor that links car-
diac UCP-3 (but not UCP-2) expression with these
diverse animal models is the transcription factor
PPAR␣ (see Table 4). Although the present study has
not directly measured PPAR␣ protein or activity, previ-
ous studies have shown that increased fatty acid avail-
ability results in PPAR␣ activation (30, 32, 33) and that
reduced PPAR␣ transcription—for example, during
cardiac hypertrophy—is associated with reduced activ-
ity and expression of PPAR␣-regulated genes (50).
CONCLUSIONS
In the adult rodent heart, expression of UCP-3 but not
UCP-2 is regulated by PPAR␣. In contrast to skeletal
muscle, in which UCP expression alters dramatically in
response to substrate availability, heart UCP expression
843CARDIAC UCP GENE EXPRESSION
does not fluctuate very much. The expression of UCP-2,
the major UCP isoform in the rat heart (100-fold
higher expression compared to UCP-3), changes rela-
tively little in response to dramatic alterations in sub-
strate availability. Uncoupling of skeletal muscle mito-
chondria may be important for the utilization of fatty
acids, whereas the uncoupling of cardiac mitochondria
might play more of a role in an antioxidant capacity,
preventing over-reduction of the electron transport
chain. In situations in which increased cardiac effi-
ciency is required, such as unloading, overloading, and
decreased fatty acid availability, cardiac UCP expression
is decreased. Whether other factors are able to signifi-
cantly increase the expression of uncoupling proteins
in the adult heart is unknown.
We wish to thank Patrick H. Guthrie and Wenhao Chen for
technical assistance. This work was supported in part by
grants from the NIH (HL-43133 and HL-61483) and from the
American Heart Association National Center. C.D. was a
Daland Fellow of the American Philosophical Society.
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Received for publication May 24, 2000.
Revised for publication July 27, 2000.
845CARDIAC UCP GENE EXPRESSION
