Regulation of cardiac and skeletal muscle malonyl-CoA
decarboxylase by fatty acids
Martin E.
Young,
Gary W.
Goodwin,
Jun
Ying,
Patrick
Guthrie,
Christopher R.
Wilson,
Frank A.
Laws, and
Heinrich
Taegtmeyer
Division of Cardiology, Department of Internal
Medicine, University of Texas-Houston Medical School, Houston, Texas
77030
 |
ABSTRACT |
Malonyl-CoA decarboxylase (MCD) catalyzes the
degradation of malonyl-CoA, an important modulator of fatty acid
oxidation. We hypothesized that increased fatty acid availability would
increase the expression and activity of heart and skeletal muscle MCD, thereby promoting fatty acid utilization. The results show that high-fat feeding, fasting, and streptozotocin-induced diabetes all
significantly increased the plasma concentration of nonesterified fatty
acids, with a concomitant increase in both rat heart and skeletal
muscle MCD mRNA. Upon refeeding of fasted animals, MCD expression
returned to basal levels. Fatty acids are known to activate
peroxisome proliferator-activated receptor-
(PPAR
). Specific
PPAR
stimulation, through Wy-14643 treatment, significantly increased the expression of MCD in heart and skeletal muscle. Troglitazone, a specific PPAR
agonist, decreased MCD expression. The
sensitivity of MCD induction by fatty acids and Wy-14643 was soleus > extensor digitorum longus > heart. High plasma
fatty acids consistently increased MCD activity only in solei, whereas MCD activity in the heart actually decreased with high-fat feeding. Pressure overload-induced cardiac hypertrophy, in which PPAR
expression is decreased (and fatty acid oxidation is decreased), resulted in decreased MCD mRNA and activity, an effect that was dependent on fatty acids. The results suggest that fatty acids induce
the expression of MCD in rat heart and skeletal muscle. Additional
posttranscriptional mechanisms regulating MCD activity appear to exist.
heart; malonyl-coenzyme A decarboxylase; nonesterified fatty acids; peroxisome proliferator-activated receptor-
; skeletal muscle
 |
INTRODUCTION |
FATTY ACIDS have
many essential functions in the cell. These include roles as fuels
(30, 41), as mediators of signal transduction [e.g.,
activation of various protein kinase C (PKC) isoforms, initiation of
apoptosis] (12, 38), as ligands for nuclear transcription factors [e.g., peroxisome proliferator-activated receptor-
(PPAR
)] (7, 20), and as essential
components of biological membranes (39). Because of these
many functions, the levels of intracellular fatty acids and their
derivatives are tightly regulated. Elevation of intracellular fatty
acids and lipids (and abnormalities in lipid handling) has been
associated with various pathologies, including insulin resistance
(31, 32), pancreatic dysfunction (38, 42),
and cardiotoxicity (46). One way in which mammalian
organisms respond to elevations in fatty acid levels is by increasing
the expression, in tissues such as cardiac and skeletal muscle of
various proteins involved in fatty acid utilization (17, 23,
26). Fatty acid-induced genes known to be involved in fatty acid
metabolism include the fatty acid transporter CD36, fatty acid
binding proteins, muscle-specific carnitine palmitoyltransferase 1 (CPT-1), and various enzymes in
-oxidation (e.g., the acyl-CoA
dehydrogenase isoforms) (4, 17, 43).
The flux-generating step in long-chain fatty acid oxidation appears to
be the transport of the activated, acyl-CoA derivative of fatty acids
into the mitochondrial matrix (reviewed in Refs. 11 and
27). Three proteins are involved in this process, CPT-1, a translocase,
and CPT-2. Of these, CPT-1 is rate limiting. Two isoforms of CPT-1
exist, the liver and muscle (m) isoforms. In fully differentiated
cardiac and skeletal muscle, the mCPT-1 isoform predominates
(5). mCPT-1 is highly sensitive to allosteric inhibition
by malonyl-CoA, resulting in reduced fatty acid oxidation (27). Situations in which intracellular malonyl-CoA levels
increase are associated with decreased fatty acid oxidation (14,
28, 34).
The steady-state concentration of any metabolite in the cell is
regulated by its rate of synthesis as well as its rate of degradation.
Malonyl-CoA is synthesized by acetyl-CoA carboxylase (ACC) and is
degraded by malonyl-CoA decarboxylase (MCD) (1). ACC
, the isoform which predominates in cardiac and
skeletal muscle, is regulated both allosterically and covalently
(19, 32). Little is known about the regulation of MCD,
although recent work suggests that MCD in the heart and skeletal muscle
is regulated covalently (14, 36). Whether the expression
of MCD is altered during various physiological or pathophysiological
conditions in cardiac or skeletal muscle is not known, with the
exception of insulin-dependent diabetes mellitus, in which cardiac MCD
expression and activity have recently been shown to be increased
(37).
The purpose of the present study was to test the hypothesis that
situations associated with increased plasma fatty acid levels would
increase the expression and activity of cardiac and skeletal muscle
MCD. The latter would promote increased fatty acid oxidation. In all
cases investigated, elevation of plasma fatty acid levels resulted in
increased cardiac and skeletal [extensor digitorum longus (EDL) and
soleus] muscle MCD expression. Changes in MCD mRNA were not always
paralleled by MCD activity. Specific PPAR
activation (by Wy-14643),
but not PPAR
activation (by troglitazone), increased MCD expression
and activity. Cardiac hypertrophy, which is associated with both
decreased PPAR
expression (3) and decreased fatty acid
oxidation (2, 21), resulted in decreased MCD expression
and activity, an effect which was dependent on fatty acids. We provide
evidence for the regulation of MCD both transcriptionally and posttranscriptionally.
 |
MATERIALS AND METHODS |
Animals.
Male Spraque-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; 12:12-h light-dark cycle) and received standard laboratory chow and water ad
libitum, unless otherwise stated.
Materials.
Chemicals, enzymes, isotopes, primers, and probes were obtained from
sources described previously (10, 16).
Dietary manipulation.
Rats were fed either standard laboratory chow, a
high-carbohydrate/low-fat (HC/LF) diet, or a low-carbohydrate/high-fat
(LC/HF) diet (Purina Mills). The HC/LF and LC/HF diets were isocaloric and varied only in the proportion of energy obtained from carbohydrate and fat. The contribution of carbohydrate, fat, and protein to total
energy available was 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
combination of sucrose and dextrin, and 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.
The effects of high-fat feeding on cardiac and skeletal muscle MCD
expression were investigated. Rats were fed the LC/HF diet for either
1, 4, or 8 days. Rats were then anesthetized with chloral hydrate (300 mg/kg), and the heart, EDL, and soleus muscles were isolated. The
isolated muscles were immediately freeze-clamped in liquid
N2.
The effects of fasting and refeeding were also investigated. Rats were
fasted for either 1 or 2 days, after which the heart and EDL and soleus
muscles were isolated as above. A subset of fasted rats was refed with
the HC/LF diet for an additional 4 days.
Induction of diabetes.
Diabetes was induced through a single injection of streptozotocin (STZ;
55 mg/kg iv). Control animals received buffer (Hanks' buffer; GIBCO
BRL Life Sciences) only. Two weeks after the initial injection, the
animals were anesthetized, and hearts and skeletal muscles were
removed, freeze-clamped, and stored at
80°C. Animals were
considered diabetic if their blood glucose level was >300 mg/dl (16.7 mM).
Specific PPAR
and PPAR
activation.
To test the effects of PPAR
activation, Wy-14643 was added to
standard powdered Purina rodent chow at a concentration of 0.01%
(wt/wt). Rats were fed the Wy-14643-containing diet for 4 days. To test
the effects of PPAR
activation, troglitazone was added to standard
powdered Purina rodent chow at a concentration of 0.1% (wt/wt). Rats
were fed the troglitazone-containing diet for 4 days. In both sets of
experiments, control animals received powdered rodent chow only.
Pressure overload-induced hypertrophy.
Cardiac pressure overload was induced by banding the ascending aorta as
described previously (24). In control animals, sham operations were performed without banding of the aorta. Nine days after
aortic constriction, the animals were anesthetized, and hearts were
removed, freeze-clamped, and stored at
80°C. Seven days before
surgery, the animals had received either standard laboratory chow
(control diet), the HC/LF diet, or the LC/HF diet. Specific feeding was
maintained until the completion of the experiment (i.e., isolation of hearts).
Plasma glucose and nonesterified fatty acid level determination.
Immediately before muscle isolation from the rats, 1 ml of blood was
withdrawn. The sample was placed on ice before centrifugation for 10 min at full speed with a desktop microfuge. The supernatant was
retained and stored at
80°C until glucose and nonesterified fatty
acid (NEFA) levels were determined. Plasma glucose levels were
measured for control and diabetic rats by means of a
glucose/L-lactate analyzer (2300 STAT, Yellow Springs
Instruments). Plasma NEFA levels were measured spectrophotometrically
with a commercially available kit (Wako Chemicals, Richmond, VA).
Specimen blanks were prepared for all samples to allow for possible hemolysis.
Measurement of MCD activity.
MCD activity in homogenates of pulverized heart and skeletal muscle was
measured by following the rate of 14CO2
generation from [3-14C]malonyl-CoA, as described
previously (14). The concentration of malonyl-CoA in the
assay was 200 µM; at this concentration of malonyl-CoA, the enzymatic
rate is near maximal (14). All activity values are
expressed as nanomoles of malonyl-CoA decarboxylated per minute per
milligram of protein. Homogenate protein concentrations were determined
using the Bradford assay.
RNA extraction and quantitative RT-PCR.
RNA extraction and quantitative RT-PCR of samples were performed using
previously described methods (6, 10, 13). Specific quantitative assays for 36B4, cyclophilin, and MCD were designed from
the rat sequences available in GenBank. Primers and probes (shown in
Table 1) were designed around specific
splice junctions, preventing the recognition of any contaminating
genomic DNA. The correlation between the number of PCR cycles required
for the fluorescent signal to reach a detection threshold and the
amount of standard was linear over a 5-log range of RNA for all assays (data not shown). The level of transcripts for the constitutive housekeeping gene products 36B4 and cyclophilin were quantitatively measured in each sample to control for sample-to-sample differences in
RNA concentration. Expression is reported as either the number of MCD
transcripts per number of 36B4 molecules or the number of MCD
transcripts per number of cyclophilin molecules.
Statistical analysis.
Data are presented as means ± SE. Statistically significant
differences between groups were calculated by the Student's
t-test. A value of P < 0.05 was considered significant.
 |
RESULTS |
Higher MCD expression and activity in heart vs. skeletal muscle.
Figure 1 shows the differential
expression and activity of MCD among the heart, EDL, and soleus
muscles. When normalized to the housekeeping gene encoding for
ribosomal 36B4 (whose level of expression, when normalized to total RNA
content, is the same among the heart, EDL, and soleus muscles; data not
shown), the level of MCD expression is 2.1- and 2.7-fold greater in the
heart compared with EDL and soleus muscles, respectively (Fig.
1A). When normalized to the housekeeping gene encoding for
cyclophilin, the level of MCD expression is similar among the three
muscles (Fig. 1B), due to differential expression of
cyclophilin among the heart, EDL, and soleus (data not shown). However,
unlike 36B4, the expression of cyclophilin does not change upon any of
the interventions within this study (data not shown). Thus the
remaining MCD expression data are normalized to cyclophilin. The
maximal activity of MCD measured in the heart was 9.0- and 4.6-fold
greater compared with the activity in EDL and soleus muscles,
respectively (Fig. 1C).

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Fig. 1.
Comparison of malonyl-CoA decarboxylase (MCD) expression
(A and B) and activity (C) between
heart, extensor digitorum longus (EDL), and soleus muscle. Values shown
are means ± SE for 6 separate observations. MCD expression values
are normalized either against 36B4 (A) or cyclophilin
(B). Units for enzyme activity are
nmol · min 1 · mg
protein 1.
|
|
High-fat feeding increases plasma NEFA levels and alters cardiac
and skeletal muscle MCD expression and activity.
The time-dependent effects of increased dietary fatty acids on cardiac
and skeletal (EDL and soleus) muscle MCD mRNA levels, MCD activity, and
plasma NEFA levels were investigated. Feeding on the LC/HF diet caused
a rapid elevation in plasma NEFA levels (within 1 day), an effect that
was sustained for the duration of the experiment (Fig.
2A). LC/HF feeding
significantly increased MCD mRNA in heart, EDL, and soleus muscle
within 1 day (Fig. 2B). The sensitivity of MCD mRNA
induction was soleus > EDL > heart. EDL and soleus muscle
MCD mRNA remained significantly elevated throughout the time course of
the experiment. Although heart MCD mRNA was elevated 4 and 8 days after
the initiation of high-fat feeding, this increase was not statistically
significant at these time points. The effects of high-fat feeding on
MCD activity are shown in Table 2. In
contrast, feeding a LC/HF diet decreased MCD activity in the heart by
17%, increased the activity in the soleus muscle by 30%, and had no
effect on the EDL muscle.

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Fig. 2.
Time course for altered plasma nonesterified fatty acid
(NEFA) levels (A), as well as for heart, EDL, and soleus
muscle MCD expression (B), during feeding on the
low-carbohydrate/high-fat (LC/HF) diet. Values shown are means ± SE for 5 separate observations. All MCD expression values are
normalized against the housekeeping gene cyclophilin.
*P < 0.05, **P < 0.01, and
***P < 0.001 vs. control (day 0).
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|
Fasting and refeeding alter plasma NEFA levels and cardiac and
skeletal muscle MCD expression and activity.
Fasting significantly increased plasma NEFA levels within 24 h
(Fig. 3A). Although plasma
NEFA levels were still significantly elevated after 2 days of fasting
(compared with basal levels), they were markedly reduced compared with
the 24-h time point. Refeeding on the HC/LF diet lowered plasma NEFA
levels to basal values. Fasting also significantly increased cardiac
and skeletal muscle MCD mRNA at both time points investigated (Fig.
3B). The sensitivity of MCD mRNA induction was soleus = EDL > heart. Refeeding returned MCD mRNA to near-basal levels in
all three muscles. In contrast, fasting had no significant effects on
MCD activity in any of the muscles investigated (Table
3). Refeeding on the HC/LF diet
significantly decreased cardiac, but not EDL or soleus, muscle MCD
activity (Table 3).

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Fig. 3.
Effects of fasting and refeeding on plasma NEFA levels
(A), as well as on heart, EDL, and soleus muscle MCD
expression (B). Animals were refed the
high-carbohydrate/low-fat (HC/LF) diet. Values shown are means ± SE for 4 or 5 separate observations. All MCD expression values are
normalized against the housekeeping gene cyclophilin.
**P < 0.01 and ***P < 0.001 vs.
control (day 0).
|
|
STZ-induced diabetes alters plasma NEFA levels and cardiac and
skeletal muscle MCD expression and activity.
STZ induction of diabetes significantly increased plasma glucose
(3.18-fold; P < 0.001) and NEFA (3.7-fold; Fig.
4A) levels. Induction of
diabetes increased MCD mRNA levels in both cardiac and skeletal muscle,
particularly in the soleus (Fig. 4B). Diabetes was also
associated with increased MCD activity in both EDL and soleus muscles
(Table 4). No change in cardiac MCD
activity was observed 2 wk after diabetes induction (Table 4).

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Fig. 4.
Effects of streptozotocin (STZ)-induced diabetes on plasma NEFA
levels (A), as well as on heart, EDL, and soleus muscle MCD
expression (B). Values shown are means ± SE for 6 separate observations. All MCD expression values are normalized against
the housekeeping gene, cyclophilin. *P < 0.05, **P < 0.01, and ***P < 0.001 vs.
control.
|
|
PPAR
, but not PPAR
, activation increases the mRNA and
enzymatic activity of MCD in cardiac and skeletal muscle.
To investigate further the mechanism by which the expression and
activity of cardiac and skeletal muscle MCD are regulated, rats were
treated either with the specific PPAR
agonist Wy-14643 or the
specific PPAR
agonist troglitazone. Wy-14643 increased the
expression of MCD in both cardiac and skeletal muscle (Fig. 5). The sensitivity of MCD mRNA induction
was the same as that seen when plasma fatty acid levels were increased
(soleus > EDL > heart; Fig. 5). In addition, Wy-14643
increased the activity of MCD in both the heart and soleus but not in
the EDL muscle (Table 5). In contrast,
troglitazone decreased MCD mRNA in both cardiac and skeletal muscles
(Fig. 6). Troglitazone had no effect on
MCD activity in any of the muscles investigated (Table
6).

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Fig. 5.
Effects of specific peroxisome proliferator-activated
receptor- (PPAR ) activation on heart, EDL, and soleus muscle MCD
expression. Animals were fed either control food or powdered food
containing Wy-14643 (0.01% wt/wt). Values shown are means ± SE
for 6 separate observations. All MCD expression values are normalized
against the housekeeping gene, cyclophilin. ***P < 0.001 vs. control.
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Fig. 6.
Effects of specific PPAR activation on heart, EDL, and
soleus muscle MCD expression. Animals were fed either control food or
powdered food containing troglitazone (0.1% wt/wt). Values shown are
means ± SE for 5 separate observations. All MCD expression values
are normalized against the housekeeping gene, cyclophilin.
*P < 0.05 and **P < 0.01 vs.
control.
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|
Pressure overload-induced alterations in cardiac MCD expression and
activity: dependence on fatty acids.
We investigated whether pressure overload-induced cardiac hypertrophy
affected the expression of MCD. Aortic constriction resulted in cardiac
hypertrophy, as indicated by an increase in the heart weight-to-body
weight ratio (4.02 ± 0.09 vs. 3.13 ± 0.06 g/kg for
experimental and control groups; P < 0.001). Cardiac hypertrophy caused a significant decrease in the levels of both MCD
mRNA and activity (Fig. 7A and
Table 7, respectively).

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Fig. 7.
Effects of pressure overload-induced hypertrophy on cardiac MCD
expression. Rats were fed 1 of 3 diets: standard laboratory chow
(normal; A), HC/LF (B), or LC/HF (C)
diets. Duration of diet feeding was a total of 16 days (7 days before
and 9 days after surgery). Values shown are means ± SE for
between 5 and 13 separate observations. All MCD expression values are
normalized against the housekeeping gene, cyclophilin.
*P < 0.05 and **P < 0.01 vs. HC/LF
control. $$P < 0.01 vs. LC/HF control.
|
|
In a separate experiment, we investigated whether fatty acids play a
role in hypertrophy-associated changes in MCD expression and activity
by placing rats on diets with differing fatty acid contents. After 7 days on the special diets (either HC/LF or LC/HF), one-half of the rats
underwent aortic banding, whereas the other one-half were sham
operated. Rats were maintained on their special diets for 9 days after
surgery, at which time the hearts were isolated. Aortic constriction
caused similar increases in the heart weight-to-body weight ratio
whether rats were fed the HC/LF diet (3.91 ± 0.24 vs. 3.16 ± 0.08 g/kg for experimental and control groups; P < 0.05) or the LC/HF diet (3.90 ± 0.13 vs. 3.17 ± 0.07 g/kg
for experimental and control groups; P < 0.05). Hearts
isolated from sham-operated rats fed the LC/HF diet compared with
sham-operated rats fed the HC/LF diet possessed significantly higher
levels of MCD expression (Fig. 7B). As observed during
standard laboratory chow feeding, banding significantly reduced the MCD
mRNA when rats were fed the LC/HF diet. In contrast, pressure overload
did not decrease cardiac MCD expression for rats fed the HC/LF diet (Fig. 7B). Similar results were observed for MCD activity,
in which aortic banding significantly decreased heart MCD activity only
for rats fed the LC/HF diet (Table 7).
 |
DISCUSSION |
We tested the hypothesis that fatty acids regulate the expression
and activity of cardiac and skeletal muscle MCD. High-fat feeding,
fasting, and STZ-induced diabetes all increased MCD mRNA in cardiac and
skeletal muscle, with concomitant elevations in plasma fatty acid
levels. However, changes in MCD mRNA were not always paralleled by MCD
activity. The specific PPAR
agonist Wy-14643 increased cardiac and
skeletal muscle MCD mRNA as well as activity. PPAR
activation did
not induce MCD. The sensitivity of MCD mRNA induction by fatty acids
(and Wy-14643) was soleus > EDL > heart. In addition,
cardiac hypertrophy, which is associated with decreased PPAR
expression (3) and decreased fatty acid oxidation
(2, 21), results in decreased MCD expression and activity,
an effect which is dependent on fatty acids. These results suggest that
MCD is regulated both transcriptionally and posttranscriptionally. In
the former case, this study suggests that fatty acids induce MCD gene
expression. Whether this is due to PPAR
activation requires further investigation.
Malonyl-CoA is a key regulator of fatty acid oxidation in both the
heart and skeletal muscle by inhibiting CPT-1 and the entry of
long-chain fatty acyl-CoAs (LCFAs) into the mitochondrial matrix (27). Because malonyl-CoA is synthesized by ACC, much work
has focused on the mechanisms of regulation of ACC in both cardiac and
skeletal muscle. ACC
, the major isoform in muscle, is regulated both allosterically (by citrate and LCFAs) and through covalent modification (reversible phosphorylation by AMP-activated protein kinase) (19, 32). MCD, the enzyme which degrades
malonyl-CoA, has only recently been investigated. Initial studies
suggest that MCD is regulated at the levels of both expression and
covalent modification (14, 37), although the exact nature
of these regulatory mechanisms is unknown.
An inverse correlation often, but not always, exists between
intracellular malonyl-CoA levels and rates of fatty acid oxidation in
both heart and skeletal muscle (8, 14, 15, 28, 29, 34).
For example, in skeletal muscle, the levels of malonyl-CoA decrease
during fasting, whereas the rate of fatty acid oxidation increases
(44). This decreased malonyl-CoA level cannot be accounted for by assayable changes in ACC activity (44). This
observation could be explained through alterations in the intracellular
concentrations of the substrate and/or allosteric effectors for ACC
(32) and/or alterations in the activity of MCD. We report
here that fasting increases the expression of skeletal muscle MCD more
than fourfold (Fig. 3B). Although assayable MCD activity did
not significantly change, there was a trend for increased activity in
both EDL and soleus muscles (Table 3). In addition to fasting, diabetes
and high-fat feeding also increase plasma fatty acid levels (Figs. 2A, 3A, and 4A). All three conditions
are associated with increased muscle MCD mRNA, suggesting that fatty
acids may be involved in the regulation of MCD gene expression. It
could be hypothesized that the mechanism by which fatty acids
potentially induce MCD is mediated by PPAR
, because specific PPAR
activation led to increased skeletal muscle (and heart) MCD expression
(as well as activity; Fig. 5 and Table 5). In contrast, specific
PPAR
activation decreased muscle MCD expression. This might be due to the lipid-lowering properties of troglitazone, thus lowering the
stimulus for MCD expression.
The heart, like skeletal muscle, is able to adapt to alterations in its
environment. For example, with pressure overload, the heart undergoes
coordinated morphological and genomic changes, including reexpression
of the fetal gene program and the development of myocyte hypertrophy
(10, 35, 45). In metabolic terms, the hypertrophied heart
decreases fatty acid utilization and increases its reliance on glucose
as a fuel (2, 21). The mechanisms involved in this
substrate switching are complex. As already mentioned, one important
transcriptional regulator of this phenomenon appears to be PPAR
.
PPAR
expression decreases with hypertrophy, as do several
PPAR
-regulated genes that are known to be involved in fatty acid
metabolism (e.g., mCPT-1 and medium- and long-chain acyl-CoA
dehydrogenase) (3, 10, 33). The present study has
found that pressure overload-induced hypertrophy results in decreased
MCD expression and activity (Fig. 7A and Table 7). Furthermore, when animals are fed a low-fat (HC/LF) diet, during which
time the availability of the ligand for PPAR
(namely fatty acids)
will be reduced, the decrease in MCD expression with hypertrophy is
prevented (Fig. 7B). Therefore, hypertrophy-induced changes in MCD expression are dependent on fatty acids. This is further evidence in support of the hypothesis that PPAR
regulates the expression of MCD. From these results, we speculate that decreased MCD
expression with pressure overload may play a role in decreased fatty
acid oxidation by the hypertrophied heart (through increased inhibition
of mCPT-1 by malonyl-CoA).
As mentioned above, both the heart and skeletal muscle are able to
adapt to changes in its environment. The heart adapts to changes in
both workload and substrate availability, resulting in switching of
substrate preference, enabling constant ATP generation and therefore
maintenance of contractile performance. For example, during
insulin-dependent diabetes, the heart adapts to increased fatty acid
utilization in the presence of both increased fatty acid availability
and decreased glucose utilization (40). In contrast, not
only does skeletal muscle require adaptation for the maintenance of ATP
generation in different activity states, but, because of its sheer
mass, skeletal muscle is important in whole body fuel
homeostasis. It is essential for skeletal muscle to respond to
alterations in plasma fuel levels, either directly (e.g., fatty acids
through PPAR
) or indirectly (e.g., glucose through insulin), thereby
preventing detrimental accumulation of intermediates, as observed
during various pathologies, especially insulin resistance
(9). These differences in the roles of substrate sensing
by heart and skeletal muscle are exemplified by the present study. The
sensitivity of MCD induction by either fatty acids or specific PPAR
activation (by Wy-14643) was soleus > EDL > heart. The
skeletal muscle therefore responded to increased fatty acid
availability much more dramatically than did the heart, presumably to
prevent the detrimental accumulation of fatty acids. Furthermore, the
highly insulin-sensitive, oxidative soleus muscle was the most
sensitive to MCD induction at the level of both transcription and
activity. Because fatty acids are aerobically metabolized through
-oxidation in the mitochondrial matrix, it is not surprising that
this muscle induced MCD to the greatest extent.
Accumulation of intracellular fatty acid (and lipid) levels is
associated with various pathologies, including insulin resistance (31, 32),
-cell dysfunction (38, 42), and
cardiotoxicity (46). Although the precise mechanisms by
which fatty acid accumulation is detrimental are not known, several
possibilities exist, including initiation of apoptosis
(38) and chronic stimulation of PKC (25). A
link has been proposed between intracellular malonyl-CoA levels and
insulin resistance, as recently reviewed by Ruderman et al.
(32). Increased malonyl-CoA levels, through either
increased synthesis or decreased degradation, would inhibit fatty acid
oxidation, resulting in increased intracellular LCFA levels and insulin
resistance. One possibility is that impaired induction of MCD in
response to fatty acids would result in intracellular LCFA
accumulation. Whether impaired MCD induction occurs in
insulin-resistant skeletal muscle requires elucidation. In support of
this hypothesis is the finding that cardiac muscle isolated from the
insulin-resistant obese Zucker rat possesses decreased PPAR
expression (46).
The present results not only provide evidence for transcriptional
control of MCD but also suggest that posttranscriptional mechanisms
operate to regulate MCD activity. For example, high-fat feeding
resulted in increased MCD activity only in the soleus muscle, despite
the observation that MCD mRNA increased in all muscles investigated.
High-fat feeding actually decreased cardiac MCD activity. Furthermore,
fasting dramatically increased MCD mRNA levels in all muscles but had
only modest effects on skeletal muscle MCD activity. These results
clearly suggest that posttranscriptional mechanisms also operate to
regulate MCD activity. From the present results, it could be
hypothesized that fatty acids, in addition to MCD induction, cause
inhibition of MCD activity. Possible mechanisms of posttranscriptional
regulation might include alternative splicing, translational control,
covalent modification, or the presence of an unidentified
MCD-inhibitory protein. In support of the hypothesis that an MCD
binding protein exists, a 40-KDa protein has been found that
co-purifies with dog heart MCD through seven separate purification
steps (Goodwin GW, unpublished observations). Recent studies in both
the heart and skeletal muscle have shown that increased contractility
is associated with increased MCD activity through covalent modification
(14, 36). This alteration in MCD activity appears to be
due to phosphorylation (36).
The present study has investigated whether alterations in substrate
availability or cardiac workload are associated with changes in heart
and skeletal muscle MCD expression and activity in vivo. When plasma
fatty acid levels were elevated through high-fat feeding, fasting, and
diabetes, MCD mRNA levels increased in both cardiac and skeletal
muscle. However, these situations are associated with changes not only
in plasma fatty acid levels, but also in levels of other fuels (such as
glucose) and hormones, which may potentially play a role in the
induction of MCD. Furthermore, the present results can only speculate
as to the involvement of PPAR
in the regulation of MCD expression.
Further experiments will be required to investigate in more depth the
role of this nuclear receptor in MCD induction. Although the present
study has investigated both the expression and the activity of MCD in several in vivo models, no measurements of ACC (or AMP kinase) expression or activity have been made, nor have intracellular levels of
malonyl-CoA or fatty acid oxidation rates been determined. Therefore,
it can only be speculated whether alterations in MCD activity, as
measured in the present study, result in expected changes in
malonyl-CoA levels and fatty acid oxidation in these same samples.
However, several previously published studies have focused on
malonyl-CoA levels and/or fatty acid oxidation rates during similar
conditions (e.g., fasting, diabetes, hypertrophy) (22, 37,
44). The present results complement these previously published
studies. Furthermore, interpretation of whole cell malonyl-CoA level
measurements can be clouded by the likely compartmentation of
malonyl-CoA within the cell (18).
In conclusion, elevation of plasma fatty acids was associated with
increased expression of MCD in both heart and skeletal muscle. We
provide evidence in support of the hypothesis that fatty acid-induced
MCD expression is mediated through PPAR
, because specific PPAR
activation stimulates MCD expression in a manner similar to that seen
by fatty acids. Thus MCD might be part of the feed forward mechanism,
in which fatty acids stimulate their utilization by muscle. Whether
this feed forward mechanism is impaired in insulin-resistant muscle is
presently unknown. In addition to altered substrate availability,
pressure overload-induced cardiac hypertrophy (which is associated with
decreased PPAR
expression and decreased fatty acid utilization)
results in decreased MCD expression and activity.
 |
ACKNOWLEDGEMENTS |
The troglitazone was a kind gift from Parke, Davis, Morris Plains, NJ.
 |
FOOTNOTES |
This work was supported in part by National Institutes of Health Grants
HL-43133 and HL-61483 and by grants from the American Heart
Association, National Center and Texas Affiliate.
Address for reprint requests and other correspondence: H. Taegtmeyer, Dept. of Internal Medicine, Div. of Cardiology, Univ. of
Texas-Houston Medical School, 6431 Fannin, MSB 1.246, Houston, TX 77030 (Email Heinrich.Taegtmeyer{at}uth.tmc.edu).
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 11 May 2000; accepted in final form 8 November 2000.
 |
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