1 Departments of Biochemistry and Physiology, East Carolina University, Greenville 27858; 2 Department of Medicine, Duke University Medical School, Durham, North Carolina 27710; and 3 Diabetes Research Laboratory, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Charlestown, Massachusetts 02129
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ABSTRACT |
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Carnitine
palmitoyltransferase I (CPT I), which is expressed as two distinct
isoforms in liver () and muscle (
), catalyzes the rate-limiting
step in the transport of fatty acid into the mitochondria. Malonyl-CoA,
a potent inhibitor of CPT I, is considered a key regulator of fatty
acid oxidation in both tissues. Still unanswered is how muscle
-oxidation proceeds despite malonyl-CoA concentrations that exceed
the IC50 for CPT I
. We evaluated
malonyl-CoA-suppressible [14C]palmitate oxidation and CPT
I activity in homogenates of red (RG) and white (WG) gastrocnemius,
soleus (SOL), and extensor digitorum longus (EDL) muscles. Adding 10 µM malonyl-CoA inhibited palmitate oxidation by 29, 39, 60, and 89%
in RG, SOL, EDL, and WG, respectively. Thus malonyl-CoA resistance,
which correlated strongly (0.678) with absolute oxidation rates
(RG > SOL > EDL > WG), was greater in red than in
white muscles. Similarly, malonyl-CoA-resistant palmitate oxidation and
CPT I activity were greater in mitochondria from RG compared with WG.
Ribonuclease protection assays were performed to evaluate whether our
data might be explained by differential expression of CPT I splice
variants. We detected the presence of two CPT I
splice variants that
were more abundant in red compared with white muscle, but the relative
expression of the two mRNA species was unrelated to malonyl-CoA
resistance. These results provide evidence of a malonyl-CoA-insensitive
CPT I activity in red muscle, suggesting fiber type-specific expression
of distinct CPT I isoforms and/or posttranslational modulations that
have yet to be elucidated.
fatty acid oxidation; fiber-type specificity
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INTRODUCTION |
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AFTER ENTERING CELLS,
long-chain fatty acid is converted to acyl-CoA by acyl-CoA synthetase
(ACS), a family of integral membrane proteins that are present in
various subcellular organelles, including mitochondria
(8). Because long-chain acyl-CoAs cannot diffuse freely
across membranes, the mitochondrial carnitine shuttle system has an
obligatory role in -oxidation by permitting acyl-CoA translocation from the cytosol into the mitochondria in all mammalian cells (10). Carnitine palmitoyltransferase I (CPT I), which
spans the outer mitochondrial membrane, catalyzes the initial step in this process by transferring acyl groups from CoA to carnitine. The
acylcarnitines formed by CPT I traverse the inner membrane via a
specific translocase that is coupled to carnitine palmitoyltransferase II (CPT II), which regenerates acyl-CoA upon transporting the fatty
acyl groups into the mitochondrial matrix. Because CPT I represents the
pace-setting reaction in the carnitine shuttle system, it is widely
considered the most critical step in controlling fatty acid flux
through the
-oxidative pathway (13).
CPT I is expressed as at least two isoforms, which are the products of
different genes: a liver enzyme, CPT I (~88 kDa), and its smaller
counterpart in cardiac and skeletal muscle, CPT I
(~82 kDa), each
having distinct kinetic properties (13). A distinguishing
regulatory property of these isoenzymes is that both are inhibited by
malonyl-CoA (15), which is produced in the cytosol by
acetyl-CoA carboxylase (ACC) (26). In both liver and
muscle, physiological alterations in malonyl-CoA concentrations correlate inversely with changes in
-oxidation. For example, starvation (15) and exercise (28) decrease
tissue levels of malonyl-CoA, which presumably relieves inhibition of
CPT I and increases fatty acyl-CoA entry into mitochondria
(3). Conversely, carbohydrate feedings stimulate ACC
activity and increase production of malonyl-CoA, which corresponds with
a decrease in fatty acid oxidation and an increase in long-chain
acyl-CoA accumulation (3). In some reports, physiological
regulation of malonyl-CoA concentration appears to differ between red
and white muscle (3, 29), suggesting that
malonyl-CoA-mediated control of fatty acid metabolism might depend on
muscle fiber type. However, the role of fiber type in modulating the
malonyl-CoA/CPT I system has not been addressed. Paradoxically,
concentrations of malonyl-CoA that are measured in both red and white
muscle (1-4 µM) (3, 15) should completely inhibit
CPT I activity at all times. This is because the muscle isoform of CPT
I is ~100 times more sensitive to malonyl-CoA (IC50
~0.03 µM) than the liver isoform (IC50 ~2.7 µM)
(7, 15). Thus the question of how fatty acid oxidation proceeds in muscle despite constitutively high malonyl-CoA levels has
remained an enigma.
Importantly, several investigators have reported that a significant portion of CPT activity measured in muscle mitochondria is uninhibited by malonyl-CoA (7, 15, 23). Historically, investigators have attributed this nonsuppressible fraction to CPT II, the inner mitochondrial membrane enzyme that can be expressed if membranes are damaged during mitochondrial preparation (7, 15). We considered an alternative view, that muscle might contain a malonyl-CoA-insensitive CPT I fraction, and hypothesized that malonyl-CoA inhibition of muscle fatty acid oxidation might depend on the fiber type of the muscle. Thus discrepancies among studies reporting varying degrees of malonyl-CoA insensitivity might have arisen because the studies were performed using muscles composed of dissimilar fiber compositions. To address this hypothesis, we compared malonyl-CoA inhibition of fatty acid oxidation and CPT I activity in whole homogenates and isolated mitochondria that were prepared from red and white skeletal muscles. The results provide evidence of a malonyl-CoA-insensitive CPT I fraction that is predominantly active in red muscle.
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METHODS |
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Materials. The [3H]carnitine and the chemiluminescence detection kit were from Amersham (Piscataway, NJ). The calnexin antibody was from Santa Cruz (Santa Cruz, CA), and the polyvinylidene difluoride (PVDF) membrane was from Bio-Rad (Hercules, CA). All other reagents were obtained from Sigma (St. Louis, MO).
Animals. Male Sprague-Dawley rats from Harlan (Indianopolis, IN) were fed a chow diet and water ad libitum. Rats weighing ~300 g were used for all experiments.
Muscle homogenization. Rats were anesthetized by an intraperitoneal injection of ketamine-xylazine (90/10 mg/kg), and skeletal muscles were removed. Muscle homogenates and isolated mitochondria were prepared using white (WG) and red (RG) gastrocnemius muscles and from soleus (SOL), extensor digitorum longus (EDL) and epitrochlearis (EPI) muscles. Approximately 50-70 mg of tissue were minced thoroughly with scissors in 300 µl of a modified sucrose-EDTA medium (SET) containing 250 mM sucrose, 1 mM EDTA, and 10 mM Tris · HCl, pH 7.4 (21). SET buffer was added to a 20-fold diluted (wt/vol) suspension, and samples were homogenized in a 3.0-ml Potter-Elvehjem glass homogenizer at 10 passes across 30 s at 1,200 rpm with a Teflon pestle. Protein concentrations in muscle homogenates ranged from 0.9 to 2.3 mg/ml.
Isolation of mitochondria and microsomes. Muscles were excised and immediately placed in ice-cold modified Chapell-Perry buffer (in mM: 100 KCl, 40 Tris · HCl, 10 Tris base, 5 MgSO4, 1 EDTA, and 1 ATP, pH 7.5) and separated into red, white, or mixed gastrocnemius; only RG and WG were used for these experiments. Muscles were blotted, weighed, and placed into 2.0 ml (RG) or 4 ml (WG) of Chapell-Perry buffer. Samples were minced thoroughly on ice, diluted 10-fold (wt/vol) with Chapell-Perry buffer, and then homogenized twice for 15 s with an Ultra-turrax at ~9,500 rpm. Tissue homogenates were centrifuged at 650 g for 10 min at 4°C. The supernatant was gravity filtered through four layers of surgical gauze and centrifuged at 8,500 g for 10 min at 4°C. Microsomes were isolated from the supernatant by ultracentrifugation at 100,000 g for 1 h at 4°C. The mitochondrial pellet from the 8,500-g spin was washed to remove erythrocytes, resuspended in 1.3 ml of Chapell-Perry buffer, and centrifuged at 8,500 g for 10 min. Both the microsomal and mitochondrial pellets were suspended in 1.0 ml of SET buffer and used immediately for assaying CPT I specific activity and palmitate oxidation. Proteins were determined by the bicinchoninic acid method. For Western blots, 15 µg of protein from mitochondrial and microsomal subfractions were separated by 8-12% gradient SDS-PAGE, transferred onto PVDF membranes, and probed for 2 h with calnexin antibody per the manufacturer's protocol. Proteins were visualized by horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G with a chemiluminescence Western blotting detection kit.
Fatty acid oxidation. Palmitate oxidation rates were determined by measuring production of 14C-labeled acid-soluble metabolites (ASM), a measure of tricarboxylic acid (TCA) cycle intermediates and acetyl esters (incomplete oxidation) (27), and [14C]CO2 (complete oxidation), by use of a modified 48-well microtiter plate (Costar, Cambridge, MA), as previously described (11). Reactions were initiated by adding 40 µl of whole homogenates or isolated mitochondria to 160 µl of the incubation buffer (pH 7.4), yielding final concentrations (in mM) of 0.2 palmitate ([1-14C]palmitate at 0.5 µCi/ml), 100 sucrose, 10 Tris · HCl, 5 potassium phosphate, 80 potassium chloride, 1 magnesium chloride, 2 L-carnitine, 0.1 malate, 2 ATP, 0.05 CoA, 1 dithiothreitol, 0.2 EDTA, and 0.5% bovine serum albumin. After incubation for 60 min at 30°C, reactions were terminated by adding 100 µl of 4 N sulfuric acid, and the CO2 produced during the incubation was trapped in 200 µl of 1 N sodium hydroxide that had been added to adjacent wells (11). The acidified medium was stored at 4°C overnight, and then ASM were assayed in supernatants of the acid precipitate (27). Radioactivity of CO2 and ASM was determined by liquid scintillation counting by use of 4 ml of Uniscint BD (National Diagnostics).
CPT I assay. CPT I activity was measured using 10-20 µg of mitochondrial or microsomal protein or 40-60 µg of protein from whole homogenates. The assays were carried out with 50 µM palmitoyl-CoA and 0.2 mM [3H]carnitine (0.5 µCi) as previously described (11). After 10 min at 30°C, the assay was terminated by adding 60 µl of 1.2 mM ice-cold HCl. The [3H]palmitoyl-carnitine formed was extracted with water-saturated butanol and quantified by liquid scintillation counting.
Ribonuclease protection assay analysis.
RNA was isolated from skeletal muscle using TRIzol (GIBCO-BRL) reagent
as previously described (9). Ribonuclease protection assays (RPA) were performed as described (31) with a
complementary RNA probe generated by T7 polymerase from a linearized
template consisting of the rat CPT I2 cDNA fragment extending from
NcoI at nucleotide 238 (relative to the initiation AUG) to
EcoRI at nucleotide 423 subcloned into pBluescript.
Statistics. Statistical analyses were performed using JMP Statistical Software (SAS, Cary, NC). The correlation between malonyl-CoA-resistant activity and total oxidation rates was evaluated with a bivariate linear regression analysis, and significance was determined by ANOVA. Two- and three-way ANOVA was performed using a standard least squares model to test both the main and interaction effects of muscle type × incubation time × malonyl-CoA concentration (where appropriate) on palmitate oxidation, CPT I activity, or percent inhibition. In experiments consisting of a single time point and/or malonyl-CoA concentration, differences between red and white muscle were performed using a one-way ANOVA.
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RESULTS |
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Malonyl-CoA inhibition of palmitate oxidation in red and white
muscle.
To address the question of whether malonyl-CoA inhibits fatty acid
oxidation equally in muscles of varied fiber compositions, we first
examined the effects of a high but still physiological malonyl-CoA
concentration (10 µM) on [14C]palmitate oxidation in
homogenates of red and white muscles. Predictably, palmitate oxidation
(ASM plus CO2) was greater in homogenates of red muscle (RG
and SOL) than in homogenates of white muscle (EDL and WG) (Fig.
1A).
Addition of 10 µM malonyl-CoA, which exceeds the IC50 of
recombinant CPT I by ~100-fold, inhibited palmitate oxidation by
29, 39, 60, and 89% in RG, SOL, EDL, and WG, respectively (Fig.
1B). Relative inhibition by malonyl-CoA was similar between
measures of complete oxidation (CO2) and total oxidation
(CO2 plus ASM). Linear regression analyses indicated that
the degree of malonyl-CoA resistance correlated positively (r2 = 0.678, P < 0.005)
with absolute rates of palmitate oxidation (Fig. 1C); thus highly
oxidative red muscles were more resistant to malonyl-CoA than highly
glycolytic white muscles. Adding protease inhibitors to the
homogeneization buffer did not eliminate the differences between red
and white muscles (data not shown), suggesting that fiber type-specific
malonyl-CoA resistance was not due to in vitro protease modification of
CPT I that may have occurred during homogenate preparation and/or
incubation. Because differences in malonyl-CoA resistance were most
marked between RG and WG, these muscles were chosen for subsequent
experiments.
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Peroxisomal oxidation in red and white muscle.
Others have shown that both mitochondria and peroxisomes contribute to
total palmitate oxidation in muscle homogenate systems (18). To quantify the contribution of peroxisomes to
palmitate oxidation in red and white muscle, we blocked mitochondrial
respiration by incubating muscle homogenates in the presence of the
electron transport chain inhibitors KCN and rotenone. In both RG and
WG, peroxisomes accounted for ~20% of total palmitate oxidation
(Table 1), similar to previous reports
(18). These results suggest that fiber type-dependent
differences in malonyl-CoA resistance are unrelated to differences in
peroxisomal oxidation. Table 1 also shows that palmitate oxidation to
CO2 depended fully on the presence of carnitine, indicating
that mitochondrial membrane integrity was not compromised during
homogenate preparations. Similarly, subtracting carnitine from the
buffer diminished production of ASM by 90%; however, since a small
portion of the 14C-labeled acid-soluble products appeared
to be carnitine independent, oxidation results from subsequent
experiments are presented as CO2 only.
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Malonyl-CoA sensitivity in homogenates of RG and WG.
To better evaluate fiber type-dependent differences in malonyl-CoA
sensitivity, palmitate oxidation to CO2 was studied in the
presence of increasing malonyl-CoA in whole homogenates of RG and WG
(Fig. 3A). In the presence of
1 and 5 µM malonyl-CoA, concentrations that fall within the range
previously reported in rat muscle (3), palmitate oxidation
was inhibited only 32-51% in RG and 52-72% in WG
(P < 0.001; Fig. 3B). When the
malonyl-CoA-resistant fraction was subtracted from the total activity,
as previously described (15), the IC50 values
appeared similar between red and white muscle (Fig. 3C),
suggesting differences in efficacy but not in the potency of
malonyl-CoA inhibition. Figure 4 shows that CPT I activity, measured in whole homogenates, was 1.7-fold greater (P < 0.01) in SOL (red) than in EPI (white)
muscle. In EPI, 10 µM malonyl-CoA inhibited CPT I activity by 62%
compared with only 34% in SOL (P < 0.01). These data
recapitulate the results from oxidation experiments and demonstrate
that fiber type-selective malonyl-CoA inhibition of CPT I activity is
consistent with inhibition of palmitate oxidation, lending further
support to our hypothesis that malonyl-CoA inhibition of muscle CPT I
depends on fiber composition.
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Malonyl-CoA sensitivity and palmitate oxidation in isolated
mitochondria.
Previous reports have suggested that the nonsuppressible component of
CPT I activity might be related to mitochondrial damage. To address
this possibility, we tested the integrity of mitochondria that were
isolated from our homogenate preparations. Similar to the results in
whole homogenates (Fig. 1), we found that oxidation rates were greater
and the inhibitory effect of malonyl-CoA was less (P < 0.001) in mitochondria from RG compared with WG (Fig. 5). Importantly, in the isolated
mitochondrial preparations, palmitate oxidation to CO2
fully required the presence of both CoA and carnitine, respective
substrates for the outer mitochondrial enzymes, ACS and CPT I, which
catalyze the first two reactions of palmitate oxidation. These data
support results obtained from whole homogenates (Table 1) and confirm
that mitochondrial membranes were intact.
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Expression of CPT I splice variants in red and white muscle.
Next, we investigated the possibility that our observations might be
related to fiber type-selective mRNA abundance of a recently identified
CPT I splice variant that is preferentially expressed in rat heart
and skeletal muscle (30, 31). Relative expression of
alternatively spliced mRNAs in different muscles was quantified by RPA
(Fig. 7). Expression of the predominant
splice variant CPT I
1 was approximately threefold greater in red
muscles (SOL and RG) than in white muscles (EDL and WG) (Fig.
7B). Likewise, expression of the alternatively spliced mRNA
species CPT I
2 was also more abundant in red muscles (Fig.
7C). Thus CPT I gene expression across fiber types is
consistent with the higher enzyme activity and fatty acid oxidative
capacity measured in red compared with white muscles. However, because
the ratio of CPT I
1 to CPT I
2 abundance was similar between red
and white muscles, mRNA expression of CPT I
2 appeared to be
unrelated to fiber type-selective differences in malonyl-CoA
resistance. In separate experiments, we used cRNA probe for CPT I
and found that abundance of this isoform was negligible in skeletal
muscle (data not shown), as previously reported (7).
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DISCUSSION |
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Malonyl-CoA is presumed to be a key regulator of muscle fatty acid
oxidation by virtue of its potent inhibition of CPT I and because
muscle malonyl-CoA content changes reciprocally with -oxidation (3). Unexplained, however, is how fatty acid oxidation
proceeds despite muscle concentrations of malonyl-CoA that should
completely inhibit CPT I
. This enigma might be at least partly
reconciled by our data, which provide evidence of a
malonyl-CoA-insensitive CPT I activity in red skeletal muscle. Similar
to previous reports, we found that, in homogenates of WG, physiological
concentrations of malonyl-CoA inhibited palmitate oxidation by
75-90%. Conversely, in RG, these same concentrations inhibited
palmitate oxidation by only 35-54%. Furthermore, 100 µM
malonyl-CoA, which exceeds the reported IC50 by several
orders of magnitude, inhibited palmitate oxidation by only 62%,
suggesting that CPT I in red muscle is resistant to full inhibition by
malonyl-CoA. Additionally, a comparison across muscle types showed that
malonyl-CoA resistance correlated positively with the muscle's fatty
acid oxidative capacity. These results provide the first reported
evidence of fiber type-specific differences in malonyl-CoA-mediated
regulation of muscle lipid oxidation.
Although the present investigation did not evaluate fiber type by parameters other than fatty acid oxidative capacity, a recent study of rat hindlimb muscles provided detailed analyses of fiber composition based on the protein expression profile of four distinct myosin heavy-chain isoforms (22). Investigators found that RG, SOL, EDL, and WG consisted of 24, 84, 5, and 0.8% type I fibers, respectively. Type II fibers were further classified into IIA, IIB, IIC, IID, IIAD, and IIDB subtypes. We combined their data on fiber type with our results presented in Fig. 1 to evaluate the relationship between histochemical fiber type and malonyl-CoA resistance. The only significant correlation detected by linear regression (r2 = 0.63, P < 0.01) was that between malonyl-CoA resistance and the proportion of type IIA fibers, which was 18, 9, 15, and <1% in RG, SOL, EDL, and WG, respectively (22). Surprisingly, these correlative findings across studies suggest that expression of the malonyl-CoA-resistant CPT I subfraction might be more closely linked to type IIA than type I fibers, although we acknowledge that this result should be interpreted cautiously because of the small number of animals that was used for the analysis. Still, in experiments using larger populations (data not shown), both fatty acid oxidative capacity and malonyl-CoA resistance were consistently greater in RG than in SOL (other muscle types were not evaluated), again implying that these metabolic properties are unrelated to the proportion of type I fibers. This finding is reflective of the poor association that is often reported between histochemical fiber type, delineated by expression of specific myosin isoforms, and the metabolic fiber type of a muscle.
Consistent with our findings, previous studies, including those that
first described the kinetic properties of muscle CPT I
(15), have also reported residual CPT activity in muscle
mitochondria exposed to high concentrations of malonyl-CoA.
Investigators have attributed this activity to inner mitochondrial
membrane CPT II, which can be exposed during mitochondrial isolation
(7). However, exposure of CPT II cannot explain our data
because, unlike previous investigations, the present study evaluated
malonyl-CoA inhibition of palmitate oxidation. We consider it unlikely
that mitochondrial damage occurred in a manner that differed
systematically between red and white muscle, and in such a way as to
permit CPT I-independent acyl-CoA entry into the matrix without
accompanying disruption of the -oxidative and TCA pathways. Thus our
observation that palmitate oxidation to CO2 was retained
indicates that mitochondria were physiologically intact. Additionally,
demonstration that palmitate oxidation was fully dependent on the
presence of both carnitine and CoA further confirms the integrity of
mitochondrial membranes.
Importantly, the present investigation evaluated the inhibitory effects of malonyl-CoA in whole muscle homogenates. This represents another key distinction between our data and previous studies, because skeletal muscle possesses two mitochondrial subpopulations, intermyofibrillar and subsarcolemmal, which exhibit distinct biochemical properties and respond differently to physiological stimuli (12). Thus, in contrast to previous studies, our experiments using whole homogenates eliminated potential problems associated with poor mitochondrial yield and disproportionate recovery of mitochondrial subpopulations, either of which might result in mischaracterization of muscle mitochondrial enzymes. In a homogenate system, both mitochondria and peroxisomes contribute to total oxidative capacity (18). We found that peroxisomes contributed equally (~19%) to total oxidation in red compared with white muscle, suggesting that differences in peroxisomal oxidation cannot explain fiber type-selective malonyl-CoA resistance.
Taken together, our findings provide strong evidence that red muscle
expresses a malonyl-CoA-resistant CPT I subfraction. CPT I has not
been isolated in a catalytically active form that would allow kinetic
characterization of the purified enzyme; however, the full-length cDNA
has been expressed in yeast (20) and mammalian COS cells
(25). The recombinant enzyme is completely inhibited by
1.0-10 µM malonyl-CoA (20, 25), which appears to
contradict our finding that, in RG, 10-100 µM malonyl-CoA
inhibited palmitate oxidation by only 35-62%. This discrepancy
suggests that red muscle might express a novel CPT I isoform that
confers a modified malonyl-CoA-regulatory site, a possibility that is
supported by evidence indicating that both rat (31) and
human (30) muscle expresses multiple mRNA splice variants
of the CPT I
transcript. The deduced polypeptide sequence of CPT
I
2, a novel mRNA species that is expressed in rat muscle
(31), predicts an isoform of the enzyme that omits putative malonyl-CoA- regulatory regions. In the present investigation, we found that both the CPT I
1 and CPT I
2 transcripts were about three times more abundant in red than in white muscles. To our knowledge, these data are the first to show that differences in CPT I
gene expression across muscle fiber types are consistent with similar
differences observed in enzyme activity and fatty acid oxidation rates.
However, relative expression of the two CPT I mRNA species was similar
in red and white muscles and thus appeared to be unrelated to fiber
type-specific differences in malonyl-CoA sensitivity. These results do
not exclude the possibility that protein expression of
2 relative to
1 might differ among muscle fiber types, although direct
demonstration that the CPT I
2 transcript is in fact translated into
a distinct isoenzyme is still lacking. Protein expression and
characterization of the catalytic and regulatory properties of novel
CPT1 splice variants should provide further insight into their
potential role in conferring malonyl-CoA insensitivity.
The emerging model of CPT I topology predicts that the catalytic site
resides in the large carboxy-terminal domain facing the cytosol and
that the smaller cytosolic amino-terminal domain is crucial for
maintaining a confirmation that permits optimal malonyl-CoA binding and
inhibition (10). When CPT I is expressed in yeast,
deletion of the conserved first 28 NH2-terminal amino acids
abolishes malonyl-CoA sensitivity and increases catalytic activity
2.5-fold, indicating that an intact NH2-terminal domain is
required for malonyl-CoA inhibition (20). Furthermore,
several investigators have proposed that it is the interaction between the COOH and NH2 domains that determines malonyl-CoA
sensitivity (25). According to this model,
posttranslational modification of either domain and/or other factors
that alter CPT I confirmation might contribute to physiological
modulation of malonyl-CoA sensitivity in vivo. Consistent with this
premise, malonyl-CoA sensitivity of hepatic CPT I decreases in response
to physiological stresses that increase
-oxidation (e.g., starvation
and diabetes) (5). The precise mechanism of this
desensitization is unknown, but several laboratories have implicated
changes in the lipid environment of the mitochondrial membrane
(2, 14). Although only limited data suggest that a similar
phenomenon might occur in muscle (10), membrane
phospholipid composition does differ markedly between red and white
fibers (1), thus leaving open the possibility that
distinctions in mitochondrial membrane properties might contribute to
fiber-specific variations in CPT I kinetics.
Voluminous evidence indicates that skeletal muscle, by virtue of its
highly heterogeneous composition, can vary considerably with respect to
its metabolic properties. Compared with white muscle, red muscle
exhibits greater insulin responsiveness and a higher capacity to
oxidize fatty acids (4, 6, 16). Clinical relevance of
these fiber-specific properties is strongly suggested by correlative
data linking a disproportionately high number of white muscle fibers to
metabolic disorders such as cardiovascular disease, obesity, and type
II diabetes (4, 24). Interestingly, these diseases have
also been associated with malonyl-CoA/CPT I-mediated alterations in
muscle lipid homeostasis (11, 19). By inhibiting CPT I and
preventing acyl-CoA entry into mitochondria, malonyl-CoA not only
decreases -oxidation but also increases muscle accumulation of
long-chain acyl-CoA (3). Because long-chain acyl-CoA and
its derivatives can serve as signaling and/or gene regulatory
molecules, this putative malonyl-CoA-acyl-CoA axis is thought to
mediate several physiological and pathophysiological processes
(19, 32). Thus, when taken together with the
aforementioned reports, our observation of fiber type-specific
malonyl-CoA resistance not only presents implications for muscle cell
functioning but also suggests a novel link between muscle fiber
composition and disorders of energy homeostasis.
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ACKNOWLEDGEMENTS |
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We thank Donghai Zheng for assistance with RNA isolations.
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FOOTNOTES |
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* These authors contributed equally to the study.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46121-06 (G. L. Dohm) and F32 DK-10017-01 (D. M. Muoio) and a grant from the North Carolina Institute of Nutrition (T. R. Koves).
Address for reprint requests and other correspondence: D. M. Muoio, PO Box 3327, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: muoio{at}duke.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.
First published December 11, 2001;10.1152/ajpendo.00233.2001
Received 29 May 2001; accepted in final form 6 December 2001.
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