Evidence of a malonyl-CoA-insensitive carnitine palmitoyltransferase I activity in red skeletal muscle

Jong-Yeon Kim1,*, Timothy R. Koves1,*, Geng-Sheng Yu3, Tod Gulick3, Ronald N. Cortright1, G. Lynis Dohm1, and Deborah M. Muoio1,2,*

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Carnitine palmitoyltransferase I (CPT I), which is expressed as two distinct isoforms in liver (alpha ) and muscle (beta ), 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 beta -oxidation proceeds despite malonyl-CoA concentrations that exceed the IC50 for CPT Ibeta . 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 Ibeta 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -oxidative pathway (13).

CPT I is expressed as at least two isoforms, which are the products of different genes: a liver enzyme, CPT Ialpha (~88 kDa), and its smaller counterpart in cardiac and skeletal muscle, CPT Ibeta (~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 beta -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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Ibeta 2 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Ibeta 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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Fig. 1.   Fiber type-dependent palmitate oxidation and malonyl-CoA resistance. RG, red gastrocnemius; WG, white gastrocnemius; SOL, soleus; EDL, extensor digitorum longus. Whole muscle homogenates were incubated for 1 h at 29°C in a medium containing 200 µM [14C]palmitic acid (0.1 µCi/well) in the presence or absence of 10 µM malonyl-CoA. Palmitate oxidation (nmol · g protein-1 · min-1) was determined by measuring production of 14C-labeled CO2 and acid-soluble metabolites (ASM) as described in METHODS. Data are means ± SE from 2-3 animals assayed in quadruplicate and presented as total oxidation (CO2 plus ASM, A), malonyl-CoA-resistant palmitate oxidation (total and CO2) in the presence of 10 µM malonyl-CoA, expressed relative to the activity at 0 µM malonyl-CoA (B), and relationship between malonyl-CoA resistance and total oxidation (C).

Figure 2 shows the results of separate experiments that were conducted to determine whether malonyl-CoA inhibition of palmitate oxidation remains linear during the course of a 1-h incubation. In this experiment, 10 µM of malonyl-CoA inhibited palmitate oxidation by 67 and 92% (P < 0.001) in homogenates of RG and WG, respectively, and the relative inhibition was similar (P = 0.71) at 15-, 30-, 45-, and 60-min time points.


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Fig. 2.   Time course of malonyl-CoA resistance in red and white muscle. Whole muscle homogenates from RG and WG gastrocnemius were incubated 15-60 min at 29°C in a medium containing 200 µM [14C]palmitic acid (0.1 µCi/well) in the presence or absence of 10 µM malonyl-CoA. Palmitate oxidation (nmol/g protein) was determined by measuring production of 14C-labeled CO2 and ASM, as described in METHODS. Data are means ± SE from 3 animals, assayed in quadruplicates, and were analyzed by 3-way ANOVA. * P < 0.001, time points at which relative malonyl-CoA inhibition is significantly different between red and white muscles.

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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Table 1.   Peroxisomal and carnitine-dependent palmitate oxidation in muscle homogenates

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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Fig. 3.   Malonyl-CoA inhibition of palmitate oxidation in red and white muscles. Whole muscle homogenates were incubated for 1 h at 29°C in a medium containing 200 µM [14C]palmitic acid (0.1 µCi/well) in the presence of 0-100 µM malonyl-CoA. Palmitate oxidation (nmol · g protein-1 · min-1) was determined by measuring production of 14C-labeled CO2 as described in METHODS and is presented as absolute rates of palmitate oxidized to CO2 (A), relative inhibition expressed as a percentage of the total activity at 0 µM malonyl-CoA (B), and relative inhibition expressed as a percentage of the total activity after subtraction of the residual activity at 100 µM malonyl-CoA. Data are means ± SE from 10 animals, assayed in quadruplicates, and were analyzed by 2-way ANOVA. Significant differences (* P < 0.001) between red and white muscles.



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Fig. 4.   Malonyl-CoA inhibition of carnitine palmitoyltransferase I (CPT I) activity in red and white muscles. Malonyl-CoA inhibition of CPT I activity (nmol · g protein-1 · min-1) was measured in muscle homogenates of red soleus (SOL) and white epitrochlears (EPI) muscles, as described in METHODS. Data are means ± SE from 10 animals assayed in triplicate. Values that are significantly different compared with controls (without malonyl-CoA) (* P < 0.01) and/or between red and white muscles (§P < 0.01) were analyzed by 1-way ANOVA.

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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Fig. 5.   Malonyl-CoA inhibition of palmitate oxidation in mitochondria from red and white muscles. Mitochondria isolated from homogenates of red and white gastrocnemius were incubated for 10 min at 29°C in a in a modified Chappell-Perry buffer containing 200 µM [14C]palmitic acid (0.1 µCi/well) in the presence or absence of 10 µM malonyl-CoA, 1 mM L-carnitine, or 50 µM CoA. Palmitate oxidation (nmol · g protein-1 · min-1) was determined by measuring production of 14C-labeled CO2, as described in METHODS. Data are means ± SE from 5 animals assayed in quadruplicates. Values that are significantly different compared with controls (* P < 0.01) and/or between red and white muscles (§P < 0.01) were analyzed by 2-way ANOVA.

Additionally, others have suggested that contamination of mitochondria with sarcoplasmic reticulum (SR) might contribute to changes in malonyl-CoA sensitivity (17). We evaluated this possibility by assessing the presence of the SR-specific marker protein calnexin in mitochondrial compared with microsomal subfractions. Western blot analyses indicated that SR contamination was similarly low in mitochondria from red and white muscles (Fig. 6A). Taken together, the data shown in Figs. 5 and 6 provide evidence that mitochondrial CPT I contributes to malonyl-CoA resistance in red muscle and that this fiber type-dependent property is unlikely to be an artifact due to SR contamination. Interestingly, we found that the CPT I specific activity in microsomes was similar to that in mitochondria (Fig. 6B). Palmitate oxidation to CO2 was undetectable in our microsomal preparations, indicating negligible levels of mitochondrial contamination (data not shown). The high CPT I activities in muscle microsomes, which is consistent with previous observations in liver microsomes, indicate that both mitochondrial and SR CPT I could have contributed to malonyl-CoA resistance in our homogenate system. How acylcarnitines that are synthesized in extramitochondrial organelles might be subsequently shuttled to the mitochondria for beta -oxidation remains uncertain.


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Fig. 6.   Microsomal CPT I activity in red and white muscles. A: microsomal and mitochondrial protein (15 µg) from RG and WG gastrocnemius was separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, probed with an antibody against the sarcoplasmic reticulum (SR) marker protein calnexin, and visualized using a chemiluminescence Western blot detections kit. B: mitochondrial and microsomal subfractions were prepared by differential centrifugation and then used immediately for assaying CPT I activity (µmol · g protein-1 · min-1), as described in METHODS. Data are means ± SE from 3 animals, assayed in triplicate, and were analyzed by 2-way ANOVA. Values that are significantly different (§P < 0.05) between red and white muscles.

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 Ibeta 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 Ibeta 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 Ibeta 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 Ibeta 1 to CPT Ibeta 2 abundance was similar between red and white muscles, mRNA expression of CPT Ibeta 2 appeared to be unrelated to fiber type-selective differences in malonyl-CoA resistance. In separate experiments, we used cRNA probe for CPT Ialpha and found that abundance of this isoform was negligible in skeletal muscle (data not shown), as previously reported (7).


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Fig. 7.   Fiber type-dependent mRNA expression of CPT Ibeta splice variants. Alternatively spliced CPT Ibeta mRNAs in red and white rat skeletal muscle. A: location of the cRNA probe used in ribonuclease protection assay (RPA) analyses is superimposed on a schematic of a partial CPT I mRNA sequence. White lines indicate splice junctions, and lengths of protected mRNA fragments are shown. B: expression of CPT Ibeta 1 shown from short-exposure RPAs performed with total RNA from rat EDL, RG, SOL, and WG muscles and mixed skeletal muscle from Clonetics (SkM) by use of the cRNA probe shown in A. C: expression of CPT Ibeta 2 shown from a longer exposed RPA performed with total RNA described in B by use of the cRNA probe shown in A. Protected fragments corresponding to each isoform are indicated to the right of each panel.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -oxidation (3). Unexplained, however, is how fatty acid oxidation proceeds despite muscle concentrations of malonyl-CoA that should completely inhibit CPT Ibeta . 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 beta -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 Ibeta 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 Ibeta transcript. The deduced polypeptide sequence of CPT Ibeta 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 Ibeta 1 and CPT Ibeta 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 beta 2 relative to beta 1 might differ among muscle fiber types, although direct demonstration that the CPT Ibeta 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 Ibeta 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 beta -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 beta -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.


    ACKNOWLEDGEMENTS

We thank Donghai Zheng for assistance with RNA isolations.


    FOOTNOTES

* 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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