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AJP-Endo Articles in PresS. Published on December 11, 2001 as DOI 10.1152/ajpendo.00233.2001 E-0233-2001-R2 Evidence of a malonyl-CoA-insensitive carnitine palmitoyltransferase 1 activity in red skeletal muscle Jong-Yeon Kim*†, Timothy R. Koves*†, Geng-Sheng Yu§ , Tod Gulick§, Ronald N. Cortright*, G. Lynis Dohm* and Deborah M. Muoio*‡† * Departments of Biochemistry and Physiology, East Carolina University, Greenville, NC, 27858 ‡ Department of Medicine, Duke University Medical School, Durham NC, 27710 § Diabetes Research Laboratory, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Charlestown MA, 02129 † authors contributed equally to the manuscript Short title: Malonyl-CoA resistance in red muscle Corresponding author: Deborah M. Muoio, PhD PO Box 3327 Duke University Medical Center Durham, NC 27710 Tel. 919-684-3644 FAX 919-684-8907 Email: [email protected] Copyright 2001 by the American Physiological Society. E-0233-2001-R2 2 ABSTRACT CPT1, which is expressed as two distinct isoforms in liver (α) and muscle (β), catalyzes the rate-limiting step in the transport of fatty acid into the mitochondria. Malonyl-CoA, a potent inhibitor CPT1, is considered a key regulator of fatty acid oxidation in both tissues. Still unanswered is how muscle β-oxidation proceeds despite malonyl-CoA concentrations that exceed the IC50 for CPT1β. We evaluated malonyl-CoA suppressible [14C]palmitate oxidation and CPT1 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 29%, 39%, 60%, and 89% in RG, SOL, EDL and WG, respectively. Thus, malonylCoA resistance, which correlated strongly (0.678) with absolute oxidation rates (RG>SOL>EDL>WG), was greater in red than in white muscles. Similarly, malonyl-CoAresistant palmitate oxidation and CPT1 activity were greater in mitochondria from RG compared to WG. Ribonuclease protection assays were performed to evaluate whether our data might be explained by differential expression of CPT1 splice variants. We detected the presence of two CPT1β splice variants that were more abundant in red compared to 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 CPT1 activity in red muscle, suggesting fibertype specific expression of distinct CPT1 isoforms and/or post-translational modulations that have yet to be elucidated. Key words: fatty acid oxidation, fiber-type specificity E-0233-2001-R2 3 Abbreviations: ACC, acyl-CoA carboxylase; ACS, acyl-CoA synthetase; ASM, acid soluble metabolites; CPT1, carnitine palmitoyltransferase 1; EPI, epitrochleas; EDL, extensor digitorum longus; RG, red gastrocnemius; SR, sarcoplasmic reticulum; SOL, soleus; WG, white gastrocnemius. E-0233-2001-R2 4 INTRODUCTION 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 plays an obligatory role in βoxidation by permitting acyl-CoA translocation from the cytosol into the mitochondria in all mammalian cells (10). Carnitine palmitoyltransferase 1 (CPT1), 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 CPT1 traverse the inner membrane via a specific translocase that is coupled to carnitine palmitoyltransferase 2 (CPT2), which regenerates acyl-CoA upon transporting the fatty acyl groups into the mitochondrial matrix. Because CPT1 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 β-oxidative pathway (13). CPT1 is expressed as at least two isoforms that are the products of different genes; a liver enzyme (CPT1α) (~88 kDa) and its smaller counterpart in cardiac and skeletal muscle (CPT1β) (~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 β-oxidation. For example, starvation (15) and exercise (28) decrease tissue levels of malonyl-CoA, which presumably relieves inhibition of CPT1 and increases fatty acyl-CoA entry into mitochondria (3). Conversely, carbohydrate feedings stimulate ACC activity and increase production of E-0233-2001-R2 5 malonyl-CoA, which corresponds with a decrease in fatty acid oxidation and an increase in longchain 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 malonylCoA-mediated control of fatty acid metabolism might depend on muscle fiber-type. However, the role of fiber-type in modulating the malonyl-CoA/CPT1 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 CPT1 activity at all times. This is because the muscle isoform of CPT1 is approximately 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 non-suppressible fraction to CPT2, 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 CPT1 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 CPT1 activity in whole homogenates and isolated mitochondria that were prepared from red and white skeletal E-0233-2001-R2 6 muscles. The results provide evidence of a malonyl-CoA insensitive CPT1 fraction that is predominately active in red muscle. METHODS Materials. The 3H-carnitine and the chemiluminescent detection kit were from Amersham, (Piscataway, NJ). The calnexin antibody was from Santa Cruz (Santa Cruz, CA) and the polyvinylidene fluoride membrane (PVDF) 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 anesthestized 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 muscles (EDL) and epitrochlears (EPI). Approximately 50-70 mg of tissue was minced thoroughly with scissors in 300 µL of a modified sucrose-EDTA medium (SET) containing 250 mM sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4 (21). SET buffer was added to a 20-fold diluted (m:v) suspension and samples were homogenized in 3.0 mL Potter-Elvehjem glass homogenizer at 10 passes across 30 seconds at 1200 RPM with a Teflon pestle. Protein concentrations in muscle homogenates ranged from 0.9 to 2.3 mg/ml. E-0233-2001-R2 7 Isolation of Mitochondria and Microsomes. Muscles were excised and immediately placed in ice-cold modified Chapell-Perry buffer (100 mM KCL, 40 mM Tris-HCl, 10 mM Tris-Base, 5mM MgSO4, 1mM EDTA, 1mM 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 (w:v) with Chapell-Perry buffer and then homogenized 2 x 15 sec using an Ultra-turrax at approximately 9,500 rpm. Tissue homogenates were centrifuged at 650 x g for 10 min at 4°C. The supernatant was gravity filtered through 4 layers of surgical gauze and centrifuged at 8,500 x g for 10 min at 4°C. Microsomes were isolated from the supernatant by ultracentrifugation at 100,000 x g for 1 h at 4°C. The mitochondrial pellet from the 8,500 x g spin was washed to remove erythrocytes, resuspended in 1.3 ml Chapell-Perry buffer, and centrifuged at 8,500 x g for 10 min. Both the microsomal and mitochondrial pellets were suspended in 1.0 ml SET buffer and used immediately for assaying CPT1 specific activity and palmitate oxidation. Proteins were determined by the BCA method. For Western blots, 15 µg protein from mitochondrial and microsomal subfractions were separated by 8-12% gradient SDS-PAGE, transferred onto PVDF membranes and probed 2 h with calnexin antibody per the manufacturer’s protocol. Proteins were visualized by horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G with a chemiluminescent Western blotting detection kit. Fatty acid oxidation. 14 Palmitate oxidation rates were determined by measuring production of C-labeled acid soluble metabolites (ASM), a measure of TCA cycle intermediates and acetyl esters (incomplete oxidation) (27), and [14C]CO2 (complete oxidation) using a modified 48-well E-0233-2001-R2 8 microtiter plate (Costar, Cambridge, MA) as previously described (11). Reactions were initiated by adding 40 µl whole homogenates or isolated mitochondria to 160 ul of the incubation buffer (pH 7.4), yielding final concentrations of 0.2 mM palmitate ([1-14C]palmitate at 0.5 µCi/ml), 100 mM sucrose, 10 mM Tris.HCl, 5 mM potassium phosphate, 80 mM potassium chloride, 1 mM magnesium chloride, 2 mM L-carnitine, 0.1 mM malate, 2 mM ATP, 0.05 mM coenzyme A, 1 mM dithiothreitol, 0.2 mM EDTA and 0.5% bovine serum albumin. After incubating 60 min at 30°C, reactions were terminated by adding100 µl of 4 N sulfuric acid and the CO2 produced during the incubation was trapped in 200 µl of 1N sodium hydroxide that had been added to adjacent wells (11). The acidified media was stored at 4°C overnight and then ASM were assayed in supernatants of the acid precipitate (27). Radioactivity of CO2 and ASM were determined by liquid scintillation counting using 4 ml Uniscint BD (National Diagnostics). Carnitine palmitoyltransferase I Assay. CPT1 activity was measured using 10-20 µg mitochondrial or microsomal protein, or 40-60 µg 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. RPA analysis. RNA was isolated from skeletal muscle using TRIzol (Gibco-BRL) reagent as previously described (9). Ribonuclease protection assays were performed as described in (31) using a complementary RNA probe generated by T7 polymerase from a linearized template E-0233-2001-R2 9 consisting of the rat CPT-Iβ2 cDNA fragment extending from NcoI at nucleotide 238 (relative to the initiation AUG) to EcoRI at nucleotide 423 subcloned into pBluescipt. 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 using a bivariate linear regression analysis and significance was determined by ANOVA. Twoand three-way ANOVA were performed using a Standard Least Squares model to test both the main and interaction effects of muscle type x incubation time x malonyl-CoA concentration (where appropriate) on palmitate oxidation, CPT1 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 Malonyl-CoA inhibition of palmate 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) (Figure 1A). Adding 10 µM malonyl-CoA, which exceeds the IC50 of rCPT1 by ~100-fold, inhibited palmitate oxidation 29%, 39%, 60% and 89% in RG, SOL, EDL and WG, respectively (Figure 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 E-0233-2001-R2 10 resistance correlated positively (R2=0.678, P<0.005) with absolute rates of palmitate oxidation (Figure 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 fibertype specific malonyl-CoA resistance was not due to in vitro protease modification of CPT1 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. 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 one-hour incubation. In this experiment, 10 µM malonyl-CoA inhibited palmitate oxidation 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-minute time points. 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 red and white gastrocnemius, peroxisomes accounted for approximately 20% of total palmitate oxidation (Table 1), similar to previous reports (18). These results suggest that fiber-type-dependent differences in malonylCoA 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 E-0233-2001-R2 11 mitochondrial membrane integrity was not compromised during homogenate preparations. Similarly, subtracting carnitine from the buffer diminished production of ASM 90%, however, since a small portion of the [14C]-labeled acid soluble products appeared to be carnitineindependent, oxidation results from subsequent experiments are presented as CO2 only. Malonyl-CoA sensitivity in homogenates of red and white gastrocnemius. 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 red and white gastrocnemius (Figure 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) (Figure 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 (Figure 3C), suggesting differences in efficacy but not the potency of malonyl-CoA inhibition. Figure 4 shows that CPT1 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 CPT1 activity 62% compared to 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 CPT1 activity is consistent with inhibition of palmitate oxidation, lending further support to our hypothesis that malonyl-CoA inhibition of muscle CPT1 depends on fiber composition. Malonyl-CoA sensitivity and palmitate oxidation in isolated mitochondria. Previous reports have suggested that the non-suppressible component of CPT1 activity might be related to E-0233-2001-R2 12 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 (Figure 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 to WG (Figure 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 CPT1, 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. 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 to microsomal subfractions. Western blot analyses indicated that SR contamination was similarly low in mitochondria from red and white muscles (Figure 6a). Taken together, the data shown in Figures 5 and 6 provide evidence that mitochondrial CPT1 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 CPT1 specific activity in microsomes was similar to that in mitochondria (Figure 6b). Palmitate oxidation to CO2 was undetectable in our microsomal preparations, indicating negligible levels of mitochondrial contamination (data not shown). The high CPT1 activities in muscle microsomes, which is consistent with previous observations in liver microsomes, indicate that both mitochondrial and SR CPT1 could have contributed to malonyl-CoA resistance in our E-0233-2001-R2 13 homogenate system. How acylcarnitines that are synthesized in extramitochondrial organelles might be subsequently shuttled to the mitochondria for β-oxidation remains uncertain. Expression of CPT1 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 CPT1β 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 (Figure 7). Expression of the predominant splice variant, CPT1β1, was approximately 3-fold greater in red muscles (SOL and RG) than in white muscles (EDL and WG) (Figure 7B). Likewise, expression of the alternatively spliced mRNA species, CPT1β2, was also more abundant in red muscles (Figure 7C). Thus, CPT1 gene expression across fibertypes is consistent with the higher enzyme activity and fatty acid oxidative capacity measured in red compared to white muscles. However, since the ratio of CPT1β1 to CPT1β2 abundance was similar between red and white muscles, mRNA expression of CPT1β2 appeared to be unrelated to fiber-type selective differences in malonyl-CoA resistance. In separate experiments using a cRNA probe for CPT1α, this mRNA showed very low abundance in skeletal muscle (data not shown), as previously reported (7). DISCUSSION Malonyl-CoA is presumed to be a key regulator of muscle fatty acid oxidation by virtue of its potent inhibition of CPT1, and because muscle malonyl-CoA content changes reciprocally with β-oxidation (3). Unexplained, however, is how fatty acid oxidation proceeds despite E-0233-2001-R2 14 muscle concentrations of malonyl-CoA that should completely inhibit CPT1β. This enigma might be at least partly reconciled by our data, which provide evidence of a malonyl-CoA insensitive CPT1 activity in red skeletal muscle. Similar to previous reports, we found that in homogenates of WG, physiological concentrations of malonyl-CoA inhibited palmitate oxidation 75-90%. Conversely, in RG, these same concentrations inhibited palmitate oxidation only 3554%. Further, 100 µM malonyl-CoA, which exceeds the reported IC50 by several orders of magnitude, inhibited palmitate oxidation only 62%, suggesting that CPT1 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 Figure 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-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 CPT1 subfraction might be E-0233-2001-R2 15 more closely linked to type IIA than type I fibers, although we acknowledge that this result should be interpreted cautiously due to the small number of animals that were 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 CPT1 (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 CPT2, which can be exposed during mitochondrial isolation (7). However, exposure of CPT2 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 CPT1-independent acyl-CoA entry into the matrix without accompanying disruption the β-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. E-0233-2001-R2 16 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 (approximately 19%) to total oxidation in red compared to 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 CPT1 subfraction. CPT1β 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 only 35-62%. This discrepancy suggests that red muscle might express a novel CPT1 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 CPT1β transcript. The deduced polypeptide sequence of CPT1β2, a novel mRNA species that is E-0233-2001-R2 17 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 CPT1β1 and CPT1β2 transcripts were ~3 times more abundant in red than in white muscles. To our knowledge, these data are the first to show that differences in CPT1 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 CPT1 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 β2 relative to β1 might differ among muscle fiber-types, although direct demonstration that the CPT1β2 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 CPT1 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 CPT1β is expressed in yeast, deletion of the conserved first 28 N-terminal amino acids abolishes malonyl-CoA sensitivity and increases catalytic activity 2.5-fold, indicating that an intact N-terminal domain is required for malonyl-CoA inhibition (20). Further, several investigators have proposed that it is the interaction between the C- and N-domains that determines malonyl-CoA sensitivity (25). According to this model, post-translational modifications of either domain and/or other factors that alter CPT1 confirmation might contribute to physiological modulation of malonyl-CoA sensitivity in vivo. Consistent with this E-0233-2001-R2 18 premise, malonyl-CoA sensitivity of hepatic CPT1 decreases in response to physiological stresses that increase β-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 CPT1 kinetics. Voluminous evidence indicates that skeletal muscle, by virtue of its highly heterogenous composition, can vary considerably with respect to its metabolic properties. Compared to 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/CPT1-mediated alterations in muscle lipid homeostasis (11;19). By inhibiting CPT1 and preventing acyl-CoA entry into mitochondria, malonyl-CoA not only decreases β-oxidation but also increases muscle accumulation of longchain 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 E-0233-2001-R2 19 resistance not only presents implications for muscle cell functioning, but also suggests a novel link between muscle fiber composition and disorders of energy homeostasis. E-0233-2001-R2 ACKNOWLEDGEMENTS This work was supported by DK 46121-06 (GLD) and F32DK 10017-01 (DMM) from the National Institutes of Health, and the North Carolina Institute of Nutrition (TMK). We thank Donghai Zheng for assistance with RNA isolations. 20 E-0233-2001-R2 21 REFERENCES 1. Blackard, W.G., J. Li, J.N. Clore, and W.B. Rizzo. Phospholipid fatty acid composition in type I and type II rat muscle. Lipids 32: 193-198, 1997. 2. Broadway, N.M. and E.D. Saggerson. Effect of membrane environment on the activity and inhabitability by malonyl-CoA of the carnitine acyltransferase of hepatic microsomal membranes. Biochem. J. 322: 435-440, 1997. 3. Chien, D., D. Dean, A.K. Saha, J.P. Flatt, and N.B. Ruderman. Malonyl-CoA content and fatty acid oxidation in rat muscle and liver in vivo. Am. J. Physiol. 279: E259-E655, 2000. 4. Cortright, R.N., D.M. Muoio, and G.L. Dohm. Skeletal muscle lipid metabolism: A fronteir for new insights into fuel homeostasis. Nutr. Biochem. 8: 228-245, 1997. 5. Drynan, L., P.A. Quant, and V.A. Zammit. The role of changes in the sensitivity of hepatic mitochondrial overt carnitine palmitoyltransferase in determining the onset of the ketosis of starvation in the rat. Biochem. J. 318: 767-770, 1996. 6. Dyck, D.J., S.J. Peters, J. Glatz, J. Gorski, H. Keizer, B. Kiens, S. Liu, E.A. Richter, L.L. Spriet, d. van, V, and A. Bonen. Functional differences in lipid metabolism in resting skeletal muscle of various fiber types. Am. J. Physiol. 272: E340-E351, 1997. 7. Esser, V., N.F. Brown, A.T. Cowan, D.W. Foster, and J.D. McGarry. Expression of cDNA isolated from rat brown adipose tissue and heart identifies the product as the muscle isoform of carnitine palmitoyltransferase I (M-CPT I). M-CPT I is the predominant CPT I E-0233-2001-R2 22 isoform expressed in both white (epididymal) and brown adipocytes. J. Biol. Chem. 271: 6972-6977, 1996. 8. Iijima, H., T. Fujino, H. Minekura, H. Suzuki, M.J. Kang, and T. Yamamoto. Biochemical studies of two rat acyl-CoA synthetases, ACS1 and ACS2. Eur. J. Biochem. 242: 186-190, 1996. 9. Jones, J.P. and G.L. Dohm. Regulation of glucose transporter GLUT-4 and hexokinase II gene transcription by insulin and epinephrine. Am. J. Physiol. 273: E682-E687, 1997. 10. Kerner, J. and C. Hoppel. Fatty acid import into mitochondria. Biochim. Biophys. Acta. 1486: 1-17, 2000. 11. Kim, J.-Y., R.C. Hickner, R.N. Cortright, G.L. Dohm, and J.A. Houmard. Lipid oxidation is reduced in obese human skeletal muscle. Am. J. Physiol. 297: E1039-E1044, 2000. 12. Lombardi, A., M. Damon, A. Vincent, F. Goglia, and P. Herpin. Characterization of oxidative phosphorylation in skeletal muscle mitochondria subpopulations in pig: a study using top-down elasticity analysis. FEBS Lett. 475: 84-88, 2000. 13. McGarry, J.D. and N.F. Brown. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur. J. Biochem. 244: 1-14, 1997. 14. McGarry, J.D. and N.F. Brown. Reconstitution of purified, active and malonyl-CoAsensitive rat liver carnitine palmitoyltransferase I: relationship between membrane environment and malonyl-CoA sensitivity. Biochem. J. 349: 179-187, 2000. E-0233-2001-R2 23 15. McGarry, J.D., S.E. Mills, C.S. Long, and D.W. Foster. Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Biochem. J. 214: 21-28, 1983. 16. Megeney, L.A., P.D. Neufer, G.L. Dohm, M.H. Tan, C.A. Blewett, G.C.B. Elder, and A. Bonen. Effects of muscle activity and fiber composition on glucose transport and GLUT-4. Am. J. Physiol. 264: E583-E5931993. 17. Niot, I., F. Pacot, P. Bouchard, J. Gresti, A. Bernard, J. Bezard, and P. Clouet. Involvement of microsomal vesicles in part of the sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition in mitochondrial fractions of rat liver. Biochem. J. 304: 577-584, 1994. 18. Piot, C., J.H. Veerkamp, C. Bauchart, and J. Hocquette. Contribution of mitochondria and peroxisomes to palmitate oxidation in rat and bovine tissues. Comp. Biochem. Physiol. 121: 185-194, 1998. 19. Ruderman, N.B., A.K. Saha, D. Vavvas, and L.A. Witters. Malonyl-CoA, fuel sensing, and insulin resistance. Am. J. Physiol. E1-E18, 1998. 20. Shi, J., H. Zhu, D.N. Arvidson, and G. Woldegiorgis. The first 28 N-terminal amino acid residues of human heart muscle carnitine palmityoltransferase I are essential for malonylCoA sensitivity and high-affinity binding. Biochemistry 39: 712-717, 2000. 21. Sholte, H., Y. Yu, J. Ross, H. Oosterkamp, A. Boonman, and H. Busch. Rapid isolation of muscle and heart mitochondria, the liability of oxidative phosphorylation and attempts to E-0233-2001-R2 24 stabilize the process in vitro by taurine, carnitine and other compounds. Mol. Cell. Biochem. 174: 61-66, 1997. 22. Staron, R.S., W.J. Kraemer, R.S. Hikida, A.C. Fry, J.D. Murray, and G.E. Campos. Fiber type composition of four hindlimb muscles of adult Fisher 344 rats. Histochem. Cel.l Biol. 111: 117-123, 1999. 23. Starritt, E.C., R.A. Howlett, G.J. Heigenhauser, and L.L. Spriet. Sensitivity of CPT I to malonyl-CoA in trained and untrained human skeletal muscle. Am. J. Physiol. 278: E462E468, 2000. 24. Storlien, L.H., D.A. Pan, A.D. Kriketos, J. O'Connor, I.D. Caterson, G.J. Cooney, A.B. Jenkins, and L.A. Baur. Skeletal muscle membrane lipids and insulin resistance. Lipids Suppl: S261-S265, 1996. 25. Swanson, S.T., D.W. Foster, J.D. McGarry, and N.F. Brown. Roles of the N- and Cterminal domains of carnitine palmitoyltransferase I isoforms in malonyl-CoA sensitivity of the enzymes: insights from expression of chimaeric proteins and mutation of conserved histidine residues. Biochem. J. 335: 513-519, 1998. 26. Trumble, G.E., M.A. Smith, and W.W. Winder. Purification and characterization of rat skeletal muscle acetyl-CoA carboxylase. Eur. J. Biochem. 231: 192-198, 1995. 27. Veerkamp, J.H., H. van Moerkerk, J.F. Glatz, J. Zuurveld, A. Jacobs, and A.J.M. Wagenmakers. 14CO2 production is no adequate measure of [14C]fatty acid oxidation. Biochem. Med. Met. Biol. 35: 248-259, 1984. E-0233-2001-R2 25 28. Winder, W.W. and D.G. Hardie. Activation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am. J. Physiol. 270: E299-E304. 1996. 29. Winder, W.W., B.F. Holmes, D.S. Rubink, J.O. Holloszy, and M. Chen. Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle. J. Appl. Physiol. 88: 2219-2226, 2000. 30. Yu, G., Y. Lu, and T. Gulick. Expression of novel isoforms of carnitine palmitoyltransferase I (CPT-I) generated by alternative splicing of the CPT-Iβ gene. Biochem. J. 334: 225-231, 1998. 31. Yu, G., Y. Lu, and T. Gulick. Rat carnitine palmitoyltransferase I β mRNA splicing isoforms. Biochim. Biophys. Acta. 1393: 166-172, 1998. 32. Zammit, V.A. The malonyl-CoA-long-chain acyl-CoA axis in the maintenance of mammalian cell function. Biochem. J. 343: 505-515, 1999. E-0233-2001-R2 26 Figure Legends Figure 1. Fiber-type dependent palmitate oxidation and malonyl-CoA resistance. Whole muscle homogenates were incubated 1 h at 29oC in a media containing 200 µM [14C]palmitic acid (0.1µCi/well) in the presence or absence of 10µM malonyl-CoA. Palmitate oxidation (nmol/g protein/min) was determined by measuring production of 14C-labeled CO2 and ASM as described in METHODS. Data are means ± SEM from 2-3 animals assayed in quadruplicates and presented as (A) Total oxidation (CO2 plus ASM) (B) 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, and (C) relationship between malonyl-CoA resistance and total oxidation. Figure 2. Time course of malonyl-CoA resistance in red and white muscle. Whole muscle homogenates from red (RG) and white (WG) gastrocnemius were incubated 15-60 min at 29oC in a media containing 200 µM [14C]palmitic acid (0.1µCi/well) in the presence or absence of 10µM malonyl-CoA. production of 14 Palmitate oxidation (nmol/g protein) was determined by measuring C-labeled CO2 and ASM as described in METHODS. Data are means ± SEM from 3 animals, assayed in quadruplicates, and were analyzed by three-way ANOVA. Time points at which relative malonyl-CoA inhibition is significantly different (P<0.001) between red and white muscles are indicated by *. Figure 3. Malonyl-CoA inhibition of palmitate oxidation in red and white muscles. Whole muscle homogenates were incubated 1 h at 29oC in a media containing 200 µM [14C]palmitic E-0233-2001-R2 27 acid (0.1µCi/well) in the presence of 0-100 µM malonyl-CoA. Palmitate oxidation (nmol/g protein/min) was determined by measuring production of 14 C-labeled CO2 as described in METHODS and is presented as (A) Absolute rates of palmitate oxidized to CO2, (B) Relative inhibition expressed as a percent of the total activity at 0 uM malonyl-CoA, and (C) Relative inhibition expressed as a percent of the total activity after subtracting the residual activity at 100 µM malonyl-CoA. Data are means ± SEM from 10 animals, assayed in quadruplicates, and were analyzed by two-way ANOVA. Significant differences (P<0.001) between red and white muscles are indicated by *. Figure 4. Malonyl-CoA inhibition of carnitine palmitoyltransferase I activity in red and white muscles. Malonyl-CoA inhibition of CPT1 activity (nmol/g protein/min) was measured in muscle homogenates of red soleus (SOL) and white epitrochlears (EPI) muscles as described in the METHODS. Data are means ± SEM from 10 animals assayed in triplicate. Values that are significantly different compared to controls (without malonyl-CoA) * and/or between red and white muscles § (P<0.01) were analyzed by one-way ANOVA. Figure 5. Malonyl-CoA inhibition of palmitate oxidation in mitochondria from red and white muscles. Mitochondria isolated from homogenates of red and white gastrocnemius were incubate 10 min at 29oC in a in a modified Chappell-Perry buffer containing 200 µM [14C]palmitic acid (0.1µCi/well) in the presence or absence of 10 µM malonyl-CoA, 1 mM LCarnitine or 50 µM coenzyme A. Palmitate oxidation (nmol/g protein/min) was determined by measuring production of 14C-labeled CO2 as described in Methods. Data are means ± SEM from E-0233-2001-R2 28 5 animals assayed in quadruplicates. Values that are significantly different compared to controls * and/or between red and white muscles § (P<0.01) were analyzed by two-way ANOVA. Figure 6. Microsomal carnitine palmitoyltransferase I activity in red and white muscles. A) Microsomal and mitochondrial protein (15 µg) from red (RG) and white (WG) gastrocnemius was separated by SDS-PAGE, transferred onto polyvinylidene fluoride membranes, probed with an antibody against the 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 CPT1 activity (µmol/g protein/min) as described in METHODS. Data are means ± SEM from 3 animals, assayed in triplicate, and were analyzed by two-way ANOVA. Values that are significantly different (P<0.05) between red and white muscles are indicated by §. Figure 7. Fiber-type dependent mRNA expression of CPT1β splice variants. Alternatively spliced CPT1β mRNAs in red and white rat skeletal muscle. (A) Location of the cRNA probe used in RPA analyses is superimposed on a schematic of a partial CPT1 mRNA sequence. White lines indicate splice junctions and lengths of protected mRNA fragments are shown. (B) Expression of CPT1β1 shown from short exposure RPAs performed with total RNA from rat extensor digitorum longus (EDL), red gastrocnemius (RG), soleus (SOL) and white gastrocnemius (WG), and mixed skeletal muscle from Clonetics (SkM) using the cRNA probe shown in A. (C). Panel 3 represents expression of CPT1β2 shown from a longer exposed RPA performed with total RNA described in B using the cRNA probe shown in A. Protected fragments corresponding to each isoform are indicated to the right of each panel. E-0233-2001-R2 29 E-0233-2001-R2 30 Red Gastrocnemius White Gastrocnemius palmitate oxidation (nmol/g/h) ASM CO2 ASM CO2 2615 ± 270 969 ± 79 1137 ± 110 682 ± 60 ETS inhibitors 686 ± 43 ND 350 ± 56 ND no carnitine 281 ± 31 ND 175 ±25 ND control Table 1 . Peroxisomal and carnitine-dependent palmitate oxidation in muscle homogenates. Whole muscle homogenates were incubated 1 h at 29oC in a media containing 200 µM [14C]palmitic acid (0.1µCi/well) in the presence or absence of mitochondrial electron transport sytem (ETS) inhibitors (2.2mM KCN and 40mg/L rotenone) and 1 mM L-carnitine. Production of 14 C-labeled CO2 and ASM were assayed as described in Methods. Data are means ± SEM from 3 animals assayed in quadruplicates. background could not be detected. ND indicates that [14C]CO2 production over (Percent activity at 10 µM malonyl-CoA) Malonyl-CoA-resistant palmitate oxidation Palmitate oxidation (nmol/g/min) E-0233-2001-R2 31 Figure 1. A. 60 50 40 30 20 10 0 RG RG SOL SOL Muscle EDL 100 EDL WG Muscle B. Total CO2 80 60 40 20 0 WG E-0233-2001-R2 32 Figure 1. Malonyl-CoA-resistant palmitate oxidation (Percent activity at 10 µM malonyl-CoA) C. 100 R2= 0.678 80 60 40 20 0 10 20 30 40 50 Total Palmitate oxidation (nmol/g/min) 60 E-0233-2001-R2 33 Figure 2. 300 RG control RG 10 µM mal-CoA Palmitate oxidation (nmol/g) 250 WG control WG 10 µM mal-CoA 200 150 100 50 * 0 15 * * * 30 45 60 Time (min) E-0233-2001-R2 34 Figure 3. red white 15 (nmol/g/min) Palmitate Oxidation to CO2 A. 10 * * * * 5 0 20 40 * 60 80 100 Malonyl-CoA (µM) 100 (%of control) Palmitate Oxidation to CO2 B. red white 75 * * 50 * * * 25 0 20 40 60 80 Malonyl CoA (µM) 100 E-0233-2001-R2 35 Figure 3. C. Palmitate oxidation to CO2 (% of control) 100 red white 80 60 40 20 0 0 10 20 30 40 Malonyl-CoA (µM) 50 E-0233-2001-R2 36 Carnitine palmitoyltransferase I activity (nmo/g/min) Figure 4. 70 control 60 10 µM malonyl-CoA 50 * § 40 30 20 *§ 10 0 SOL EPI E-0233-2001-R2 37 Figure 5. red (nmol/mg/min Palmitate oxidation to CO2 1.60 1.20 0.80 white § * 0.40 *§ 0 control 10 µM malonylCoA * * minus carnitine * * minus CoA E-0233-2001-R2 38 Figure 6. A. RG mt mcs WG mt mcs Carnitine palmitoyltransferase I activity (µmol/g/min) B. 7.0 mitochondria 6.0 microsomes 5.0 4.0 § 3.0 § 2.0 1.0 0 RG WG E-0233-2001-R2 39 Figure 7. A EcoRI NcoI 174 β1 β2 ATG 2 3 B 5 6 C RPA β 2 EDL RG SOL WG Sk M RPA β 2 EDL RG SOL WG rCPT1 β 2 RPA β 2 2 185 Probe β1 β2 β1