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. Author manuscript; available in PMC: 2013 Dec 15.
Published in final edited form as: Biochem J. 2012 Nov 15;448(1):10.1042/BJ20121110. doi: 10.1042/BJ20121110

Identification of a novel malonyl-CoA IC50 for CPT-1: implications for predicting in vivo fatty acid oxidation rates

Brennan K Smith *, Christopher GR Perry *, Timothy R Koves †, David C Wright *, Jeffrey C Smith ‡, P Darrell Neufer §, Deborah M Muoio †, Graham P Holloway *
PMCID: PMC3863641  NIHMSID: NIHMS428635  PMID: 22928974

Synopsis

Published values regarding the sensitivity (IC50) of carnitine palmitoyl transferase I (CPT-I) to malonyl-CoA (M-CoA) inhibition in isolated mitochondria are inconsistent with predicted in vivo rates of fatty acid oxidation. Therefore, we have re-examined M-CoA inhibition kinetics under varying palmitoyl-CoA (P-CoA) concentrations in both isolated mitochondria and permeabilized muscle fibres (PMF). PMF have an 18-fold higher IC50 (0.61 vs 0.034 μM) in the presence of 25 μM P-CoA and a 13-fold higher IC50 (6.3 vs 0.49 μM) in the presence of 150 μM P-CoA compared to isolated mitochondria. M-CoA inhibition kinetics determined in PMF predicts that CPT-I activity is inhibited by 33% in resting muscle compared to >95% in isolated mitochondria. Additionally, the ability of M-CoA to inhibit CPT-I appears to be dependent on P-CoA concentration, as the relative inhibitory capacity of M-CoA is decreased with increasing P-CoA concentrations. Altogether, the use of PMF appears to provide a M-CoA IC50 that better reflects the predicted in vivo rates of fatty acid oxidation. These findings also demonstrate the ratio of [P-CoA]/[M-CoA] is critical for regulating CPT-I activity and may partially rectify the in vivo disconnect between M-CoA content and CPT-I flux within the context of exercise and type II diabetes.

Keywords: skeletal muscle, carnitine palmitoyl transferase-I, malonyl-CoA, palmitoyl-CoA, isolated mitochondria, permeabilized fibres

Introduction

The regulation of long chain fatty acid (LCFA) oxidation is a central field of study in the context of exercise and metabolic disease. The rate limiting step in LCFA oxidation has been attributed mainly to its transport across the mitochondrial outer membrane via carnitine palmitoyltransferase I (CPT-I) [1,2]. Carnitine-dependent transport of LCFA into the mitochondria was first conceptualized in the 1960s and strong evidence has now accumulated to indicate that CPT-I has an obligatory role in LCFA oxidation [3-5].

All previous work investigating the regulatory mechanisms surrounding CPT-I have been performed in isolated mitochondria. Although isolated mitochondria are a standard tool for certain measurements (i.e. P/O ratios), several physiologically relevant mitochondrial characteristics are altered during the isolation procedure [6-8]. Additionally, the removal of the cytoskeleton during the isolation procedure may alter the inherent regulatory mechanisms associated with LCFA oxidation as the cytoskeleton has previously been shown to influence CPT-I activity [9,10] and other mitochondrial regulatory processes [11,12]. In contrast to isolated mitochondria, permeabilized skeletal muscle fibres (PMF) maintain the inherent cytoskeletal architecture and mitochondrial morphology while the sarcolemma is selectively permeabilzed [13,14]. This approach enables in situ investigations into mitochondrial physiology as substrates/chemicals added to the media have direct access to the mitochondria within their native state. In addition, the provision of a myosin ATPase inhibitor (blebbistatin) to the PMF preparation enables the analysis of mitochondrial parameters under more physiological conditions (i.e. 37°C), and better represents the in vivo situation accordingly [15,16]

In skeletal muscle, malonyl-coenzyme A (M-CoA) inhibits CPT-I activity, and therefore the content of M-CoA is considered an important regulator of skeletal muscle LCFA oxidation [2,17]. However, a number of discrepancies currently exist within the literature surrounding M-CoA inhibition kinetics of CPT-I. Firstly, the reported concentration of M-CoA required to inhibit CPT-I activity 50% (IC50) in isolated mitochondria (∼0.025-0.49 μM) [2,18,19] is lower than resting M-CoA content [2,20,21] suggesting that CPT-I activity and rates of LCFA oxidation should be negligible at rest This is inconsistent with the well characterized reliance on LCFA oxidation at rest in vrvo [22,23]. Secondly, during exercise, a decrease in M-CoA content is thought to “release the brake” on CPT-I and increase LCFA transport into the mitochondria for subsequent oxidation [24,25]. However, previous studies in humans have reported unchanged [26,27] or negligible decreases [28] in skeletal muscle M-CoA concentrations during exercise despite pronounced increases in LCFA oxidation. In addition, the role of M-CoA in regulating mitochondrial LCFA entry in type II diabetes has shown disparate findings as M-CoA levels are elevated in the skeletal muscle of type II diabetic humans [29] and rats (ZDF rats) [30,31] yet LCFA entry into the mitochondria is increased in both species [32,33]. Of potential importance, during exercise and in type II diabetes, LCFA-CoA levels within skeletal muscle are increased [34-36] and LCFA-CoA levels have been previously shown to decrease the effectiveness of M-CoA inhibition on CPT-I [37,38]. Therefore, any change in LCFA-CoA content can influence CPT-I activity independent of changes in M-CoA content.

Therefore, to address the controversies surrounding M-CoA inhibition kinetics of CPT-I, we aimed to determine the sensitivity of CPT-I to M-CoA in isolated mitochondria and in PMF under varying concentrations of palmitoyl-CoA (P-CoA, a LCFA-CoA moiety). We report that PMF have a 13 tol8-fold higher IC50 than isolated mitochondria and that the ability of M-CoA to inhibit CPT-I is dependent on the concentration of P-CoA in both preparations.

Experimental

Animals

Ten-week-old female Sprague-Dawley rats (274±8 g) were bred on site at the University of Guelph, and housed in a climate control facility on a 12 h light/dark cycle and provided rat chow and water ad libitum. Malonyl-CoA decarboxylase knockout (mcd-/-) mice [39] were bred onsite at Duke University. All facets of this study were approved by the University of Guelph Animal Care Committee and the Duke University Institutional Animal Care and conform to the guide for the care and use of laboratory animals published by the US National Institutes of Health. The red gastrocnemius muscle was used for all experiments.

Preparation of permeabilized fibres

The preparation of PMF was adopted from prior publications [15,40], as we have previously reported [41]. Following dissection of red gastrocnemius (n=6), fibre bundles (∼2 mg) were separated with fine forceps under a binocular dissecting microscope in BIOPS buffer containing, CaK2EGTA (2.77 mM), K2EGTA (7.23 mM), Na2ATP (5.77 mM), MgCl2*6H20 (6.56 mM), Na2Phosphocreatine (15 mM), Imidazole (20 mM), Dithiothreitol (0.5 mM) and MES (50 mM). Following separation, fibre bundles were placed in BIOPS containing 50μg/ml saponin, agitated for 30min and then washed in respiration buffer (MIR05) containing EGTA (0.5 mM), MgC12*6H20 (3 mM), K-lactobionate (60 mM), Taurine (20 mM), KH2P04 (10 mM), HEPES (20 mM), Sucrose (110 mM) and fatty acid free BSA (1 g/L). Fibres were left in cold MIR05 until respiration analysis.

Permeabilized muscle fibre respiration

Mitochondrial respiration was measured in PMF by high-resolution respirometry (Oroboros Oxygraph-2 k, Innsbruck, Austria) at 37°C and room air saturated oxygen tension in the presence of 25 μM blebbistatin to ensure PMF relaxation. Separate fibres from the same animal were used to determine (in duplicate) the kinetic properties of P-CoA supported respiration and the sensitivity of P-CoA respiration to M-CoA inhibition. To measure P-CoA supported respiration, MIR05, 5 mM ADP, 2 mM malate, and 2 mM L-carnitine were used as the respiration medium. Once a baseline respiration was determined, various concentrations of P-CoA (25, 50, 75, 100, 150 and 200 μM) were titrated into the chambers. To measure M-CoA sensitivity, respiration was determined in the presence of 150 μM and 25 μM P-CoA, and various concentrations of M-CoA (0.25, 0.5, 1, 2, 10, 25 and 50 μM) were subsequently titrated into the respiration chambers. Additionally, the impact of a fixed M-CoA concentration (7 μM) on respiration supported by varying P-CoA concentrations (25, 50, 75, and 100 μM) was performed. Separate fibres were used to determine respiratory control ratios in the presence of malate (2 mM) + glutamate (10 mM) +/- 5 mM ADP in the presence and absence of exogenous cytochrome c (10 μM) and oligomycin (2 μg/ml). P-CoA and palmitoylcarnitine were made up in water whereas the palmitate was made up in MIR05 supplemented with 10% BSA representing a 6.6:1 palmitate: BSA ratio. Optimization experiments of the various substrates have previously been done to ensure high quality, consistent experiments.

Carnitine palmitoyl transferase-I activity

CPT-I activity was determined as described by McGarry et al [2] with minor modifications as we have previously reported [19,42]. CPT-I activity is defined as the rate at which tritiated palmitoylcarnitine is formed from tritiated L-carnitine following the addition of isolated mitochondria to the reaction medium. The assay buffer consisted of 117 mM Tns-HCl, 0.28 mM reduced glutathione, 4.4 mM ATP, 2.22 mM KCN, 0.1 mM rotenone, 0.5% BSA, 5 mM L-carnitine and 1 μCi tritiated L-carnitine (L-[3H]carnitine Amersham Bioscience, Buckinghamshire, England). Briefly, the assay was conducted at 37°C and initiated by the addition of 10 μl of mitochondrial suspension (final protein content of 5 μg/reaction) to 10 μl of varying P-CoA concentrations (18.75, 37.5, 75, 150, and 200 μM) and 80 μl of a standard reaction medium. The reaction containing 150 μM P-CoA was also carried out in the presence of various M-CoA concentrations (0.5, 1, 2, 10, 25 and 50 μM). The reaction was stopped after 6 min with the addition of ice-cold HCL. Palmitoyl-[3H] carnitine was extracted in water-saturated butanol in a process involving three washes with distilled water and subsequent re-centrifugation steps to separate the butanol phase, in which the radioactivity was counted.

Isolated mitochondrial respiration

Differential centrifugation was used to isolate mitochondria from the red gastrocnemius muscle as we have previously published [41,43]. To measure M-CoA sensitivity, MIR05, 800 μM ADP, 2 mM malate, and 750 μM L-carnitine was used as the respiration medium and respiration was stimulated with 25 μM P-CoA. Once a baseline respiration was determined, various concentrations of M-CoA (0.03, 0.05, 0.1, 0.25 μM) were titrated into the chambers. Quality control experiments were also performed with isolated mitochondria to determine respiratory control ratios and mitochondrial coupling (ADP/O). In brief, state IV respiration was determined in the presence of 10 mM pyruvate+5 mM malate and state III respiration was initiated with the addition of 100 μM ADP. Maximal state HI respiration was determined in the presence of 2 mM ADP.

One phase exponential decacy predictive equation

One phase exponential decacy is defined as: Y = Y0-Plateau*e(-K*X) + Plateau Where:

Y = respiration rate

Y0 = respiration rate without exogenous M-CoA

Plateau = where any subsequent addition of M-CoA has no effect on respiration

K = rate constant

X = M-CoA concentration

Plateau was constrained to zero (Graphpad Prism 5).

Statistics

Michaelis-Menten kinetics and IC50 values for M-CoA were determined by plotting data points in GraphPad Prism 5 software following the subtraction of baseline values. The IC50 is defined as the concentration of M-CoA where CPT-I activity and/or P-CoA supported respiration were reduced by half. Unpaired 2-tailed t-tests were used to compare isolated mitochondria and permeabilized fibres P-CoA Km and M-CoA IC50. Statistical significance was accepted with a p value ≤ 0.05.

Results and Discussion

The current manuscript evaluated M-CoA inhibition kinetics (IC50) of CPT-I in isolated mitochondria and PMF. The integrity of the isolated mitochondria was ensured by determining pyruvate/malate respiratory control ratio measurements (5.4±0.4 nmol O2/mg/min) and appropriate P/O ratios (2.9±0.1). In the PMF preparation, the malate (2 mM) + glutamate (10 mM) +/- 5 mM ADP respiratory control ratio was 9.5±0.2. In addition, the exogenous provision of cytochrome c during ADP-supported respiration did not increase respiration >10%, and the addition of oligomycin fully prevented P-CoA respiration (indicating coupled respiration; data not shown). Combined, these quality control experiments indicate that the isolated mitochondria and the mitochondria within the PMF preparation were fully coupled and intact and therefore appropriate for the current study.

CPT-I remains rate-limiting for fatty-acid supported respiration in permeabilzed muscle fibres

We first determined that CPT-I represented a rate-limiting step in state 3 mitochondrial fatty acid supported respiration within the PMF preparation. This was accomplished by examining the kinetics of various lipids species proximal and distal to CPT-I. The sensitivity of the PMF preparation to palmitate (Km = 90 μM) and P-CoA (Km = 80 μM), substrates that require CPT-I, were very similar (Figure 1). In contrast, palmitoyl carnitine supported respiration, which does not require CPT-I, displayed a marked increase in sensitivity (Km = 23 μM). Altogether, these data indicate that similar to isolated mitochondria, CPT-I is rate limiting for lipid-supported respiration in PMF.

Figure 1.

Figure 1

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Palmitate, palmitoyl-CoA (P-CoA) and palmitoyl carnitine (PC) respiration kinetics in permeabilized muscle fibres. Palmitate was titrated in the presence of malate (2mM), ADP (5mM), Co-enzyme A (1mM) and L-carnitine (2mM). P-CoA was titrated in the presence of malate (2mM), ADP (5mM) and L-carnitine (2mM). PC was titrated in the presence of malate (2mM) and ADP (5mM). All respiration chambers contained 25 μM blebbistatin. All R2 values are above 0.98. P-CoA was inhibitory on respiration at ∼220 μM and PC was inhibitory on respiration at 100 μM. N=6 and each independent experiment was performed in duplicate, and data is expressed as mean ± SEM.

Use of permeabilzed muscle fibres yields a higher IC50 for malonyl-CoA

Prior to performing experiments to elucidate the M-CoA inhibitory kinetics on CPT-I in PMF, we determined the kinetic response of isolated mitochondria and PMF to titrations of P-CoA, a substrate for CPT-I. High P-CoA concentrations (>40 μM) prevent respiration in isolated mitochondria and therefore we determined the sensitivity of isolated mitochondria to P-CoA utilizing a CPT-I activity assay as classically performed [2]. The sensitivities of PMF and isolated mitochondria to the substrate P-CoA were similar as the Km values were 80 μM and 75 μM, respectively (Figure 2A and B). In addition, the Lineweaver-Burk plots located as insets in Figures 2A and B, also displayed similar slopes in isolated mitochondria and permeabilized fibres. These data suggest that a diffusion limitation does not exist within PMF for saturating fatty acid concentrations, and validate the use of identical P-CoA concentrations between methodologies in our subsequent experiments. Additionally, we performed experiments without saponin in PMF. In these experiments, although the respiration rates were ∼30-50% lower, M-CoA sensitivity was again not different with or without saponification indicating that the saponin permeabilization step does not affect the IC50 value observed in PMF. Altogether, these data support the use of PMF to assess M-CoA sensitivity.

Figure 2.

Figure 2

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Carnitine palmitoyltransferase I (CPT-I) activity in isolated mitochondria (A) and oxygen consumption in permeabilized muscle fibres (B) in the presence of various palmitoyl-CoA (P-CoA) concentrations. The Km values shown on the figures represent those determined for the mean regression lines and they are not different between methodologies. These values were used to generate the Lineweaver-Burk plots for enzyme kinetics, which are located as an inset in each graph. The slopes for these graphs were 0.41 and 0.39 for isolated mitochondria and permeabilized fibres, respectively. N=4 in isolated mitochondria, and N=6 in permeabilized fibres. Each independent experiment was performed in duplicate, and data is expressed as mean ±SEM.

Therefore, we next investigated the kinetic properties of M-CoA inhibition of oxygen consumption in isolated mitochondria and PMF in the presence of resting concentrations of P-CoA (25 μM) [34]. Maximal inhibition by M-CoA was similar between methodologies (-50%; Figure 3A, B), however this occurred at vastly different M-CoA concentrations (∼10-fold higher in PMF). In these highly controlled conditions, and in the presence of a physiologically relevant P-CoA concentration, the IC50 for M-CoA was 0.034 μM and 0.61 μM in isolated mitochondria and PMF, respectively (Figure 3A and B), suggesting M-CoA inhibition of CPT-I is considerably attenuated in an in situ model. In addition, the kinetics of M-CoA inhibition on P-CoA supported respiration in isolated mitochondria in the current study are virtually identical to previous reports examining M-CoA inhibition of CPT-I activity [2,2,38,44] further suggesting that CPT-I is rate-limiting for fatty acid oxidation under these experimental conditions.

Figure 3.

Figure 3

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Malonyl-CoA inhibition kinetics in isolated mitochondria (A) and in permeabilized muscle fibres (B) in the presence of 25 μM palmitoyl-CoA The IC50 values shown represent those determined for the mean regression line. Permeabilzed fibres displayed an ∼18-fold higher IC50 compared to isolated mitochondria. N=4 in isolated mitochondria and N=4 in permeabilized fibres. Each independent experiment was performed in duplicate and data is expressed as mean ± SEM. * significantly different from isolated mitochondria.

We next determined the kinetic properties of M-CoA inhibition in the presence of exercise concentrations of P-CoA (150 μM) [34]. We found that the sensitivity to M-CoA remained substantially lower in PMF, as the IC50 were 0.49 μM and 6.30 μM in isolated mitochondria and PMF, respectively (Figure 4A and B). However, compared to the IC50 values found using resting concentrations of P-CoA, these exercise values represent 14-fold and 10-fold increases in isolated mitochondria and PMF, respectively.

Figure 4.

Figure 4

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Malonyl-CoA inhibition kinetics in isolated mitochondria (A) and in permeabilized muscle fibres (B) in the presence of 150 μM palmitoyl-CoA The IC50 values shown represent those determined for the mean regression line. Permeabilzed muscle fibres display a ∼13-fold higher IC50 compared to isolated mitochondria. N=4 in isolated mitochondria and N=6 in permeabilized fibres. Each independent experiment was performed in duplicate, and data is expressed as mean ± SEM. * significantly different from isolated mitochondria.

Altogether, these data indicate that the sensitivity for M-CoA inhibition is vastly lower in PMF compared to isolated mitochondria, as the IC50 is higher in PMF in the presence of both resting (18-fold) and exercise (13-fold) P-CoA concentrations.

Role of palmitoyl-CoA in regulating malonyl-CoA inhibition kinetics

The pronounced difference in M-CoA IC50 values in the presence of higher P-CoA concentrations in both isolated mitochondria and PMF (Figures 3 and 4) led us to further examine the notion that P-CoA concentrations can alter M-CoA sensitivity in PMF. Previous work in isolated mitochondria has highlighted the interaction between M-CoA and P-CoA [2,37,38] but considering the marked differences observed between PMF and isolated mitochondria, we decided to re-examine this concept in PMF. This was done by determining the ability of 7 μM M-CoA to inhibit P-CoA respiration at varying (25-100 μM) concentrations of P-CoA in PMF [34]. With this approach, M-CoA inhibited respiration -63% in the presence of 25μM P-CoA, but only -26% in the presence of 100 μM P-CoA (Figure 5A and B). These data clearly show that increasing P-CoA concentration attenuates M-CoA inhibition of CPT-I suggesting that the ratio of M-CoAP-CoA is important in determining the overall catalytic activity of CPT-I. This implies when LCFA-CoA levels are elevated, such as during exercise or in type II diabetes, a reduction in M-CoA is not required to alter CPT-I flux. This could explain the pronounced increase in LCFA oxidation that occurs during exercise in humans in the face of little [28] or no change [26] in M-CoA levels. Additionally, a hallmark of chronic exercise training is the ability to increase fatty acid oxidation quickly at the onset of exercise, yet, following exercise training in rats, the normal exercise decline in M-CoA content is attenuated [45], suggesting additional mechanisms regulate CPT-I. In the context of the present data and others [2,37,38], the increase in the rate of fatty acid supply in the trained state would negate the need for M-CoA to substantially decrease in order to increase CPT-I flux and therefore fatty acid oxidation.

Figure 5.

Figure 5

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Palmitoyl-CoA (P-CoA) respiration in permeabilzed muscle fibres in the presence of 7 μM malonyl-CoA (M-CoA). Respiration of varying concentrations of P-CoA was inhibited by the addition of 7 μM. At each increased concentration of P-CoA, inhibition by M-CoA was decreased. N=4 and each independent experiment was performed in duplicate. Data is expressed as mean ± SEM.

The interaction between P-CoA and M-CoA may also explain how in type II diabetes mitochondrial matrix fatty acid oversupply exists in the presence of elevated M-CoA concentrations [29-33,46]. In the context of the present data and others [2,37,38], this disconnect may be explained by the increase in LCFA-CoA levels observed in type II diabetes which would render M-CoA inhibition less effective and potentially account for the increase in CPT-I flux and resultant mitochondrial matrix fatty acid oversupply present in this disease [35,36]. Considering fatty acid oversupply to the mitochondria has been associated with impaired insulin sensitivity [33,40], LCFA-CoA levels within the context of CPT-I regulation may be an important factor to consider in type II diabetes.

Perspectives and significance

Predicted physiological M-CoA inhibition of fatty acid oxidation

As previously documented [2,18,19,38,44], the IC50 determined in isolated mitochondria would suggest that LCFA oxidation is substantially inhibited at rest. Considering the respiratory quotient across a leg muscle at rest is ∼0.77 – 0.83 [22,23], these current values do not appear to represent the predicted in vivo situation. To put our results into physiological context, we applied the data from our “at rest” M-CoA inhibition curves derived from isolated mitochondria and PMF (Figure 3A and B) to a one phase exponential decay equation to predict the % inhibition of LCFA oxidation in the presence of resting concentrations of M-CoA (Table 1). We assumed resting M-CoA concentrations to be 0.7 μM as previously documented in two independent studies using HPLC/MS [47] and HPLC/MS/MS [48]. As seen in Table 1, inhibition of LCFA oxidation is predicted to be >95% in isolated mitochondria at rest. However, in PMF, the predicted inhibition of LCFA oxidation is ∼33% which appears to better reflect the in vivo state as reported respiratory quotients across a leg muscle [22,23] indicates that fatty acid oxidation would contribute ∼56-77% of the necessary energy at rest. In addition, independent of potential minor reductions in M-CoA content, the increase in P-CoA content (150 μM) within muscle during exercise attenuates M-CoA inhibition of LCFA oxidation to a predicted 2.1% in PMF compared to 44% in isolated mitochondria (Table 1).

Table 1. Predicted in vivo fatty acid oxidation inhibition.

Method P-CoA concentration Rate constant (K) M-CoA concentration Predicated rate of fat oxidation Predicted % inhibition
Isolated mitochondria 25 μM 4.076 0.7 μM 2.20 nmol/min/mg mito pr >95%
PMF 25 μM 0.03707 0.7 μM 14.1 pmol/s/mg dw 33%
Isolated mitochondria 150 μM 1.417 0.7 μM 8.98 nmol/min/mg mito pr 44%
PMF 150 μM 0.03026 0.7 μM 150 pmol/s/mg dw 2.1%
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The rate constant (K) used in the one phase exponential decay equation was derived from Graphpad Prism 5. The malonyl-CoA concentration was adopted from two previous publications [47,48].

Altogether, these data suggest that in an in situ model the predicted % inhibition of fatty acid oxidation appears to better reflect the predicted in vivo state compared to isolated mitochondria. However, it should be acknowledged that we have not been able to account for subcellular or regional differences in M-CoA concentrations. It has been previously suggested that the β-isoform of acetyl-CoA carboxylase located on the mitochondrial outer membrane “channels” M-CoA to CPT-I [49-51] and additionally, a mitochondrial enzyme in mammals capable of synthesizing M-CoA within the mitochondria has been recently characterized [52]. Therefore, it is possible that the use of total cellular content of M-CoA (0.7 μM) as we have done is not reflective of the in vivo exposure of CPT-I to M-CoA. While we acknowledge limitations pertaining to selecting the biologically relevant M-CoA concentration, these do not affect our interpretations, as these same limitations exist for both isolated mitochondrial and PMF preparations. We therefore hypothesize that the presence of the inherent cellular architecture within PMF may help explain the differences between methodologies. We also conclude that physiological increases in P-CoA may represent a key regulator of CPT-I activity. The mechanism by which P-CoA can alter M-CoA inhibition appears to be by increasing the dissociation constant (Kd) of M-CoA for CPT-I (∼10-fold) [37]. Therefore, P-CoA binding to CPT-I potentially alters the conformation of CPT-I such that M-CoA binding is less favorable [37]. These experiments [37] were done in the presence of maximal P-CoA and therefore the 10-fold increase in the Kd appears to match our data with “maximal” (exercise) concentrations of P-CoA as we report a 13-fold increase in the IC50 in the presence of exercise P-CoA concentrations [37].

Literature comparison of IC50 values in skeletal muscle

Previous literature displays ∼70-fold range in M-CoA IC50 values creating difficulty in interpreting the importance of M-CoA even within an isolated mitochondrial preparation. Table 2 compares our present results in the context of previous literature and highlights the vast range of reported IC50 values. The apparent disparities within the previous literature appear to be explained by the concentration of P-CoA, as the highest values were all generated with ≥150 μM P-CoA (Table 2). Additionally, the IC50 values between rat and human skeletal muscle are similar, suggesting the M-CoA kinetic properties are evolutionary conserved.

Table 2. Literature comparison of skeletal muscle IC50 values.

Method Species P-CoA μM used IC50 Reference
Isolated mitochondria: CPT-I activity Rat 50 0.023 [38]
Human 50 0.025 [2]
Rat 50 0.034 [2]
Rat 50 0.041 [44]
Rat 40 0.07 [58]
Human 300 0.490 [18]
Human 300 1.600 [19]
Rat 150 0.43 Present study
Isolated mitochondria: respiration Rat 25 0.034 Present study
Permeabilzed muscle fibres: respiration Rat 25 0.61 Present study
Rat 150 6.3 Present study
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Potential explanations for differences between methodologies

It is currently unknown why PMF have a higher M-CoA IC50 value compared to isolated mitochondria. However, isolating mitochondria from skeletal muscle requires homogenization followed by a series of differential centrifugation steps, a process that likely exerts substantial sheer stress on the mitochondrial membranes, and therefore CPT-I [7]. This process has been shown to alter several characteristics of mitochondria, including respiration of specific substrates and susceptibility of calcium induced opening of the permeability transition pore [6]. Current models of CPT-I propose a hairpin structure, with both N and C termini located in the cytosol [53] and the interaction of the N and C termini is essential for modulating and preserving M-CoA sensitivity [7,54]. Therefore, isolating mitochondria may alter the interaction of these two cytosolic loops. However, this proposition is unlikely as current literature suggests that altering this interaction prevents M-CoA binding and therefore a challenge to this interaction would increase the IC50 [7,54]. Currently, no published literature exists to explain structure/function alterations in CPT-I that increase M-CoA binding/sensitivity.

Alternatively, M-CoA has been previously hypothesized to be sequestered into specific compartments within the skeletal muscle and it therefore remains possible that whatever mechanism promoting the sequestering and compartmentalization of M-CoA in skeletal muscle could still be present in PMF and not in isolated mitochondria [20,55,56].

The presence of M-CoA binding proteins in PMF may also explain the discrepancy in IC50 values between methodologies. In support of this proposal, the presence of liver M-CoA binding proteins has been previously documented [57]. Additionally, the presence of M-CoA binding proteins would be expected to decrease the effect of M-CoA in muscle at lower concentrations, while at supraphysiological concentrations of M-CoA these binding proteins would become saturated, and therefore CPT-I would be inhibited. This description recapitulates the observed trends in PMF as M-CoA inhibition was attenuated at all concentrations <10 μM in comparison to isolated mitochondria but inhibition was similar to isolated mitochondria at higher M-CoA concentrations (25 and 50 μM). Therefore, the current data supports the idea that M-CoA binding proteins exist in muscle although the identification of these proteins and the potential interaction with the cytoskeleton remains to be investigated.

Conclusion

In conclusion, we provide evidence that the IC50 value for M-CoA in PMF is much higher than in isolated mitochondria and appears to better reflect in vivo fatty acid oxidation rates. Therefore, we hypothesize that these functional differences between PMF and isolated mitochondria underscore the important influence of mitochondrial morphology and/or the extra-mitochondrial environment in regulating LCFA oxidation via M-CoA inhibition of CPT-1. Additionally, P-CoA levels can alter M-CoA inhibition kinetics of CPT-I which may explain some of the previous literature discrepancies. Lastly, within the context of exercise and type II diabetes, the impact of altering P-CoA concentrations may address the disconnect between M-CoA levels and CPT-I flux previously observed.

Acknowledgments

Funding: This work was funded by The Natural Sciences and Engineering Research Council of Canada (NSERC; GH, JS and DW) and infrastructure was purchased with the assistance of the Canadian Foundation for Innovation (GH, JS and DW) as well as the Ontario Research Fund (GH, JS and DW). BS is supported by an NSERC graduate scholarship and DW is a Canada Research Chair. Funding from Carleton University (JS) is acknowledged. Additional support was provided by grants from the United States Public Health Service: [R01 DK073488] and [DK074825] (PDN) and [R01 AG028930], [AK089312], and [HL101189] (DMM).

Abbreviations

CPT-I

carnitine palmitoyl transferase-I

P-CoA

palmitoyl-CoA

M-CoA

malonyl-CoA

PmFB

permeabilzed muscle fibres

IC50

concentration of inhibitor that decreases enzyme activity by 50%

Km

concentration of substrate that stimulates enzyme activity by 50%

LCFA

long chain fatty acids

Reference List

  • 1.Kerner J, Hoppel C. Fatty acid import into mitochondria. Biochim Biophys Acta. 2000;1486:1–17. doi: 10.1016/s1388-1981(00)00044-5. [DOI] [PubMed] [Google Scholar]
  • 2.McGarry JD, Mills SE, Long CS, Foster DW. Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-CoA in non-hepatic tissues of the rat. Biochem J. 1983;214:21–28. doi: 10.1042/bj2140021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bremer J. Carnitine in intermediary metabolism. The biosynthesis of palmitylcarnitine by cell subfractions. J Biol Chem. 1963;238:2774–2779. [PubMed] [Google Scholar]
  • 4.Bremer J, Norum KR. The mechanism of substrate inhibition of palmityl coenzyme A: carnitine palmityltransferase by palmityl coenzyme A. J Biol Chem. 1967;242:1744–1748. [PubMed] [Google Scholar]
  • 5.Fritz IB, Yue KT. Long-chain carnitine acyltransferase and the role of acylcarnitine derivatives in the catalytic increase of fatty acid oxidation induced by carnitine. J Lipid Res. 1963;4:279–288. [PubMed] [Google Scholar]
  • 6.Picard M, Taivassalo T, Ritchie D, Wright KJ, Thomas MM, Romestaing C, Hepple RT. Mitochondrial structure and function are disrupted by standard isolation methods. PLoS One. 2011;6:el8317. doi: 10.1371/journal.pone.0018317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Fraser F, Corstorphine CG, Zammit VA. Topology of carnitine palmitoyltransferase I in the mitochondrial outer membrane. Biochem J. 1997;323(Pt 3):711–718. doi: 10.1042/bj3230711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Saks VA, Kuznetsov AV, Khuchua ZA, Vasilyeva EV, Belikova JO, Kesvatera T, Tiivel T. Control of cellular respiration in vivo by mitochondrial outer membrane and by creatine kinase. A new speculative hypothesis: possible involvement of mitochondnal cytoskeleton interactions. J Mol Cell Cardiol. 1995;27:625–645. doi: 10.1016/s0022-2828(08)80056-9. [DOI] [PubMed] [Google Scholar]
  • 9.Velasco G, Geelen MJ, Gomez del PT, Guzman M. Malonyl-CoA-independent acute control of hepatic carnitine palmitoyltransferase I activity. Role of Ca2+/calmodulin-dependent protein kinase II and cytoskeletal components. J Biol Chem. 1998;273:21497–21504. doi: 10.1074/jbc.273.34.21497. [DOI] [PubMed] [Google Scholar]
  • 10.Velasco G, Gomez del PT, Carling D, Guzman M. Evidence that the AMP-activated protein kinase stimulates rat liver carnitine palmitoyltransferase I by phosphorylating cytoskeletal components. FEBS Lett. 1998;439:317–320. doi: 10.1016/s0014-5793(98)01400-8. [DOI] [PubMed] [Google Scholar]
  • 11.Kay L, Li Z, Mericskay M, Olivares J, Tranqui L, Fontaine E, Tiivel T, Sikk P, Kaambre T, Samuel JL, Rappaport L, Usson Y, Leverve X, Paulin D, Saks VA. Study of regulation of mitochondrial respiration in vivo. An analysis of influence of ADP diffusion and possible role of cytoskeleton. Biochim Biophys Acta. 1997;1322:41–59. doi: 10.1016/s0005-2728(97)00071-6. [DOI] [PubMed] [Google Scholar]
  • 12.Rostovtseva TK, Sheldon KL, Hassanzadeh E, Monge C, Saks V, Bezrukov SM, Sackett DL. Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration. Proc Natl Acad Sci U S A. 2008;105:18746–18751. doi: 10.1073/pnas.0806303105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, Tranqui L, Olivares J, Winkler K, Wiedemann F, Kunz WS. Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem. 1998;184:81–100. [PubMed] [Google Scholar]
  • 14.Veksler VI, Kuznetsov AV, Sharov VG, Kapelko VI, Saks VA. Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers. Biochim Biophys Acta. 1987;892:191–196. doi: 10.1016/0005-2728(87)90174-5. [DOI] [PubMed] [Google Scholar]
  • 15.Perry CG, Kane DA, Lin CT, Kozy R, Cathey BL, Lark DS, Kane CL, Brophy PM, Gavin TP, Anderson EJ, Neufer PD. Inhibiting myosin-ATPase reveals dynamic range of mitochondrial respiratory control in skeletal muscle. Biochem J. 2011;437:215–222. doi: 10.1042/BJ20110366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kashfi K, Cook GA. Temperature effects on malonyl-CoA inhibition of carnitine palmitoyltransferase I. Biochim Biophys Acta. 1995;1257:133–139. doi: 10.1016/0005-2760(95)00063-i. [DOI] [PubMed] [Google Scholar]
  • 17.Saggerson ED, Carpenter CA. Effects of fasting and malonyl CoA on the kinetics of carnitine palmitoyltransferase and carnitine octanoyltransferase in intact rat liver mitochondria. FEBS Lett. 1981;132:166–168. doi: 10.1016/0014-5793(81)81152-0. [DOI] [PubMed] [Google Scholar]
  • 18.Starritt EC, Howlett RA, Heigenhauser GJ, Spriet LL. Sensitivity of CPT I to malonyl-CoA in trained and untrained human skeletal muscle. Am J Physiol Endocrinol Metab. 2000;278:E462–E468. doi: 10.1152/ajpendo.2000.278.3.E462. [DOI] [PubMed] [Google Scholar]
  • 19.Holloway GP, Bezaire V, Heigenhauser GJ, Tandon NN, Glatz JF, Luiken JJ, Bonen A, Spriet LL. Mitochondrial long chain fatty acid oxidation, fatty acid translocase/CD36 content and carnitine palmitoyltransferase I activity in human skeletal muscle during aerobic exercise. J Physiol. 2006;571:201–210. doi: 10.1113/jphysiol.2005.102178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Chien D, Dean D, Saha AK, Flatt JP, Ruderman NB. Malonyl-CoA content and fatty acid oxidation in rat muscle and liver in vivo. Am J Physiol Endocrinol Metab. 2000;279:E259–E265. doi: 10.1152/ajpendo.2000.279.2.E259. [DOI] [PubMed] [Google Scholar]
  • 21.Maclean PS, Winder WW. Caffeine decreases malonyl-CoA in isolated perfused skeletal muscle of rats. J Appl Physiol. 1995;78:1496–1501. doi: 10.1152/jappl.1995.78.4.1496. [DOI] [PubMed] [Google Scholar]
  • 22.Kelley DE, Goodpaster B, Wing RR, Simoneau JA. Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity, and weight loss. Am J Physiol. 1999;277:E1130–E1141. doi: 10.1152/ajpendo.1999.277.6.E1130. [DOI] [PubMed] [Google Scholar]
  • 23.Wahren J, Felig P, Ahlborg G, Jorfeldt L. Glucose metabolism during leg exercise in man. J Clin Invest. 1971;50:2715–2725. doi: 10.1172/JCI106772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Winder WW, Arogyasami J, Barton RJ, Elayan IM, Vehrs PR. Muscle malonyl-CoA decreases during exercise. J Appl Physiol. 1989;67:2230–2233. doi: 10.1152/jappl.1989.67.6.2230. [DOI] [PubMed] [Google Scholar]
  • 25.Winder WW, Arogyasami J, Elayan IM, Cartmill D. Time course of exercise-induced decline in malonyl-CoA in different muscle types. Am J Physiol. 1990;259:E266–E271. doi: 10.1152/ajpendo.1990.259.2.E266. [DOI] [PubMed] [Google Scholar]
  • 26.Odland LM, Heigenhauser GJ, Lopaschuk GD, Spriet LL. Human skeletal muscle malonyl-CoA at rest and during prolonged submaximal exercise. Am J Physiol. 1996;270:E541–E544. doi: 10.1152/ajpendo.1996.270.3.E541. [DOI] [PubMed] [Google Scholar]
  • 27.Odland LM, Hewlett RA, Heigenhauser GJ, Hultman E, Spriet LL. Skeletal muscle malonyl-CoA content at the onset of exercise at varying power outputs in humans. Am J Physiol. 1998;274:E1080–E1085. doi: 10.1152/ajpendo.1998.274.6.E1080. [DOI] [PubMed] [Google Scholar]
  • 28.Roepstorff C, Halberg N, Hillig T, Saha AK, Ruderman NB, Wojtaszewski JF, Richter EA, Kiens B. Malonyl-CoA and carnitine in regulation of fat oxidation in human skeletal muscle during exercise. Am J Physiol Endocrinol Metab. 2005;288:E133–E142. doi: 10.1152/ajpendo.00379.2004. [DOI] [PubMed] [Google Scholar]
  • 29.Bandyopadhyay GK, Yu JG, Ofrecio J, Olefsky JM. Increased malonyl-CoA levels in muscle from obese and type 2 diabetic subjects lead to decreased fatty acid oxidation and increased lipogenesis; thiazolidinedione treatment reverses these defects. Diabetes. 2006;55:2277–2285. doi: 10.2337/db06-0062. [DOI] [PubMed] [Google Scholar]
  • 30.Yu X, McCorkle S, Wang M, Lee Y, Li J, Saha AK, Unger RH, Ruderman NB. Leptinomimetic effects of the AMP kinase activator AICAR in leptin-resistant rats: prevention of diabetes and ectopic lipid deposition. Diabetologia. 2004;47:2012–2021. doi: 10.1007/s00125-004-1570-9. [DOI] [PubMed] [Google Scholar]
  • 31.Beha A, Juretschke HP, Kuhlmann J, Neumann-Haefelin C, Belz U, Gerl M, Kramer W, Roden M, Herling AW. Muscle type-specific fatty acid metabolism in insulin resistance: an integrated in vivo study in Zucker diabetic fatty rats. Am J Physiol Endocrinol Metab. 2006;290:E989–E997. doi: 10.1152/ajpendo.00459.2005. [DOI] [PubMed] [Google Scholar]
  • 32.Mihalik SJ, Goodpaster BH, Kelley DE, Chace DH, Vockley J, Toledo FG, DeLany JP. Increased levels of plasma acylcarnitines in obesity and type 2 diabetes and identification of a marker of glucohpotoxicity. Obesity (Silver Spring) 2010;18:1695–1700. doi: 10.1038/oby.2009.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Koves TR, Ussher JR, Noland RC, Slentz D, Mosedale M, Ilkayeva O, Bain J, Stevens R, Dyck JR, Newgard CB, Lopaschuk GD, Muoio DM. Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance. Cell Metab. 2008;7:45–56. doi: 10.1016/j.cmet.2007.10.013. [DOI] [PubMed] [Google Scholar]
  • 34.Watt MJ, Heigenhauser GJ, O'Neill M, Spriet LL. Hormone-sensitive lipase activity and fatty acyl-CoA content in human skeletal muscle during prolonged exercise. J Appl Physiol. 2003;95:314–321. doi: 10.1152/japplphysiol.01181.2002. [DOI] [PubMed] [Google Scholar]
  • 35.Chalkley SM, Hettiarachchi M, Chisholm DJ, Kraegen EW. Long-term high-fat feeding leads to severe insulin resistance but not diabetes in Wistar rats. Am J Physiol Endocrinol Metab. 2002;282:E1231–E1238. doi: 10.1152/ajpendo.00173.2001. [DOI] [PubMed] [Google Scholar]
  • 36.Ellis BA, Poynten A, Lowy AJ, Furler SM, Chisholm DJ, Kraegen EW, Cooney GJ. Long-chain acyl-CoA esters as indicators of lipid metabolism and insulin sensitivity in rat and human muscle. Am J Physiol Endocrinol Metab. 2000;279:E554–E560. doi: 10.1152/ajpendo.2000.279.3.E554. [DOI] [PubMed] [Google Scholar]
  • 37.Kolodziej MP, Zammit VA. Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes. Biochem J. 1990;267:85–90. doi: 10.1042/bj2670085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Mills SE, Foster DW, McGarry JD. Interaction of malonyl-CoA and related compounds with mitochondria from different rat tissues. Relationship between ligand binding and inhibition of carnitine palmitoyltransferase I. Biochem J. 1983;214:83–91. doi: 10.1042/bj2140083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Dyck JR, Hopkins TA, Bonnet S, Michelakis ED, Young ME, Watanabe M, Kawase Y, Jishage K, Lopaschuk GD. Absence of malonyl coenzyme A decarboxylase in mice increases cardiac glucose oxidation and protects the heart from ischemic injury. Circulation. 2006;114:1721–1728. doi: 10.1161/CIRCULATIONAHA.106.642009. [DOI] [PubMed] [Google Scholar]
  • 40.Anderson EJ, Lustig ME, Boyle KE, Woodlief TL, Kane DA, Lin CT, Price JW, III, Kang L, Rabinovitch PS, Szeto HH, Houmard JA, Cortright RN, Wasserman DH, Neufer PD. Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans. J Clin Invest. 2009;119:573–581. doi: 10.1172/JCI37048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Smith BK, Jain SS, Rimbaud S, Dam A, Quadrilatero J, Ventura-Clapier R, Bonen A, Holloway GP. FAT/CD36 is located on the outer mitochondrial membrane, upstream of long-chain acyl-CoA synthetase, and regulates palmitate oxidation. Biochem J. 2011;437:125–134. doi: 10.1042/BJ20101861. [DOI] [PubMed] [Google Scholar]
  • 42.Benton CR, Holloway GP, Campbell SE, Yoshida Y, Tandon NN, Glatz JF, Luiken JJ, Spriet LL, Bonen A. Rosiglitazone increases fatty acid oxidation and fatty acid translocase (FAT/CD36) but not carnitine palmitoyltransferase I in rat muscle mitochondria. J Physiol. 2008;586:1755–1766. doi: 10.1113/jphysiol.2007.146563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Lally JS, Snook LA, Han XX, Chabowski A, Bonen A, Holloway GP. Subcellular lipid droplet distribution in red and white muscles in the obese Zucker rat. Diabetologia. 2012;55:479–488. doi: 10.1007/s00125-011-2367-2. [DOI] [PubMed] [Google Scholar]
  • 44.Mills SE, Foster DW, McGarry JD. Effects of pH on the interaction of substrates and malonyl-CoA with mitochondrial carnitine palmitoyltransferase I. Biochem J. 1984;219:601–608. doi: 10.1042/bj2190601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Hutber CA, Rasmussen BB, Winder WW. Endurance training attenuates the decrease in skeletal muscle malonyl-CoA with exercise. J Appl Physiol. 1997;83:1917–1922. doi: 10.1152/jappl.1997.83.6.1917. [DOI] [PubMed] [Google Scholar]
  • 46.Bell JA, Volpi E, Fujita S, Cadenas JG, Rasmussen BB. Dysregulation of muscle fatty acid metabolism in type 2 diabetes is independent of malonyl-CoA. Diabetologia. 2006;49:2144–2152. doi: 10.1007/s00125-006-0362-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Minkler PE, Kerner J, Kasumov T, Parland W, Hoppel CL. Quantification of malonyl-coenzyme A in tissue specimens by high-performance liquid chromatography/mass spectrometry. Anal Biochem. 2006;352:24–32. doi: 10.1016/j.ab.2006.02.015. [DOI] [PubMed] [Google Scholar]
  • 48.Gilibili RR, Kandaswamy M, Sharma K, Giri S, Rajagopal S, Mullangi R. Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of acetyl-CoA and malonyl-CoA in animal tissues. Biomed Chromatogr. 2011;25:1352–1359. doi: 10.1002/bmc.1608. [DOI] [PubMed] [Google Scholar]
  • 49.Abu-Elheiga L, Jayakumar A, Baldini A, Chirala SS, Wakil SJ. Human acetyl-CoA carboxylase: characterization, molecular cloning, and evidence for two isoforms. Proc Natl Acad Sci U S A. 1995;92:4011–4015. doi: 10.1073/pnas.92.9.4011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Lopez-Casillas E, Bai DH, Luo XC, Kong IS, Hermodson MA, Kim KH. Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A carboxylase. Proc Natl Acad Sci U S A. 1988;85:5784–5788. doi: 10.1073/pnas.85.16.5784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Widmer J, Fassihi KS, Schlichter SC, Wheeler KS, Crute BE, King N, Nutile-McMenemy N, Noll WW, Daniel S, Ha J, Kim KH, Witters LA. Identification of a second human acetyl-CoA carboxylase gene. Biochem J. 1996;316(Pt 3):915–922. doi: 10.1042/bj3160915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Witkowski A, Thweatt J, Smith S. Mammalian ACSF3 protein is a malonyl-CoA synthetase that supplies the chain extender units for mitochondrial fatty acid synthesis. J Biol Chem. 2011;286:33729–33736. doi: 10.1074/jbc.M111.291591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Zammit VA, Price NT, Fraser F, Jackson VN. Structure-function relationships of the liver and muscle isoforms of carnitine palmitoyltransferase I. Biochem Soc Trans. 2001;29:287–292. doi: 10.1042/0300-5127:0290287. [DOI] [PubMed] [Google Scholar]
  • 54.Shi J, Zhu H, Arvidson DN, Woldegiorgis G. The first 28 N-terminal amino acid residues of human heart muscle carnitine palmitoyltransferase I are essential for malonyl CoA sensitivity and high-affinity binding. Biochemistry. 2000;39:712–717. doi: 10.1021/bi9918700. [DOI] [PubMed] [Google Scholar]
  • 55.McGarry JD, Brown NF. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur J Biochem. 1997;244:1–14. doi: 10.1111/j.1432-1033.1997.00001.x. [DOI] [PubMed] [Google Scholar]
  • 56.Ruderman NB, Saha AK, Vavvas D, Witters LA. Malonyl-CoA fuel sensing, and insulin resistance. Am J Physiol. 1999;276:E1–E18. doi: 10.1152/ajpendo.1999.276.1.E1. [DOI] [PubMed] [Google Scholar]
  • 57.Dugan RE, Osterlund BR, Drong RF, Swenson TL. A malonyl-CoA-binding protein from liver. Biochem Biophys Res Commun. 1987;147:234–241. doi: 10.1016/s0006-291x(87)80111-0. [DOI] [PubMed] [Google Scholar]
  • 58.Guzman M, Castro J. Effects of endurance exercise on carnitine palmitoyltransferase I from rat heart, skeletal muscle and liver mitochondria. Biochim Biophys Acta. 1988;963:562–565. doi: 10.1016/0005-2760(88)90327-x. [DOI] [PubMed] [Google Scholar]

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