malonyl-CoA Sensitivity Research Articles

High intensity exercise inhibits carnitine palmitoyltransferase-I sensitivity to l-carnitine.

The decline in fat oxidation at higher power outputs of exercise is a complex interaction between several mechanisms; however, the influence of mitochondrial bioenergetics in this process remains elusive. Therefore, using permeabilized muscle fibers from mouse skeletal muscle, we aimed to determine if acute exercise altered mitochondrial sensitivity to (1) adenosine diphosphate (ADP) and inorganic phosphate (Pi), or (2) carnitine palmitoyltransferase-I (CPT-I) independent (palmitoylcarnitine, PC) and dependent [palmitoyl-CoA (P-CoA), malonyl-CoA (M-CoA), and l-carnitine] substrates, in an intensity-dependent manner. As the apparent ADP Km increased to a similar extent following low (LI) and high (HI) intensity exercise compared with sedentary (SED) animals, and Pi sensitivity was unaltered by exercise, regulation of phosphate provision likely does not contribute to the well-established intensity-dependent shift in substrate utilization. Mitochondrial sensitivity to PC and P-CoA was not influenced by exercise, while M-CoA sensitivity was attenuated similarly following LI and HI. In contrast, CPT-I sensitivity to l-carnitine was only altered following HI, as HI exercise attenuated l-carnitine sensitivity by ∼40%. Moreover, modeling the in vivo concentrations of l-carnitine and P-CoA during exercise suggests that CPT-I flux is ∼25% lower following HI, attributed equally to reductions in l-carnitine content and l-carnitine sensitivity. Altogether, these data further implicate CPT-I flux as a key event influencing metabolic interactions during exercise, as a decline in l-carnitine sensitivity in addition to availability at higher power outputs could impair mitochondrial fatty acid oxidation.

Mitochondrial-derived reactive oxygen species influence ADP sensitivity, but not CPT-I substrate sensitivity.

The mechanisms regulating oxidative phosphorylation during exercise remain poorly defined; however, key mitochondrial proteins, including carnitine palmitoyltransferase-I (CPT-I) and adenine nucleotide translocase, have redox-sensitive sites. Interestingly, muscle contraction has recently been shown to increase mitochondrial membrane potential and reactive oxygen species (ROS) production; therefore, we aimed to determine if mitochondrial-derived ROS influences bioenergetic responses to exercise. Specifically, we examined the influence of acute exercise on mitochondrial bioenergetics in WT (wild type) and transgenic mice (MCAT, mitochondrial-targeted catalase transgenic) possessing attenuated mitochondrial ROS. We found that ablating mitochondrial ROS did not alter palmitoyl-CoA (P-CoA) respiratory kinetics or influence the exercise-mediated reductions in malonyl CoA sensitivity, suggesting that mitochondrial ROS does not regulate CPT-I. In contrast, while mitochondrial protein content, maximal coupled respiration, and ADP (adenosine diphosphate) sensitivity in resting muscle were unchanged in the absence of mitochondrial ROS, exercise increased the apparent ADP Km (decreased ADP sensitivity) ∼30% only in WT mice. Moreover, while the presence of P-CoA decreased ADP sensitivity, it did not influence the basic response to exercise, as the apparent ADP Km was increased only in the presence of mitochondrial ROS. This basic pattern was also mirrored in the ability of ADP to suppress mitochondrial H2O2 emission rates, as exercise decreased the suppression of H2O2 only in WT mice. Altogether, these data demonstrate that while exercise-induced mitochondrial-derived ROS does not influence CPT-I substrate sensitivity, it inhibits ADP sensitivity independent of P-CoA. These data implicate mitochondrial redox signaling as a regulator of oxidative phosphorylation.

Increased cardiac fatty acid oxidation in a mouse model with decreased malonyl-CoA sensitivity of CPT1B.

Mitochondrial fatty acid oxidation (FAO) is an important energy provider for cardiac work and changes in cardiac substrate preference are associated with different heart diseases. Carnitine palmitoyltransferase 1B (CPT1B) is thought to perform the rate limiting enzyme step in FAO and is inhibited by malonyl-CoA. The role of CPT1B in cardiac metabolism has been addressed by inhibiting or decreasing CPT1B protein or after modulation of tissue malonyl-CoA metabolism. We assessed the role of CPT1B malonyl-CoA sensitivity in cardiac metabolism. We generated and characterized a knock in mouse model expressing the CPT1BE3A mutant enzyme, which has reduced sensitivity to malonyl-CoA. In isolated perfused hearts, FAO was 1.9-fold higher in Cpt1bE3A/E3A hearts compared with Cpt1bWT/WT hearts. Metabolomic, proteomic and transcriptomic analysis showed increased levels of malonylcarnitine, decreased concentration of CPT1B protein and a small but coordinated downregulation of the mRNA expression of genes involved in FAO in Cpt1bE3A/E3A hearts, all of which aim to limit FAO. In vivo assessment of cardiac function revealed only minor changes, cardiac hypertrophy was absent and histological analysis did not reveal fibrosis. Malonyl-CoA-dependent inhibition of CPT1B plays a crucial role in regulating FAO rate in the heart. Chronic elevation of FAO has a relatively subtle impact on cardiac function at least under baseline conditions.

Controlling skeletal muscle CPT-I malonyl-CoA sensitivity: the importance of AMPK-independent regulation of intermediate filaments during exercise.

The obligatory role of carnitine palmitoyltransferase-I (CPT-I) in mediating mitochondrial lipid transport is well established, a process attenuated by malonyl-CoA (M-CoA). However, the necessity of reducing M-CoA concentrations to promote lipid oxidation has recently been challenged, suggesting external regulation on CPT-I. Since previous work in hepatocytes suggests the involvement of the intermediate filament fraction of the cytoskeleton in regulating CPT-I, we investigated in skeletal muscle if CPT-I sensitivity for M-CoA inhibition could be regulated by the intermediate filaments, and whether AMP-activated protein kinase (AMPK) could be involved in this process. Chemical disruption (3,3'-iminodipropionitrile, IDPN) of the intermediate filaments did not alter mitochondrial respiration or sensitivity for numerous substrates (palmitoyl-CoA, ADP, palmitoyl carnitine and pyruvate). In contrast, IDPN reduced CPT-I sensitivity for M-CoA inhibition in permeabilized muscle fibers, identifying M-CoA kinetics as a specific target for intermediate filament regulation. Importantly, exercise mimicked the effect of IDPN on M-CoA sensitivity, suggesting that intermediate filament disruption in vivo is physiologically important for CPT-I regulation. To ascertain a potential mechanism, since AMPK is activated during exercise, AMPK β1β2-KO mice were utilized in an attempt to ablate the observed exercise response. Unexpectedly, these mice displayed drastic attenuation in resting M-CoA sensitivity, such that exercise and IDPN could not further alter M-CoA sensitivity. These data suggest that AMPK is not required for the regulation of the intermediate filament interaction with CPT-I. Altogether, these data highlight that M-CoA sensitivity is important for regulating mitochondrial lipid transport. Moreover, M-CoA sensitivity appears to be regulated by intermediate filament interaction with CPT-I, a process that is important when metabolic homeostasis is challenged.

An Environment-dependent Structural Switch Underlies the Regulation of Carnitine Palmitoyltransferase 1A

The enzyme carnitine palmitoyltransferase 1 (CPT1), which is anchored in the outer mitochondrial membrane (OMM), controls the rate-limiting step in fatty acid β-oxidation in mammalian tissues. It is inhibited by malonyl-CoA, the first intermediate of fatty acid synthesis, and it responds to OMM curvature and lipid characteristics, which reflect long term nutrient/hormone availability. Here, we show that the N-terminal regulatory domain (N) of CPT1A can adopt two complex amphiphilic structural states, termed Nα and Nβ, that interchange in a switch-like manner in response to offered binding surface curvature. Structure-based site-directed mutageneses of native CPT1A suggest Nα to be inhibitory and Nβ to be noninhibitory, with the relative Nα/Nβ ratio setting the prevalent malonyl-CoA sensitivity of the enzyme. Based on the amphiphilic nature of N and molecular modeling, we propose malonyl-CoA sensitivity to be coupled to the properties of the OMM by Nα-OMM associations that alter the Nα/Nβ ratio. For enzymes residing at the membrane-water interface, this constitutes an integrative regulatory mechanism of exceptional sophistication.

Packing of transmembrane domain 2 of carnitine palmitoyltransferase‐1A affects oligomerization and malonyl‐CoA sensitivity of the mitochondrial outer membrane protein

The purpose of this study was to investigate the sequence-dependence of oligomerization of transmembrane domain 2 (TM2) of rat carnitine palmitoyltransferase 1A (rCPT1A), to elucidate the role of this domain in the function of the full-length enzyme. Oligomerization of TM2 was studied qualitatively using complementary genetic assays that facilitate measurement of helix-helix interactions in the Escherichia coli inner membrane, and multiple quantitative biophysical methods. The effects of TM2-mutations on oligomerization and malonyl-CoA inhibition of the full-length enzyme (expressed in the yeast Pichia pastoris) were quantified. Changes designed to disrupt close-packing of the GXXXG(A) motifs reduced the oligomeric state of the corresponding TM2 peptides from hexamer to trimer (or lower), a reduction also observed on mutation of the TM2 sequence in the full-length enzyme. Disruption of these GXXXG(A) motifs had a parallel effect on the malonyl-CoA sensitivity of rCPT1A, reducing the IC(50) from 30.3 ± 5.0 to 3.0 ± 0.6 μM. For all measurements, wild-type rCPT1A was used as a control alongside various appropriate (e.g., molecular mass) standards. Our results suggest that sequence-determined, TM2-mediated oligomerization is likely to be involved in the modulation of malonyl-CoA inhibition of CPT1A in response to short- and long-term changes in protein-protein and protein-lipid interactions that occur in vivo.

Increased Mitochondrial Fatty Acid Oxidation Is Sufficient to Protect Skeletal Muscle Cells from Palmitate-induced Apoptosis

The mechanisms underlying the protective effect of monounsaturated fatty acids (e.g. oleate) against the lipotoxic action of saturated fatty acids (e.g. palmitate) in skeletal muscle cells remain poorly understood. This study aimed to examine the role of mitochondrial long-chain fatty acid (LCFA) oxidation in mediating oleate's protective effect against palmitate-induced lipotoxicity. CPT1 (carnitine palmitoyltransferase 1), which is the key regulatory enzyme of mitochondrial LCFA oxidation, is inhibited by malonyl-CoA, an intermediate of lipogenesis. We showed that expression of a mutant form of CPT1 (CPT1mt), which is active but insensitive to malonyl-CoA inhibition, in C2C12 myotubes led to increased LCFA oxidation flux even in the presence of high concentrations of glucose and insulin. Furthermore, similar to preincubation with oleate, CPT1mt expression protected muscle cells from palmitate-induced apoptosis and insulin resistance by decreasing the content of deleterious palmitate derivates (i.e. diacylglycerols and ceramides). Oleate preincubation exerted its protective effect by two mechanisms: (i) in contrast to CPT1mt expression, oleate preincubation increased the channeling of palmitate toward triglycerides, as a result of enhanced diacylglycerol acyltransferase 2 expression, and (ii) oleate preincubation promoted palmitate oxidation through increasing CPT1 expression and modulating the activities of acetyl-CoA carboxylase and AMP-activated protein kinase. In conclusion, we demonstrated that targeting mitochondrial LCFA oxidation via CPT1mt expression leads to the same protective effect as oleate preincubation, providing strong evidence that redirecting palmitate metabolism toward oxidation is sufficient to protect against palmitate-induced lipotoxicity.

Functional effects of protein kinases and peroxynitrite on cardiac carnitine palmitoyltransferase-1 in isolated mitochondria

We have previously shown that metoprolol can inhibit carnitine palmitoyltransferase-1 catalytic activity and decrease its malonyl CoA sensitivity within 30 min, suggesting the importance of a covalent modification. The aim of this study was to characterize the effects of PTMs on CPT-1 in the heart. Mitochondria were isolated from the hearts of male Wistar rats and incubated with kinases of interest (protein kinase A, CAMK-II, p38 MAPK, Akt) or with peroxynitrite and sodium nitroprusside. PKA decreased CPT-1 malonyl CoA sensitivity, associated with phosphorylation of CPT-1A, whereas CAMK-II increased malonyl CoA sensitivity by phosphorylating CPT-1B. p38 bound to CPT-1B and stimulated CPT-1 activity. The association of CPT-1 with these kinases and their scaffolding proteins was confirmed in co-localization studies. Peroxynitrite and sodium nitroprusside reversibly stimulated CPT-1 activity, and the change in CPT-1B activity was most consistently associated with glutathiolation of CPT-1B. These studies have identified a new regulatory system of kinases, scaffolding proteins and thiol redox chemistry which can control cardiac CPT-1 in vitro.

Modulation of the hepatic malonyl-CoA–carnitine palmitoyltransferase 1A partnership creates a metabolic switch allowing oxidation of de novo fatty acids

Liver mitochondrial beta-oxidation of LCFAs (long-chain fatty acids) is tightly regulated through inhibition of CPT1A (carnitine palmitoyltransferase 1A) by malonyl-CoA, an intermediate of lipogenesis stimulated by glucose and insulin. Moreover, CPT1A sensitivity to malonyl-CoA inhibition varies markedly depending on the physiopathological state of the animal. In the present study, we asked whether an increase in CPT1A activity solely or in association with a decreased malonyl-CoA sensitivity could, even in the presence of high glucose and insulin concentrations, maintain a sustained LCFA beta-oxidation and/or protect from triacylglycerol (triglyceride) accumulation in hepatocytes. We have shown that adenovirus-mediated expression of rat CPT1wt (wild-type CPT1A) and malonyl-CoA-insensitive CPT1mt (CPT1AM593S mutant) in cultured fed rat hepatocytes counteracted the inhibition of oleate beta-oxidation induced by 20 mM glucose/10 nM insulin. Interestingly, the glucose/insulin-induced cellular triacylglycerol accumulation was prevented, both in the presence and absence of exogenous oleate. This resulted from the generation of a metabolic switch allowing beta-oxidation of de novo synthesized LCFAs, which occurred without alteration in glucose oxidation and glycogen synthesis. Moreover, CPT1mt expression was more effective than CPT1wt overexpression to counteract glucose/insulin effects, demonstrating that control of CPT1A activity by malonyl-CoA is an essential driving force for hepatic LCFA metabolic fate. In conclusion, the present study highlights that CPT1A is a prime target to increase hepatic LCFA beta-oxidation and that acting directly on the degree of its malonyl-CoA sensitivity may be a relevant strategy to prevent and/or correct hepatic steatosis.

A characteristic Glu17 residue of pig carnitine palmitoyltransferase 1 is responsible for the low Km for carnitine and the low sensitivity to malonyl-CoA inhibition of the enzyme

Human carnitine palmitoyltransferase 1B (CPT1B) is a highly malonyl-CoA-sensitive enzyme (IC50=0.097 microm) and has a positive determinant (residues 18-28) of malonyl-CoA inhibition. By contrast, rat carnitine palmitoyltransferase 1A is less sensitive to malonyl-CoA inhibition (IC(50)=1.9 microm), and has both a positive (residues 1-18) and a negative (residues 18-28) determinant of its inhibition. Interestingly, pig CPT1B shows a low degree of malonyl-CoA sensitivity (IC(50)=0.804 microm). Here, we examined whether any additional molecular determinants affect malonyl-CoA inhibition of CPT1B. We show that the malonyl-CoA sensitivity of CPT1B is determined by the length (either 50 or 128 residues) of the N-terminal region constructed by recombining pig and human enzymes. We also show that the N-terminal region of pig CPT1B carries a single positive determinant of malonyl-CoA sensitivity, but that this is located between residues 1 and 18 of the N-terminal segment. Importantly, we found a single amino acid variation (D17E) relevant to malonyl-CoA sensitivity. Thus, Asp17 is specifically involved, under certain assay conditions, in the high malonyl-CoA sensitivity of the human enzyme, whereas the naturally occurring variation, Glu17, is responsible for both the low malonyl-CoA sensitivity and high carnitine affinity characteristics of the pig enzyme. This is the first demonstration that a single naturally occurring amino acid variation can alter CPT1B enzymatic properties.

Phosphorylation of carnitine palmitoyltransferase‐1 in heart mitochondria

Aim: Carnitine palmitoyltransferase‐1 (CPT‐1) catalyzes the entry of long‐chain acyl CoA's to the cytoplasm, an important control step in fatty acid oxidation. Previous studies from our laboratory indicate that the maximum activity and malonyl CoA sensitivity of CPT‐1 are acutely regulated by metoprolol in the rat heart. The aim of the present study was to determine whether incubation of isolated mitochondria from left ventricular tissue with protein kinase A, Akt or calcium/ calmodulin‐dependent protein kinase alters the maximum activity or malonyl CoA sensitivity of CPT‐1. Methods: Isolated mitochondria from normal Wistar rats were incubated with purified preparations of PKA, CAMK II or Akt for 10 minutes. Phosphorylation of CPT‐1 isoforms was measured by co‐immunoprecipitation with phosphothreonine and phosphoserine antibodies, and the activity and malonyl CoA sensitivity of CPT‐1 was assayed. Results: PKA and CAMK‐II phosphorylated CPT‐1. PKA decreased CPT‐1 malonyl CoA sensitivity whereas CAMK‐II increased CPT‐1 malonyl CoA sensitivity. There was no effect on the maximum activity of CPT‐1. Conclusions: PKA and CAMK‐II regulate CPT‐1 sensitivity in isolated heart mitochondria in vitro. These kinases may be counter‐regulatory in the control of CPT‐1 malonyl CoA sensitivity.

The role of protein-mediated transport in regulating mitochondrial long-chain fatty acid oxidation

This thesis is an investigation of the role of fatty acid translocase (FAT/CD36), plasma membrane associated fatty acid binding protein (FABPpm), and carnitine palmitoyltransferase I (CPTI) in transporting long-chain fatty acids (LCFAs) across mitochondrial membranes. Maximal CPTI activity, as well as the sensitivity of CPTI for its substrate palmitoyl-CoA (P-CoA) and its inhibitor malonyl-CoA (M-CoA), were measured in mitochondria isolated from human vastus lateralis muscles at rest and following muscle contraction. Exercise did not alter maximal CPTI activity or the sensitivity of CPTI for P-CoA. In contrast, exercise progressively attenuated the ability of M-CoA to inhibit CPTI activity. Mitochondrial FAT/CD36 protein content was also measured at rest, during, and following 2 h of cycling at ~60% maximal oxygen uptake. Exercise progressively increased the content of mitochondrial FAT/CD36 (+59%), which was significantly (p < 0.05) correlated with palmitate oxidation during exercise (r = 0.52), while palmitate oxidation was inhibited ~80% by the administration of a specific FAT/CD36 inhibitor. These data suggest that alterations in CPTI M-CoA sensitivity and increases in mitochondrial FAT/CD36 coordinate exercise-induced increases in fatty acid oxidation. FABPpm, another plasma membrane transport protein, has identical amino acid sequence to mitochondrial aspartate aminotransferase (mAspAT). Since FABPpm contributes to plasma membrane fatty acid transport, the role of FABPpm with respect to mitochondrial LCFA transport was investigated. However, unlike FAT/CD36, muscle contraction did not induce an increase in mitochondrial FABPpm protein in rat or human skeletal muscle. In addition, electrotransfecting FABPpm cDNA into rat skeletal muscle upregulated this protein in mitochondria by 80% without altering mitochondrial palmitate oxidation. In contrast, electrotransfection increased mAspAT activity by 90%, and this was correlated (r = 0.75; p < 0.01) with FABPpm protein. These data suggest that FABPpm does not contribute to the regulation of mitochondrial LCFA transport. Previously, it has been suggested that mitochondria from obese individuals contain an inherent dysfunction to oxidize LCFAs. In age-matched lean (BMI = 23.3 ± 0.7 kg·m–2) and obese (BMI = 37.6 ± 2.2 kg·m–2) individuals, isolated mitochondrial palmitate oxidation was not altered. In addition, mitochondrial FAT/CD36 content was not different in lean and obese individuals. In contrast, citrate synthase and β-hydroxyacyl-CoA dehydrogenase, common markers of total mitochondrial content, were decreased with obesity. Therefore, the decrease in mitochondrial content appeared to account for the observed reductions in whole-muscle LCFA oxidation.

Rat Liver Carnitine Palmitoyltransferase 1 Forms an Oligomeric Complex within the Outer Mitochondrial Membrane

Carnitine palmitoyltransferase (CPT) 1A catalyzes the rate-limiting step in the transport of long chain acyl-CoAs from cytoplasm to the mitochondrial matrix by converting them to acylcarnitines. Located within the outer mitochondrial membrane, CPT1A activity is inhibited by malonyl-CoA, its allosteric inhibitor. In this study, we investigate for the first time the quaternary structure of rat CPT1A. Chemical cross-linking studies using intact mitochondria isolated from fed rat liver or from Saccharomyces cerevisiae expressing CPT1A show that CPT1A self-assembles into an oligomeric complex. Size exclusion chromatography experiments using solubilized mitochondrial extracts suggest that the fundamental unit of its quaternary structure is a trimer. When studied in blue native-PAGE, the CPT1A hexamer could be observed, however, suggesting that under these native conditions CPT1A trimers might be arranged as dimers. Moreover, the oligomeric state of CPT1A was found unchanged by starvation and by streptozotocin-induced diabetes, conditions characterized by changes in malonyl-CoA sensitivity of CPT1A. Finally, gel filtration analysis of several yeast-expressed chimeric CPTs demonstrates that the first 147 N-terminal residues of CPT1A, encompassing its two transmembrane segments, trigger trimerization independently of its catalytic C-terminal domain. Deletion of residues 1-82, including transmembrane 1, did not abrogate oligomerization, but the latter is limited to a trimer by the presence of the large catalytic C-terminal domain on the cytosolic face of mitochondria. Based on these findings, we proposed that the oligomeric structure of CPT1A would allow the newly formed acylcarnitines to gain direct access into the intermembrane space, hence facilitating substrate channeling.

Definition by Functional and Structural Analysis of Two Malonyl-CoA Sites in Carnitine Palmitoyltransferase 1A

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine in the presence of l-carnitine, thus facilitating the entry of fatty acids to mitochondria, in a process that is physiologically inhibited by malonyl-CoA. To examine the mechanism of CPT1 liver isoform (CPT1A) inhibition by malonyl-CoA, we constructed an in silico model of both its NH2- and COOH-terminal domains. Two malonyl-CoA binding sites were found. One of these, the "CoA site" or "A site," is involved in the interactions between NH2- and COOH-terminal domains and shares the acyl-CoA hemitunnel. The other, the "opposite-to-CoA site" or "O site," is on the opposite side of the enzyme, in the catalytic channel. The two sites share the carnitine-binding locus. To prevent the interaction between NH2- and COOH-terminal regions, we produced CPT1A E26K and K561E mutants. A double mutant E26K/K561E (swap), which was expected to conserve the interaction, was also produced. Inhibition assays showed a 12-fold decrease in the sensitivity (IC50) toward malonyl-CoA for CPT1A E26K and K561E single mutants, whereas swap mutant reverts to wild-type IC50 value. We conclude that structural interaction between both domains is critical for enzyme sensitivity to malonyl-CoA inhibition at the "A site." The location of the "O site" for malonyl-CoA binding was supported by inhibition assays of expressed R243T mutant. The model is also sustained by kinetic experiments that indicated linear mixed type malonyl-CoA inhibition for carnitine. Malonyl-CoA alters the affinity of carnitine, and there appears to be an exponential inverse relation between carnitine Km and malonyl-CoA IC50.

The Mitochondrial Intermembrane Loop Region of Rat Carnitine Palmitoyltransferase 1A Is a Major Determinant of Its Malonyl-CoA Sensitivity

Carnitine palmitoyltransferase (CPT) 1A adopts a polytopic conformation within the mitochondrial outer membrane, having both the N- and C-terminal segments on the cytosolic aspect of the membrane and a loop region connecting the two transmembrane (TM) segments protruding into the inter membrane space. In this study we demonstrate that the loop exerts major effects on the sensitivity of the enzyme to its inhibitor, malonyl-CoA. Insertion of a 16-residue spacer between the C-terminal part of the loop sequence (i.e. between residues 100 and 101) and TM2 (which is predicted to start at residue 102) increased the sensitivity to malonyl-CoA inhibition of the resultant mutant protein by more than 10-fold. By contrast, the same insertion made between TM1 and the loop had no effects on the kinetic properties of the enzyme, indicating that effects on the catalytic C-terminal segment were specifically induced by loop-TM2 interactions. Enhanced sensitivity was also observed in all mutants in which the native TM2-loop pairing was disrupted either by making chimeras in which the loops and TM2 segments of CPT 1A and CPT 1B were exchanged or by deleting successive 9-residue segments from the loop sequence. The data suggest that the sequence spanning the loop-TM2 boundary determines the disposition of this TM in the membrane so as to alter the conformation of the C-terminal segment and thus affect its interaction with malonyl-CoA.

Effects of cardiolipin on carnitine palmitoyltransferase kinetics

We previously reported [Biochem. J. (1994) 299, 761-767] that certain phospholipids added to intact rat liver mitochondria increased sensitivity of carnitine palmitoyltransferase-I (CPT1A) to inhibition by malonyl-CoA. The most effective phospholipid was cardiolipin, which enhanced inhibition so that it mimicked the normal change in malonyl-CoA sensitivity occurring in the fasting to fed transition. Since cardiolipin is a normal component of eukaryotic mitochondrial membranes, we used mitochondria from Saccharomyces cerevisiae to test the hypothesis that the absence of cardiolipin in mitochondria in vivo would change the enzyme kinetics of CPT1. Yeast do not oxidize fatty acids in mitochondria. They do not express any of the components of the CPT system for facilitating transport of fatty acids into mitochondria. From the Greenberg lab we obtained a cardiolipin synthase knock-out strain of S. cerevisiae lacking cardiolipin and having mitochondrial function deficiencies [J. Biol. Chem. (2000) 275, 22387-94], and we obtained its parent strain for use as controls. We transformed both strains with an expression vector for CPT1A, and harvested mitochondria from the cells after breaking cells with glass beads. CPT1A in mitochondria from the parent wild-type yeast had a Km for carnitine of 0.65 ± 0.05 mM, while mitochondria from the cardiolipin synthase knockout yeast had a Km of 0.31 ± 0.03 mM. We also tested malonyl-CoA sensitivity and found mitochondria from wild-type yeast to have an I-50 (concentration of malonyl-CoA producing 50% inhibition) = 0.50 ± 0.07 microM, while mitochondria from cardiolipin synthase knockout yeast had an I-50 = 2.0 ± 0.3 microM. These results suggest that membrane cardiolipin in vivo alters CPT1A to increase its sensitivity to inhibition by malonyl-CoA, and this may be a physiologically significant mechanism for regulating fatty acid oxidation. (Supported by NIH Grant HL-66924)

Mitochondrial long chain fatty acid oxidation, fatty acid translocase/CD36 content and carnitine palmitoyltransferase I activity in human skeletal muscle during aerobic exercise

Mitochondrial fatty acid transport is a rate-limiting step in long chain fatty acid (LCFA) oxidation. In rat skeletal muscle, the transport of LCFA at the level of mitochondria is regulated by carnitine palmitoyltransferase I (CPTI) activity and the content of malonyl-CoA (M-CoA); however, this relationship is not consistently observed in humans. Recently, fatty acid translocase (FAT)/CD36 was identified on mitochondria isolated from rat and human skeletal muscle and found to be involved in LCFA oxidation. The present study investigated the effects of exercise (120 min of cycling at approximately 60% V(O2peak)) on CPTI palmitoyl-CoA and M-CoA kinetics, and on the presence and functional significance of FAT/CD36 on skeletal muscle mitochondria. Whole body fat oxidation rates progressively increased during exercise (P < 0.05), and concomitantly M-CoA inhibition of CPTI was progressively attenuated. Compared to rest, 120 min of cycling reduced (P < 0.05) the inhibition of 0.7, 2, 5 and 10 microM M-CoA by 16%, 21%, 30% and 34%, respectively. Whole body fat oxidation and palmitate oxidation rates in isolated mitochondria progressively increased (P < 0.05) during exercise, and were positively correlated (r = 0.78). Mitochondrial FAT/CD36 protein increased by 63% (P < 0.05) during exercise and was significantly (P < 0.05) correlated with mitochondrial palmitate oxidation rates at all time points (r= 0.41). However, the strongest (P < 0.05) correlation was observed following 120 min of cycling (r = 0.63). Importantly, the addition of sulfo-N-succimidyloleate, a specific inhibitor of FAT/CD36, reduced mitochondrial palmitate oxidation to approximately 20%, indicating FAT/CD36 is functionally significant with respect to LCFA oxidation. We hypothesize that exercise-induced increases in fatty acid oxidation occur as a result of an increased ability to transport LCFA into mitochondria. We further suggest that decreased CPTI M-CoA sensitivity and increased mitochondrial FAT/CD36 protein are both important for increasing whole body fatty acid oxidation during prolonged exercise.

Carnitine Palmitoyltransferase I in Liver of Periparturient Dairy Cows: Effects of Prepartum Intake, Postpartum Induction of Ketosis, and Periparturient Disorders

Thirty-five multiparous Holstein cows were used to determine the role of mitochondrial carnitine palmitoyltransferase I (CPT I) in liver on peripartal adaptations of fatty acid metabolism. From dry-off to parturition, cows were fed a diet at either ad libitum (n=17) or restricted intake (RI, 80% of calculated requirements for net energy; n=18). After parturition, all cows were fed a lactation diet. At 4 d in milk (DIM), cows underwent a physical examination and were classified as healthy (n=15) or having at least one periparturient disorder (PD; n=17). Cows in the healthy group were assigned to either a control (n=6) group or a ketosis induction (KI; n=9) group. Cows with periparturient disorders were assigned to a third (PDC; n=17) group. Cows in control and PDC groups were fed for ad libitum intake. Cows in KI were fed at 50% of their respective intake at d 4 postpartum starting from 5 DIM and continuing to signs of clinical ketosis or until 14 DIM; cows then were returned to ad libitum intake. Liver was biopsied at −30 d, 1 d, at signs of clinical ketosis or 14 d, and 28 d relative to parturition. Mitochondria were isolated by differential centrifugation. Activity of CPT I was 5.4 and 7.6nmol of palmitoylcarnitine formed per min/mg of protein for ad libitum and RI, respectively, at −30 DIM. Sensitivity of CPT I to its inhibitor, malonyl CoA, did not differ between ad libitum and RI cows. Differences in CPT I activity between ad libitum and RI were no longer significant at 1 DIM. Postpartum CPT I activity and malonyl CoA sensitivity at 1 DIM, onset of clinical ketosis or 14 DIM, and 28 DIM were not affected by prepartum intake (ad libitum vs. RI), postpartum health status (healthy vs. PD), or ketosis induction status (control vs. KI vs. PDC). Activity of CPT I was positively correlated with liver total lipid, liver triglyceride, liver triglyceride to glycogen ratio, and serum nonesterified fatty acids. Activity of CPT I and dry matter intake were not correlated. Prepartum intake affected prepartum CPT I activity but not malonyl CoA sensitivity. Neither induction of primary ketosis nor periparturient disorders greatly affected CPT I activity or sensitivity, which indicates that alterations of CPT I may not be a major factor in the etiology of primary ketosis or other periparturient disorders.

Structure-Function Studies with the Mitochondrial Carnitine Palmitoyltransferases I and II

Mitochondrial carnitine palmitoyltransferases I and II (CPTI and CPTII), together with the carnitine carrier, transport long-chain fatty acyl-CoAs from the cytosol to the mitochondrial matrix for β-oxidation. As an enzyme that catalyzes the rate-limiting step in fatty acid oxidation, CPTI is inhibited by malonyl-CoA, the first intermediate in fatty acid synthesis. Our development of a high level of expression for CPTI and CPTII in P. pastoris, a yeast with no endogenous CPT activity has enabled us to map the malonyl-CoA and substrate binding sites by mutational analysis. Using deletion and substitution mutants of L-CPTI expressed in P. pastoris, we have shown that Glu3 and His5 are necessary for malonyl-CoA inhibition and high-affinity binding of L-CPTI but not for catalysis. Similar studies of M-CPTI clearly establish that the N-terminal residues Glu3, Val19, Leu23, and Ser24 in M-CPTI are important for malonyl-CoA inhibition and binding, but not for catalysis. Furthermore, using chimeras between rat and pig L-CPTI, and deletion mutation analysis, we demonstrated that the differences in malonyl-CoA sensitivity observed between the pig and rat L-CPTI were due to differences in the interaction of the first 18 N-terminal amino acid residues with the C-terminal region of the respective enzymes. Consistent with this, the conserved C-terminal residues R601, E603, R606, and K560 were found to be important for L-CPTI activity, malonyl-CoA inhibition and binding, because mutation of these residues decreased malonyl-CoA sensitivity and enzyme activity. We also identified two conserved C-terminal residues in L-CPTI, D567, and E590, that when mutated to alanine cause a substantial increase in malonyl-CoA sensitivity, suggesting a structural basis for the differences in malonyl-CoA sensitivity between L-CPTI and M-CPTI. Our cysteine-scanning mutagenesis of M-CPTI revealed that a single Cys residue, Cys305, was essential for catalysis. In addition, deletion and substitution analysis of the extreme C-terminal region of M-CPTI, suggest that L764 may be important for proper folding and optimal activity. In summary, our structure-function studies with the mitochondrial carnitine palmitoyltransferases I and II have identified critical residues for inhibitor and substrate binding and catalysis.

Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity

We have previously proposed that changes in malonyl-CoA sensitivity of rat L-CPT1 (liver carnitine palmitoyltransferase 1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains. By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we have now shown the existence of such N-C (N- and C-terminal domain) intramolecular interactions both in wild-type L-CPT1 expressed in Saccharomyces cerevisiae and in the native L-CPT1 in fed rat liver mitochondria. These N-C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity. Moreover, increasing the outer membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitization, attenuated the N-C interactions. This indicates that the changes in malonyl-CoA sensitivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N-C interactions. Finally, we show that mutations in the regulatory regions of the N-terminal domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased [S24A (Ser24-->Ala)/Q30A] or abrogated (E3A) malonyl-CoA sensitivity. Moreover, we have identified the region immediately N-terminal to transmembrane domain 1 (residues 40-47) as being involved in the chemical N-C cross-linking. These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains, and that this determines its degree of malonyl-CoA sensitivity depending on the physiological state.