Target size analysis by radiation inactivation of carnitine palmitoyltransferase activity and malonyl-CoA binding in outer membranes from rat liver mitochondria.

The functional molecular sizes of the protein(s) mediating the carnitine palmitoyltransferase I (CPT I) activity and the [14C]malonyl-CoA binding in purified outer-membrane preparations from rat liver mitochondria were determined by radiation-inactivation analysis. In all preparations tested the dose-dependent decay in [14C]malonyl-CoA binding was less steep than that for CPT I activity, suggesting that the protein involved in malonyl-CoA binding may be smaller than that catalysing the CPT I activity. The respective sizes computed from simultaneous analysis for molecular-size standards exposed under identical conditions were 60,000 and 83,000 DA for malonyl-CoA binding and CPT I activity respectively. In irradiated membranes the sensitivity of CPT activity to malonyl-CoA inhibition was increased, as judged by malonyl-CoA inhibition curves for the activity in control and in irradiated membranes that had received 20 Mrad radiation and in which CPT activity had decayed by 60%. Possible correlations between these data and other recent observations on the CPT system are discussed.     

Carnitine palmitoyltransferase activities: Effects of serum albumin,acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP acyl-CoA binding protein - BSA bovine serum albumin - CPT carnitine palmitoyltransferase - CPT₀ malonyl-CoA sensitive CPT of the outer mitochondrial membrane - CPT malonyl-CoA insensitive CPT of the inner mitochondrial membrane - OG octylglucoside - OMV outer membrane vesicles - IMV inner membrane vesicles Affiliated to the Department of Experimental Medicine, University of Montreal     

Regulation of carnitine palmitoyltransferase (CPT) I during fasting in rainbow trout (Oncorhynchus mykiss) promotes increased mitochondrial fatty acid oxidation

Periods of fasting, in most animals, are fueled principally by fatty acids, and changes in the regulation of fatty acid oxidation must exist to meet this change in metabolic substrate use. We examined the regulation of carnitine palmitoyltransferase (CPT) I, to help explain changes in mitochondrial fatty acid oxidation with fasting. After fasting rainbow trout (Oncorhynchus mykiss) for 5 wk, the mitochondria were isolated from red muscle and liver to determine (1) mitochondrial fatty acid oxidation rate, (2) CPT I activity and the concentration of malonyl-CoA needed to inhibit this activity by 50% (IC(50)), (3) mitochondrial membrane fluidity, and (4) CPT I (all five known isoforms) and peroxisome proliferator-activated receptor (PPARα and PPARβ) mRNA levels. Fatty acid oxidation in isolated mitochondria increased during fasting by 2.5- and 1.75-fold in liver and red muscle, respectively. Fasting also decreased sensitivity of CPT I to malonyl-CoA (increased IC(50)), by two and eight times in red muscle and liver, respectively, suggesting it facilitates the rate of fatty acid oxidation. In the liver, there was also a significant increase CPT I activity per milligram mitochondrial protein and in whole-tissue PPARα and PPARβ mRNA levels. However, there were no changes in mitochondrial membrane fluidity in either tissue, indicating that the decrease in CPT I sensitivity to malonyl-CoA is not due to bulk fluidity changes in the membrane. However, there were significant differences in CPT I mRNA levels during fasting. Overall, these data indicate some important changes in the regulation of CPT I that promote the increased mitochondrial fatty acid oxidation that occurs during fasting in trout.     

Regulation of cardiac malonyl-CoA content and fatty acid oxidation during increased cardiac power

Myocardial fatty acid oxidation is regulated by carnitine palmitoyltransferase I (CPT I), which is inhibited by malonyl-CoA. Increased cardiac power causes a fall in malonyl-CoA content and accelerated fatty acid oxidation; however, the mechanism for the decrease in malonyl-CoA is unclear. Malonyl-CoA is formed by acetyl-CoA carboxylase (ACC) and degraded by malonyl-CoA decarboxylase (MCD); thus a fall in malonyl-CoA could be due to activation of MCD, inhibition of ACC, or both. This study assessed the effects of increased cardiac power on malonyl-CoA content and ACC and MCD activities. Anesthetized pigs were studied under control conditions and during increased cardiac power in response to dobutamine infusion and aortic constriction alone, under hyperglycemic conditions, or with the CPT I inhibitor oxfenicine. An increase in cardiac power was accompanied by increased myocardial O(2) consumption, decreased malonyl-CoA concentration, and increased fatty acid oxidation. There were no differences among groups in activity of ACC or AMP-activated protein kinase (AMPK), which physiologically inhibits ACC. There also were no differences in V(max) or K(m) of MCD. Previous studies have demonstrated that AMPK can be inhibited by protein kinase B (PKB); however, PKB was activated by dobutamine and the elevated insulin that accompanied hyperglycemia, but there was no effect on AMPK activity. In conclusion, the fall in malonyl-CoA and increase in fatty acid oxidation that occur with increased cardiac work were not due to inhibition of ACC or activation of MCD, suggesting alternative regulatory mechanisms for the work-induced decrease in malonyl-CoA concentration.     

Carnitine acyltransferase activities in rat brain mitochondria. Bimodal distribution, kinetic constants, regulation by malonyl-CoA and developmental pattern.

Carnitine palmitoyltransferase and carnitine octanoyltransferase activities in brain mitochondrial fractions were approx. 3-4-fold lower than activities in liver. Estimated Km values of CPT1 and CPT2 (the overt and latent forms respectively of carnitine palmitoyltransferase) for L-carnitine were 80 microM and 326 microM, respectively, and K0.5 values for palmitoyl-CoA were 18.5 microM and 12 microM respectively. CPT1 activity was strongly inhibited by malonyl-CoA, with I50 values (concn. giving 50% of maximum inhibition) of approx. 1.5 microM. In the absence of other ligands, [2-14C]malonyl-CoA bound to intact brain mitochondria in a manner consistent with the presence of two independent classes of binding sites. Estimated values for KD(1), KD(2), N1 and N2 were 18 nM, 27 microM, 1.3 pmol/mg of protein and 168 pmol/mg of protein respectively. Neither CPT1 activity, nor its sensitivity towards malonyl-CoA, was affected by 72 h starvation. Rates of oxidation of palmitoyl-CoA (in the presence of L-carnitine) or of palmitoylcarnitine by non-synaptic mitochondria were extremely low, indicating that neither CPT1 nor CPT2 was likely to be rate-limiting for beta-oxidation in brain. CPT1 activity relative to mitochondrial protein increased slightly from birth to weaning (20 days) and thereafter decreased by approx. 50%.     

C75 activates malonyl-CoA sensitive and insensitive components of the CPT system

Carnitine palmitoyltransferase I (CPT-I) and II (CPT-II) enzymes are components of the carnitine palmitoyltransferase shuttle system which allows entry of long-chain fatty acids into the mitochondrial matrix for subsequent oxidation. This system is tightly regulated by malonyl-CoA levels since this metabolite is a strong reversible inhibitor of the CPT-I enzyme. There are two distinct CPT-I isotypes (CPT-Ialpha and CPT-Ibeta), that exhibit different sensitivity to malonyl-CoA inhibition. Because of its ability to inhibit fatty acid synthase, C75 is able to increase malonyl-CoA intracellular levels. Paradoxically it also activates long-chain fatty acid oxidation. To identify the exact target of C75 within the CPT system, we expressed individually the different components of the system in the yeast Pichia pastoris. We show here that C75 acts on recombinant CPT-Ialpha, but also on the other CPT-I isotype (CPT-Ibeta) and the malonyl-CoA insensitive component of the CPT system, CPT-II.     

Re-evaluation of the interaction of malonyl-CoA with the rat liver mitochondrial carnitine palmitoyltransferase system by using purified outer membranes.

1. The interaction of malonyl-CoA with the outer carnitine palmitoyltransferase (CPT) system of rat liver mitochondria was re-evaluated by using preparations of highly purified outer membranes, in the light of observations that other subcellular structures that normally contaminate crude mitochondrial preparations also contain malonyl-CoA-sensitive CPT activity. 2. In outer-membrane preparations, which were purified about 200-fold with respect to the inner-membrane-matrix fraction, malonyl-CoA binding was largely accounted for by a single high-affinity component (KD = 0.03 microM), in contrast with the dual site (low- and high-affinity) previously found with intact mitochondria. 3. There was no evidence that the decreased sensitivity of CPT to malonyl-CoA inhibition observed in outer membranes obtained from 48 h-starved rats (compared with those from fed animals) was due to a decreased ratio of malonyl-CoA binding to CPT catalytic moieties. Thus CPT specific activity and maximal high-affinity [14C]malonyl-CoA binding (expressed per mg of protein) were increased 2.2- and 2.0-fold respectively in outer membranes from 48 h-starved rats. 4. Palmitoyl-CoA at a concentration that was saturating for CPT activity (5 microM) decreased the affinity of malonyl-CoA binding by an order of magnitude, but did not alter the maximal binding of [14C]malonyl-CoA. 5. Preincubation of membranes with either tetradecylglycidyl-CoA or 2-bromopalmitoyl-CoA plus carnitine resulted in marked (greater than 80%) inhibition of high-affinity binding, concurrently with greater than 95% inhibition of CPT activity. These treatments also unmasked an effect of subsequent treatment with palmitoyl-CoA to increase low-affinity [14C]malonyl-CoA binding. 6. These data are discussed in relation to the possible mechanism of interaction between the malonyl-CoA-binding site and the active site of the enzyme.     

Cloning and tissue distribution of a carnitine palmitoyltransferase I gene in rainbow trout (Oncorhynchus mykiss)

The carnitine palmitoyltransferase I (EC.2.3.1.21; CPT I) mediates the transport of fatty acids across the outer mitochondrial membrane. In mammals, there are two different proteins CPT I in the skeletal muscle (M) and liver (L) encoded by two genes. The carnitine palmitoyltransferase system of lower vertebrates received little attention. With the aim of improving knowledge on the CPT family in fish, we examined CPT I cDNA and CPT activity in different tissues of rainbow trout (Oncorhynchus mykiss). Using RT-PCR, we successfully cloned a partial CPT I cDNA sequence (1650 bp). The predicted protein sequence revealed identities of 63% and 61% with human L-CPT I and M-CPT I, respectively. This mRNA is expressed in liver, white and red skeletal muscles, heart, intestine, kidney and adipose tissue of trout. This is in good agreement with the measurement of the CPT activity in the same tissues. The [IC(50)] that reflects the sensitivity to malonyl-CoA inhibition was 0.116+/-0.004 microM for the liver and 0.426+/-0.041 microM for the white muscle. These results demonstrate for the first time the existence of at least one gene encoding for CPT I present in both the liver and the muscle of rainbow trout.     

Interaction of malonyl-CoA and related compounds with mitochondria from different rat tissues. Relationship between ligand binding and inhibition of carnitine palmitoyltransferase I.

The sensitivity of carnitine palmitoyltransferase I (CPT I; EC 2.3.1.21) to inhibition by malonyl-CoA and related compounds was examined in isolated mitochondria from liver, heart and skeletal muscle of the rat. In all three tissues the same order of inhibitory potency emerged: malonyl-CoA much greater than succinyl-CoA greater than methylmalonyl-CoA much greater than propionyl-CoA greater than acetyl-CoA. For any given agent, suppression of CPT I activity was much greater in skeletal muscle than in liver, with the heart enzyme having intermediate sensitivity. With skeletal-muscle mitochondria a high-affinity binding site for [14C]malonyl-CoA was readily demonstrable (Kd approx. 25 nM). The ability of other CoA esters to compete with [14C]malonyl-CoA for binding to the membrane paralleled their capacity to inhibit CPT I. Palmitoyl-CoA also competitively inhibited [14C]malonyl-CoA binding, in keeping with its known ability to overcome malonyl-CoA suppression of CPT I. For reasons not yet clear, free CoA displayed anomalous behaviour in that its competition for [14C]malonyl-CoA binding was disproportionately greater than its inhibition of CPT I. Three major conclusions are drawn. First, malonyl-CoA is not the only physiological compound capable of suppressing CPT I, since chemically related compounds, known to exist in cells, also share this property, particularly in tissues where the enzyme shows the greatest sensitivity to malonyl-CoA. Second, malonyl-CoA and its analogues appear to interact with the same site on the mitochondrial membrane, as may palmitoyl-CoA. Third, the degree of site occupancy by inhibitors governs the activity of CPT I.     

The return of malonyl-CoA to the brain: Cognition and other stories

Nutrients, hormones and the energy sensor AMP-activated protein kinase (AMPK) tightly regulate the intracellular levels of the metabolic intermediary malonyl-CoA, which is a precursor of fatty acid synthesis and a negative regulator of fatty acid oxidation. In the brain, the involvement of malonyl-CoA in the control of food intake and energy homeostasis has been known for decades. However, recent data uncover a new role in cognition and brain development. The sensing of malonyl-CoA by carnitine palmitoyltransferase 1 (CPT1) proteins regulates a variety of functions, such as the fate of neuronal stem cell precursors, the motility of lysosomes in developing axons, the trafficking of glutamate receptors to the neuron surface (necessary for proper synaptic function) and the metabolic coupling between astrocytes and neurons. We discuss the relevance of those recent findings evidencing how nutrients and metabolic disorders impact cognition. We also enumerate all nutritional and hormonal conditions that are known to regulate malonyl-CoA levels in the brain, reflect on protein malonylation as a new post-translational modification, and give a reasoned vision of the opportunities and challenges that future research in the field could address.     

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.     

Identification of conserved amino acid residues in rat liver carnitine palmitoyltransferase I critical for malonyl-CoA inhibition. Mutation of methionine 593 abolishes malonyl-CoA inhibition

Carnitine palmitoyltransferase (CPT) I, which catalyzes the conversion of palmitoyl-CoA to palmitoylcarnitine facilitating its transport through the mitochondrial membranes, is inhibited by malonyl-CoA. By using the SequenceSpace algorithm program to identify amino acids that participate in malonyl-CoA inhibition in all carnitine acyltransferases, we found 5 conserved amino acids (Thr(314), Asn(464), Ala(478), Met(593), and Cys(608), rat liver CPT I coordinates) common to inhibitable malonyl-CoA acyltransferases (carnitine octanoyltransferase and CPT I), and absent in noninhibitable malonyl-CoA acyltransferases (CPT II, carnitine acetyltransferase (CAT) and choline acetyltransferase (ChAT)). To determine the role of these amino acid residues in malonyl-CoA inhibition, we prepared the quintuple mutant CPT I T314S/N464D/A478G/M593S/C608A as well as five single mutants CPT I T314S, N464D, A478G, M593S, and C608A. In each case the CPT I amino acid selected was mutated to that present in the same homologous position in CPT II, CAT, and ChAT. Because mutant M593S nearly abolished the sensitivity to malonyl-CoA, two other Met(593) mutants were prepared: M593A and M593E. The catalytic efficiency (V(max)/K(m)) of CPT I in mutants A478G and C608A and all Met(593) mutants toward carnitine as substrate was clearly increased. In those CPT I proteins in which Met(593) had been mutated, the malonyl-CoA sensitivity was nearly abolished. Mutations in Ala(478), Cys(608), and Thr(314) to their homologous amino acid residues in CPT II, CAT, and ChAT caused various decreases in malonyl-CoA sensitivity. Ala(478) is located in the structural model of CPT I near the catalytic site and participates in the binding of malonyl-CoA in the low affinity site (Morillas, M., Gómez-Puertas, P., Rubi, B., Clotet, J., Ari?o, J., Valencia, A., Hegardt, F. G., Serra, D., and Asins, G. (2002) J. Biol. Chem. 277, 11473-11480). Met(593) may participate in the interaction of malonyl-CoA in the second affinity site, whose location has not been reported.     

Novel effect of C75 on carnitine palmitoyltransferase I activity and palmitate oxidation

C75 is a potential drug for the treatment of obesity. It was first identified as a competitive, irreversible inhibitor of fatty acid synthase (FAS). It has also been described as a malonyl-CoA analogue that antagonizes the allosteric inhibitory effect of malonyl-CoA on carnitine palmitoyltransferase I (CPT I), the main regulatory enzyme involved in fatty acid oxidation. On the basis of MALDI-TOF analysis, we now provide evidence that C75 can be transformed to its C75-CoA derivative. Unlike the activation produced by C75, the CoA derivative is a potent competitive inhibitor that binds tightly but reversibly to CPT I. IC50 values for yeast-overexpressed L- or M-CPT I isoforms, as well as for purified mitochondria from rat liver and muscle, were within the same range as those observed for etomoxiryl-CoA, a potent inhibitor of CPT I. When a pancreatic INS(823/13), muscle L6E9, or kidney HEK293 cell line was incubated directly with C75, fatty acid oxidation was inhibited. This suggests that C75 could be transformed in the cell to its C75-CoA derivative, inhibiting CPT I activity and consequently fatty acid oxidation. In vivo, a single intraperitoneal injection of C75 in mice produced short-term inhibition of CPT I activity in mitochondria from the liver, soleus, and pancreas, indicating that C75 could be transformed to its C75-CoA derivative in these tissues. Finally, in silico molecular docking studies showed that C75-CoA occupies the same pocket in CPT I as palmitoyl-CoA, suggesting an inhibiting mechanism based on mutual exclusion. Overall, our results describe a novel role for C75 in CPT I activity, highlighting the inhibitory effect of its C75-CoA derivative.     

Distinct kinetics of carnitine palmitoyltransferase i in contact sites and outer membranes of rat liver mitochondria

Carnitine palmitoyltransferase I (CPT I) of rat liver mitochondria is an integral, polytopic protein of the outer membrane that is enriched at contact sites. As CPT I kinetics are highly dependent on its membrane environment, we have measured the kinetic parameters of CPT I present in rat liver submitochondrial membrane fractions enriched in either outer membrane or contact sites. The K(m) for palmitoyl-CoA was 2.4-fold higher for CPT I in outer membranes than that for the enzyme in contact sites. In addition, whereas in contact sites malonyl-CoA behaved as a competitive inhibitor of CPT I with respect to palmitoyl-CoA, in outer membranes malonyl-CoA inhibition was non-competitive. As a result of the combination of these changes, the IC(50) for malonyl-CoA was severalfold higher for CPT I in contact sites than for the enzyme in bulk outer membrane. The K(i) for malonyl-CoA, the K(m) for carnitine, and the catalytic constant of the enzyme were all unaffected. It is concluded that the different membrane environments in outer membranes and contact sites result in an altered conformation of L-CPT I that specifically affects the long-chain acyl-CoA binding site. The accompanying changes in the kinetics of the enzyme provide an additional potent mechanism for the regulation of L-CPT I activity.     

Hypothalamic malonyl-CoA and CPT1c in the treatment of obesity

Metabolic integration of nutrient sensing in the central nervous system has been shown to be an important regulator of adiposity by affecting food intake and peripheral energy expenditure. Modulation of de novo fatty acid synthetic flux by cytokines and nutrient availability plays an important role in this process. Inhibition of hypothalamic fatty acid synthase by pharmacologic or genetic means leads to an increased malonyl-CoA level and suppression of food intake and adiposity. Conversely, the ectopic expression of malonyl-CoA decarboxylase in the hypothalamus is sufficient to promote feeding and adiposity. Based on these and other findings, metabolic intermediates in fatty acid biogenesis, including malonyl-CoA and long-chain acyl-CoAs, have been implicated as signaling mediators in the central control of body weight. Malonyl-CoA has been hypothesized to mediate its effects in part through an allosteric interaction with an atypical and brain-specific carnitine palmitoyltransferase-1 (CPT1c). CPT1c is expressed in neurons and binds malonyl-CoA, however, it does not perform the same biochemical function as the prototypical CPT1 enzymes. Mouse knockout models of CPT1c exhibit suppressed food intake and smaller body weight, but are highly susceptible to weight gain when fed a high-fat diet. Thus, the brain can directly sense and respond to changes in nutrient availability and composition to affect body weight and adiposity.     

Use of a selectively permeabilized isolated rat hepatocyte preparation to study changes in the properties of overt carnitine palmitoyltransferase activity in situ.

1. A permeabilized isolated rat liver cell preparation was developed to achieve selective permeabilization of the cell membrane to metabolites and to allow the assay of mitochondrial overt carnitine palmitoyltransferase (CPT I) activity in situ. By performing the digitonin-induced permeabilization in the presence of fluoride and bivalent-metal-cation sequestrants, it was possible to demonstrate that the activity of other enzymes, which are regulated by reversible phosphorylation, was preserved during the procedure and subsequent washing of cells before assay. 2. CPT activity at a sub-optimal palmitoyl-CoA concentration was almost totally (approximately 90%) inhibited by malonyl-CoA, indicating that mitochondrial CPT I was largely measured in this preparation. 3. The palmitoyl-CoA-saturation and malonyl-CoA-inhibition curves for CPT activity in permeabilized cells were very similar to those obtained previously for the enzyme in isolated liver mitochondria. Moreover, starvation and diabetes had the same effects on enzyme activity, affinity for palmitoyl-CoA and malonyl-CoA sensitivity of CPT I in isolated cells as found in isolated mitochondria. These physiologically induced changes persisted through the cell preparation and incubation period. 4. Neither incubation of cells with glucagon or insulin nor incubation with pyruvate and lactate before permeabilization resulted in alterations of these parameters of CPT I in isolated cells. 5. The results are discussed in relation to the temporal relationships of changes in the activity and properties of CPT I in vivo in relation to the effects of insulin and glucagon on fatty acid metabolism in vivo.