Liver Carnitine Palmitoyltransferase I Research Articles

Acyl-CoA binding proteins interact with the acyl-CoA binding domain of mitochondrial carnitine palmitoyl transferase I

Although the rate limiting step in mitochondrial fatty acid oxidation, catalyzed by carnitine palmitoyl transferase I (CPTI), utilizes long-chain fatty acyl-CoAs (LCFA-CoA) as a substrate, how LCFA-CoA is transferred to CPTI remains elusive. Based on secondary structural predictions and conserved tryptophan residues, the cytoplasmic C-terminal domain was hypothesized to be the LCFA-CoA binding site and important for interaction with cytoplasmic LCFA-CoA binding/transport proteins to provide a potential route for LCFA-CoA transfer. To begin to address this question, the cytoplasmic C-terminal region of liver CPTI (L-CPTI) was recombinantly expressed and purified. Data herein showed for the first time that the L-CPTI C-terminal 89 residues were sufficient for high affinity binding of LCFA-CoA (K (d) = 2-10 nM) and direct interaction with several cytoplasmic LCFA-CoA binding proteins (K (d) < 10 nM), leading to enhanced CPTI activity. Furthermore, alanine substitutions for tryptophan in L-CPTI (W391A and W452A) altered secondary structure, decreased binding affinity for LCFA-CoA, and almost completely abolished L-CPTI activity, suggesting that these amino acids may be important for ligand stabilization necessary for L-CPTI activity. Moreover, while decreased activity of the W452A mutant could be explained by decreased binding of lipid binding proteins, W391 itself seems to be important for activity. These data suggest that both interactions with lipid binding proteins and the peptide itself are important for optimal enzyme activity.

Effects of trans-10,cis-12 CLA on liver size and fatty acid oxidation under energy restriction conditions in hamsters

ObjectiveLittle evidence exists concerning the effects of trans-10,cis-12 conjugated linoleic acid (CLA) under energy restriction. Thus, the effects of this CLA isomer on adipose tissue size, liver composition, as well as on expression and activity of carnitine-palmitoyl transferase I (CPT-I) and acyl CoA oxidase (ACO), in hamsters fed an energy-restricted diet were analyzed. MethodsHamsters were fed a high-fat diet for 7 wk and then subjected to 25% energy-restricted diets supplemented with 0.5% linoleic acid or 0.5% trans-10,cis-12 CLA for 3 wk. Serum insulin, free-triiodothyronine and non-esterified fatty acid levels, liver triacylglycerol, protein and water contents, and CPT-I, ACO, and Peroxisome proliferator-activated receptor alpha (PPARα) expressions and enzyme activities were assessed. ResultsEnergy restriction reduced liver size, serum levels of insulin, free-triiodothyronine, and non-esterified fatty acid and increased CPT-I activity. Liver composition was not modified. No differences were found between both restricted groups, with the exception of CPT-I and ACO oxidative enzyme activities, which were greater in hamsters fed the CLA diet. ConclusionsEnergy restriction does not cause trans-10,cis-12 CLA to induce liver hyperplasia. Although this CLA isomer increases liver CPT-I and ACO activities, this effect does not result in reduced hepatic triacylglyerol content or decreased adipose tissue size. Consequently, this CLA isomer seems not to be a useful tool for inclusion in body weight loss strategies followed during obesity treatment.

Mass spectrometric demonstration of the presence of liver carnitine palmitoyltransferase-I (CPT-I) in heart mitochondria of adult rats

The carnitine palmitoyltransferase-I (CPT-I) enzymes catalyze the regulated step in overall mitochondrial fatty acid oxidation. The liver and muscle isoforms are expressed in liver and skeletal muscle respectively with the isoforms exhibiting different kinetic properties and apparent molecular weight masses. In contrast, the heart expresses both isoforms at the mRNA level. However, for the expression of the liver isoform at the protein level only indirect evidence is available, such as tagging with radiolabeled CPT-I inhibitors followed by SDS-PAGE separation and kinetic analysis using inhibitors. The importance of fatty acid oxidation in the heart and the potential regulation via the liver isoform of CPT-I demands proof of the liver isoform in the heart. Using a proteomic approach in the present study we demonstrate that rat heart mitochondria (a) contain both the muscle and liver isoforms; (b) both proteins retain their C- and N-termini; (c) the N-terminal alanine residues are acetylated; (d) and in rat heart mitochondria the liver isoform is phosphorylated on tyrosine 281. By providing amino acid sequence information this is the first unequivocal demonstration that the liver isoform of CPT-I is expressed at the protein level in adult rat heart mitochondria and that the apparent smaller molecular size of the muscle isoform is not due to proteolytic truncation.

Post‐translational Modifications of the Mitochondrial Outer Membrane Proteins: Carnitine Palmitoyltransferase‐I, Long‐chain acyl‐CoA synthetase, and Voltage Dependent Anion Channel

Long-chain fatty acids require activation and transport into the mitochondrial matrix before â-oxidation can occur. Carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage dependent anion channel (VDAC), localized in the mitochondrial outer membrane play a critical role in this transport process and are the focus of our study. We have developed a method for analysis of hydrophobic membrane proteins based on isolation of resident membranes, separation of proteins by gel electrophoresis, electroelution of the proteins, and enzymatic protein digestion by trypsin and proteinase K. The resulting peptides from the digestions were analyzed by MALDI-TOF and HPLC ESI-MS/MS. With this method, we have achieved 75% to 99% sequence coverage for the mentioned transmembrane proteins. This method provides a targeted approach for examining membrane proteins in detail including post-translational modifications and identification of less abundant isoforms. In summary using our method: (a) we documented phosphorylation sites on CPT-I, VDAC, and LCAS using peptide mass fingerprinting and ESI-MS/MS, (b) on CPT-I, we identified a nitrated tyrosine in position 589, (c) we provided evidence for acetylation of the N-terminal alanine in VDAC-1, and (d) we found that in the mature rat liver CPT-I the cDNA deduced N-terminal methionine is removed and alanine is acetylated. Studies regarding the functional/regulatory impact of these modifications are currently being addressed. Supported by DK-066107, PO1 AG15885, and VA Medical Research Service

Carnitine: a nutritional, biosynthetic, and functional perspective

Carnitine status in humans is reported to vary according to body composition, gender, and diet. Plasma carnitine concentration positively correlates with the dietary intake of carnitine. The content of carnitine in foodstuff is based on old and inadequate methodology. Nevertheless, dietary carnitine is important. The molecular biology of the enzymes of carnitine biosynthesis has recently been accomplished. Carnitine biosynthesis requires pathways in different tissues and is an efficient system. Overall biosynthesis is determined by the availability of trimethyllysine from tissue proteins. Carnitine deficiency resulting from a defect in biosynthesis has yet to be reported. The role of carnitine in long-chain fatty acid oxidation is well defined. Recent evidence supports a role for the voltage-dependent anion channel in the transport of acyl-CoAs through the mitochondrial outer membrane. The mitochondrial outer membrane carnitine palmitoyltransferase-I in liver can be phosphorylated and when phosphorylated the sensitivity to malonyl-CoA is greatly decreased. This may explain the change in sensitivity of liver carnitine palmitoyltransferase-I observed during fasting and diabetes. Recently reported data clarify the role of carnitine and the carnitine transport system in the interplay between peroxisomes and mitochondrial fatty acid oxidation. Lastly, the buffering of the acyl-CoA/CoA coupled by carnitine reflects intracellular metabolism. This mass action effect underlies the use of carnitine as a therapeutic agent. In summary, these new observations help to further our understanding of the molecular aspects of carnitine in medicine.

The Extreme C Terminus of Rat Liver Carnitine Palmitoyltransferase I Is Not Involved in Malonyl-CoA Sensitivity but in Initial Protein Folding

We previously reported that the N-terminal domain (1-147 residues) of rat liver carnitine palmitoyltransferase I (L-CPTI) was essential for import into the outer mitochondrial membrane and for maintenance of a malonyl-CoA-sensitive conformation. Malonyl-CoA binding experiments using mitochondria of Saccharomyces cerevisiae strains expressing wild-type L-CPTI or previously constructed chimeric CPTs (Cohen, I., Kohl, C., McGarry, J.D., Girard, J., and Prip-Buus, C. (1998) J. Biol. Chem. 273, 29896-29904) indicated that the N-terminal domain was unable, independently of the C-terminal domain, to bind malonyl-CoA with a high affinity, suggesting that the modulation of malonyl-CoA sensitivity occurred through N/C intramolecular interactions. To assess the role of the C terminus in malonyl-CoA sensitivity, a series of C-terminal deletion mutants was generated. The kinetic properties of Delta772-773 and Delta767-773 deletion mutants were similar to those of L-CPTI, indicating that the last two highly conserved Lys residues in all known L-CPTI species were not functionally essential. By contrast, Delta743-773 deletion mutant was totally inactive and unfolded, as shown by its sensitivity to trypsin proteolysis. Because the C terminus of the native folded L-CPTI could be cleaved by trypsin without inducing protein unfolding, we concluded that the last 31 C-terminal residues constitute a secondary structural determinant essential for the initial protein folding of L-CPTI.

Pig Liver Carnitine Palmitoyltransferase: CHIMERA STUDIES SHOW THAT BOTH THE N- AND C-TERMINAL REGIONS OF THE ENZYME ARE IMPORTANT FOR THE UNUSUAL HIGH MALONYL-CoA SENSITIVITY

Pig and rat liver carnitine palmitoyltransferase I (L-CPTI) share common K(m) values for palmitoyl-CoA and carnitine. However, they differ widely in their sensitivity to malonyl-CoA inhibition. Thus, pig l-CPTI has an IC(50) for malonyl-CoA of 141 nm, while that of rat L-CPTI is 2 microm. Using chimeras between rat L-CPTI and pig L-CPTI, we show that the entire C-terminal region behaves as a single domain, which dictates the overall malonyl-CoA sensitivity of this enzyme. The degree of malonyl-CoA sensitivity is determined by the structure adopted by this domain. Using deletion mutation analysis, we show that malonyl-CoA sensitivity also depends on the interaction of this single domain with the first 18 N-terminal amino acid residues. We conclude that pig and rat L-CPTI have different malonyl-CoA sensitivity, because the first 18 N-terminal amino acid residues interact differently with the C-terminal domain. This is the first study that describes how interactions between the C- and N-terminal regions can determine the malonyl-CoA sensitivity of L-CPTI enzymes using active C-terminal chimeras.

Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARα activation

3-Thia fatty acids are modified fatty acids that promote hepatic peroxisome proliferation and decrease serum triacylglycerol, cholesterol and free fatty acid levels in rats. In vivo administration of tetradecylthioacetic acid (TTA) to rats led to a significant decrease in liver apolipoproteins apoA-I, A-II, A-IV, and C-III mRNA levels, and to an increase of liver acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-II, and 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-CoA synthase) mRNA levels and activities. By contrast, no significant changes of lipoprotein lipase (LPL) mRNA levels were detected in rat epididymal adipose tissue. Liver carnitine palmitoyltransferase-I, apoB, apoE, and LDL receptor mRNA levels were not significantly affected. When tested in vitro, TTA increased rat ACO and carnitine palmitoyltransferase-I mRNA levels in primary rat hepatocytes and also LPL mRNA levels in 3T3-L1 preadipocytes. TTA also enhanced the transcriptional activity of chimeras containing the DNA binding domain of the yeast transcription factor Gal4 fused to the ligand binding domain of either human PPARalpha or human PPARgamma. The effect depended on the concentration tested and the cell type. In conclusion, our data suggest that in vitro, TTA activates both PPARalpha and PPARgamma, but the latter with much lower affinity. TTA affects serum lipid levels in vivo in rats by acting mainly on the liver via PPARalpha where it decreases the liver expression of genes involved in vascular lipid transport and increases the expression of genes involved in intracellular fatty acid metabolism. -Raspé, E., L. Madsen, A-M. Lefebvre, I. Leitersdorf, L. Gelman, J. Peinado-Onsurbe, J. Dallongeville, J-C. Fruchart, R. Berge, and B. Staels. Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARalpha activation.

A Single Amino Acid Change (Substitution of Glutamate 3 with Alanine) in the N-terminal Region of Rat Liver Carnitine Palmitoyltransferase I Abolishes Malonyl-CoA Inhibition and High Affinity Binding

We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.

Role of carnitine palmitoyltransferase I in the control of ketogenesis in primary cultures of rat astrocytes.

The role of carnitine palmitoyltransferase I (CPT-I) in the control of ketogenesis was studied in primary cultures of rat astrocytes. Ketone bodies were the major product of [14C]palmitate oxidation by cultured astrocytes, whereas CO2 made a minor contribution to the total oxidation products. Using tetradecylglycidate as a specific, cell-permeable inhibitor of CPT-I, a flux control coefficient of 0.77 +/- 0.07 was calculated for CPT-I over the flux of [14C]palmitate to ketone bodies. CPT-I from astrocytes was sensitive to malonyl-CoA (IC50 = 3.4 +/- 0.8 microM) and cross-reacted on western blots with an antibody raised against liver CPT-I. On the other hand, astrocytes expressed significant acetyl-CoA carboxylase (ACC) activity, and consequently they contained considerable amounts of malonyl-CoA. Western blot analysis of ACC isoforms showed that ACC in astrocytes--like in neurons, liver, and white adipose tissue--mostly comprised the 265-kDa isoform, whereas the 280-kDa isoform--which was highly expressed in skeletal muscle--showed much lower abundance. Forskolin was used as a tool to study the modulation of the ketogenic pathway in astrocytes. Thus, forskolin decreased in parallel ACC activity and intracellular malonyl-CoA levels, whereas it stimulated CPT-I activity and [14C]palmitate oxidation to both ketone bodies and CO2. Results show that in cultured astrocytes (a) CPT-I exerts a very high degree of control over ketogenesis from palmitate, (b) the ACC/malonyl-CoA/CPT-I system is similar to that of liver, and (c) the ACC/malonyl-CoA/CPT-I system is subject to regulation by cyclic AMP.

Deletion of the conserved first 18 N-terminal amino acid residues in rat liver carnitine palmitoyltransferase I abolishes malonyl-CoA sensitivity and binding.

To assess the role of the 130 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) on malonyl-CoA sensitivity and binding, we constructed a series of mutants with deletions of the 18, 35, 52, 73, 83, or 129 most N-terminal amino acid residues. The deletion mutants were expressed in the yeast Pichia pastoris. We determined the effects of these mutations on L-CPTI activity, malonyl-CoA sensitivity, and binding in isolated mitochondria prepared from the yeast strains expressing the wild-type and deletion mutants. The mutant protein that lacked the first 18 N-terminal amino acid residues, Delta18, had activity and kinetic properties similar to wild-type L-CPTI, but it was almost completely insensitive to malonyl-CoA inhibition (I50 = 380 microM versus 2.0 microM). In addition, loss of malonyl-CoA sensitivity in Delta18 was accompanied by a 70-fold decrease in affinity for malonyl CoA (KD = 70 nM versus 1.1 nM) compared to wild-type L-CPTI. Deletion of the first 35, 52, 73, and 83 N-terminal amino acid residues had a similar effect on malonyl-CoA sensitivity as did the 18-residue deletion mutant, and there was a progressive reduction in the affinity for malonyl-CoA binding. By contrast, deletion of the first 129 N-terminal amino acid residues resulted in the synthesis of an inactive protein. To our knowledge, this is the first report to demonstrate a critical role for these perfectly conserved first 18 N-terminal amino acid residues of L-CPTI in malonyl-CoA sensitivity and binding.

Differential regulation in the heart of mitochondrial carnitine palmitoyltransferase-I muscle and liver isoforms.

Carnitine palmitoyltransferase-I (CPT-I) plays a crucial role in regulating cardiac fatty acid oxidation which provides the primary source of energy for cardiac muscle contraction. CPT-I catalyzes the transfer of long chain fatty acids into mitochondria and is recognized as the primary rate controlling step in fatty acid oxidation. Molecular cloning techniques have demonstrated that two CPT-I isoforms exist as genes encoding the 'muscle' and 'liver' enzymes. Regulation of fatty acid oxidation rates depends on both short-term regulation of enzyme activity and long-term regulation of enzyme synthesis. Most early investigations into metabolic control of fatty acid oxidation at the CPT-I step concentrated on the hepatic enzyme which can be inhibited by malonyl-CoA and can undergo dramatic amplification or reduction of its sensitivity to inhibition by malonyl-CoA. The muscle CPT-I is inherently more sensitive to malonyl-CoA inhibition but has not been found to undergo any alteration of its sensitivity. Short-term control of activity of muscle CPT-I is apparently regulated by malonyl-CoA concentration in response to fuel supply (glucose, lactate, pyruvate and ketone bodies). The liver isoform is the only CPT-I enzyme present in the mitochondria of liver, kidney, brain and most other tissues while muscle CPT-I is the sole isoform expressed in skeletal muscle as well as white and brown adipocytes. The heart is unique in that it contains both muscle and liver isoforms. Liver CPT-I is highly expressed in the fetal heart, but at birth its activity begins to decline whereas the muscle isoform, which is very low at birth, becomes the predominant enzyme during postnatal development. In this paper, the differential regulation of the two CPT-I isoforms at the protein and the gene level will be discussed.

Functional Studies of Yeast-Expressed Human Heart Muscle Carnitine Palmitoyltransferase I

Long-chain fatty acids are the primary source of energy production in the heart. Carnitine palmitoyltrans- ferase I (CPT-I) catalyzes the first reaction in the transport of long-chain fatty acids from the cytoplasm to the mitochondrion, a rate-limiting step in β-oxidation. In this study, we report the functional expression of the human heart/skeletal muscle isoform of CPT-I (M-CPT-I) in the yeastPichia pastoris. Screening of a human heart cDNA library with cDNA fragments encoding the rat heart M-CPT-I resulted in the isolation of a single full-length human heart M-CPT-I cDNA clone. The clone has an open reading frame of 2316 bp with a 5′ untranslated region of 38 bp and a 256-bp 3′ untranslated region with the poly(A)+addition sequence AATAAA. The predicted protein has 772 amino acids and a molecular mass of 88 kDa. Northern blot analysis of mRNAs from different human tissues using the human M-CPT-I cDNA as a probe revealed an abundant transcript of ∼3.1 kb that was only present in human heart and skeletal muscle tissue. Expression of the human M-CPT-I cDNA inP. pastoris,a yeast with no endogenous CPT activity, produced an 80-kDa protein that was located in the mitochondria. Isolated mitochondria from the M-CPT-I expression strain exhibited a malonyl-coenzyme A (CoA)-sensitive CPT activity that was detergent labile. TheI50for malonyl-CoA inhibition of the yeast-expressed M-CPT-I was 69 nm, and theKms for carnitine and palmitoyl-CoA were 666 and 42 μm, respectively. TheI50for malonyl-CoA inhibition of the heart enzyme is 30 times lower than that of the yeast-expressed liver CPT-I, and theKmfor carnitine is more than 20 times higher than that of the liver CPT-I. This is the first report of the expression of a heart CPT-I in a system devoid of endogenous CPT activity and the functional characterization of a human heart M-CPT-I in the absence of the liver isoform and CPT-II.

Effect of feeding pattern on the sensitivity of hepatic carnitine palmitoyl-transferase to inhibition by malonyl-CoA in the rat

1. 1. The feeding pattern influences the inhibitory effects of malonyl-CoA on carnitine palmitoyltransferase-I. 2. 2. The sensitivity of liver carnitine palmitoyltransferase-I to malonyl-CoA is increased in rats meal-fed when compared to rats fed ad libitum. 3. 3. Moreover, liver carnitine palmitoyltransferase-I of meal-fed rats remains more sensitive to inhibition by malonyl-CoA during a 24 hour fast than liver carnitine palmitoyltransferase-I of rats previously fed ad libitum.