Pig Liver Carnitine Palmitoyltransferase I, with Low <i>K</i><sub>m</sub> for Carnitine and High Sensitivity to Malonyl-CoA Inhibition, Is a Natural Chimera of Rat Liver and Muscle Enzymes

Nicot, Carine; Hegardt, Fausto G.; Woldegiorgis, Gebre; Haro, Diego; Marrero, Pedro F.

doi:10.1021/bi0024106

Cited by 27 publications

(45 citation statements)

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“…In contrast, the pig IC 50 value was much lower than that determined in newborn rabbits and fasted rats (23,33). The difference may be due to the atypical chimeric structure of the porcine liver enzyme, containing a muscle-CPT I binding site for carnitine and malonyl-CoA (28). In ketogenic animals, regulation of fatty acid oxidation and ketogenesis may be mediated by both changes in the amount of malonyl-CoA and/or activity of CPT I.…”

Section: Discussionmentioning

confidence: 70%

“…Most recent studies indicate that the key regulatory enzyme-hepatic CPT I-has an atypical molecular structure with a limited expression in pigs compared with other mammalian species (28,29). The porcine CPT I protein is a natural chimera of the more typical mammalian liver (L) and muscle (M)-CPT I isotypes, containing the L-CPT I binding site for acyl-CoA and the M-CPT I binding sites for carnitine and malonyl-CoA.…”

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confidence: 99%

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Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid β-oxidation in liver of neonatal swine

Lin

Shim

Odle

2010

American Journal of Physiology-Regulatory, Integrative and Comp

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Lin X, Shim K, Odle J. Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid ␤-oxidation in liver of neonatal swine. Am J Physiol Regul Integr Comp Physiol 298: R1435-R1443, 2010. First published March 17, 2010 doi:10.1152/ajpregu.00634.2009.-To examine the regulation of hepatic acetogenesis in neonatal swine, carnitine palmitoyltransferase I (CPT I) activity was measured in the presence of varying palmitoyl-CoA (substrate) and malonyl-CoA (inhibitor) concentrations, and [1-14 C]-palmitate oxidation was simultaneously measured. Accumulation rates of 14 C-labeled acetate, ketone bodies, and citric acid cycle intermediates within the acid-soluble products were determined using radio-HPLC. Measurements were conducted in mitochondria isolated from newborn, 24-h (fed or fasted), and 5-mo-old pigs. Acetate rather than ketone bodies was the predominant radiolabeled product, and its production increased twofold with increasing fatty acid oxidation during the first 24-h suckling period. The rate of acetogenesis was directly proportional to CPT I activity. The high activity of CPT I in 24-h-suckling piglets was not attributable to an increase in CPT I gene expression, but rather to a large decrease in the sensitivity of CPT I to malonyl-CoA inhibition, which offset a developmental decrease in affinity of CPT I for palmitoyl-CoA. Specifically, the IC 50 for malonyl-CoA inhibition and Km value for palmitoyl-CoA measured in 24-h-suckling pigs were 1.8-and 2.7-fold higher than measured in newborn pigs. The addition of anaplerotic carbon from malate (10 mM) significantly reduced 14 C accumulation in acetate (P Ͻ 0.003); moreover, the reduction was much greater in newborn (80%) than in 24-h-fed (72%) and 5-mo-old pigs (55%). The results demonstrate that acetate is the primary product of hepatic mitochondrial ␤-oxidation in Sus scrofa and that regulation during early development is mediated primarily via kinetic modulation of CPT I. acetate; anaplerosis; carnitine palmitoyltransferase I activity; ketone bodies; mitochondria; Sus scrofa IT IS WELL ESTABLISHED THAT hepatic long-chain fatty acid oxidation is acutely controlled by a system in which carnitine palmitoyltransferase I (CPT I) is regulated allosterically by a change in malonyl-CoA concentration and/or the sensitivity to malonyl-CoA inhibition. This regulatory mechanism dictates fatty acid flux into mitochondria and controls the rates of ␤-oxidation and ketogenesis based on the animal's physiological status. The neonatal period represents a physiological state characterized by marked enhancement of fatty acid oxidation and ketogenesis. Both humans and rats present a significant hyperketonemia during the suckling period (40). Evidence confirms that the physiological hyperketonemia is a result of the high hepatic ketogenic rate in neonates consuming a diet (i.e., milk) that is high in fat and low in carbohydrate. In fact, more than 90% of the non-CO 2 carbon derived from fatty acid oxidation in liver homogenates is ketone bodies (21). Wh...

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Section: Discussionmentioning

confidence: 70%

mentioning

confidence: 99%

Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid β-oxidation in liver of neonatal swine

Lin

Shim

Odle

2010

American Journal of Physiology-Regulatory, Integrative and Comp

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“…Construction of Expression Plasmids-Pig L-CPTI and rat L-CPTI were subcloned into pHW010, generating PLCPTI/pHW010 and RL-CPTI/pHW010 as previously described (11,12). To generate the chimeras, a HindIII site (shown in italics in the primer sequence below) was introduced into the cDNAs of both enzymes by overlap extension.…”

Section: Methodsmentioning

confidence: 99%

“…Pig L-CPTI encodes for a 772-amino acid protein that shares 86 and 63% identity with rat L-CPTI and human M-CPTI, respectively. When expressed in the yeast Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics similar to human or rat L-CPTI (11). However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition that is characteristic of human or rat M-CPTI enzymes (10,11).…”

mentioning

confidence: 99%

“…When expressed in the yeast Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics similar to human or rat L-CPTI (11). However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition that is characteristic of human or rat M-CPTI enzymes (10,11). Therefore, pig L-CPTI behaves like a natural chimera of the L-and M-CPTI isotypes, which makes it a useful model to study the structure-function relationships of the CPTI enzymes (11).…”

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confidence: 99%

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Pig Liver Carnitine Palmitoyltransferase

Nicot¹,

Relat²,

Woldegiorgis³

et al. 2002

Journal of Biological Chemistry

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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 M. 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 Cterminal chimeras.The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) 1 catalyzes the conversion of long-chain acyl-CoAs to acylcarnitines in the presence of L-carnitine. This is the initial step in the entry of long-chain fatty acids into the mitochondrial matrix, where they undergo ␤ oxidation. This is also the first rate-limiting step in fatty acid oxidation in all tissues and is highly regulated by its physiological inhibitor, malonyl-CoA (1). Inhibition of CPTI by malonyl-CoA, the first intermediate in fatty acid synthesis, allows the cell to coordinate fatty acid oxidation and synthesis (2). Understanding the regulation of CPTI by malonyl-CoA is important since this enzyme is a potential pharmacological target in type 2 diabetes, where excessive fatty acid oxidation is undesirable and must be controlled.There are two isotypes of CPTI: a liver (L-CPTI) and a muscle (M-CPTI) isotype. Both genes encode for proteins, which share a high degree of identity but that differ markedly in their kinetic characteristics. L-CPTI has a much lower K m for carnitine and a higher IC 50 for malonyl-CoA inhibition than the muscle isotype (1). These kinetic characteristics of the liver and muscle enzymes are also retained when the cDNAs are expressed in a heterologous system (3-6). CPTI is an integral membrane protein that has two ␣-helical hydrophobic transmembrane domains, the amino and most of the carboxyl regions being exposed to the cytosolic face of the membrane (7).It has been shown that the first 18 N-terminal amino acids of L-CPTI play a critical role in malonyl-CoA sensitivity since deletion of this region leads to loss of inhibition by malonyl-CoA (8). Further work demonstrated that this region acts as a positive determinant of malonyl-CoA sensitivity, while amino acids 19 -30 act as a negative determinant (9). Interestingly, although the first 18 amino acids of rat L-CPTI and M-CPTI are identical, the role of the M-CPTI N-term...

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Lipids and Lipid Utilization in Swine

Lin

Azain

Odle

2012

Sustainable Swine Nutrition

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Pig Liver Carnitine Palmitoyltransferase I, with Low K_m for Carnitine and High Sensitivity to Malonyl-CoA Inhibition, Is a Natural Chimera of Rat Liver and Muscle Enzymes

Cited by 27 publications

References 29 publications

Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid β-oxidation in liver of neonatal swine

Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid β-oxidation in liver of neonatal swine

Pig Liver Carnitine Palmitoyltransferase

Lipids and Lipid Utilization in Swine

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Pig Liver Carnitine Palmitoyltransferase I, with Low Km for Carnitine and High Sensitivity to Malonyl-CoA Inhibition, Is a Natural Chimera of Rat Liver and Muscle Enzymes

Cited by 27 publications

References 29 publications

Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid β-oxidation in liver of neonatal swine

Carnitine palmitoyltransferase I control of acetogenesis, the major pathway of fatty acid β-oxidation in liver of neonatal swine

Pig Liver Carnitine Palmitoyltransferase

Lipids and Lipid Utilization in Swine

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Pig Liver Carnitine Palmitoyltransferase I, with Low K_m for Carnitine and High Sensitivity to Malonyl-CoA Inhibition, Is a Natural Chimera of Rat Liver and Muscle Enzymes