Review
Fatty acid import into mitochondria

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Abstract

The mitochondrial carnitine system plays an obligatory role in β-oxidation of long-chain fatty acids by catalyzing their transport into the mitochondrial matrix. This transport system consists of the malonyl-CoA sensitive carnitine palmitoyltransferase I (CPT-I) localized in the mitochondrial outer membrane, the carnitine:acylcarnitine translocase, an integral inner membrane protein, and carnitine palmitoyltransferase II localized on the matrix side of the inner membrane. Carnitine palmitoyltransferase I is subject to regulation at the transcriptional level and to acute control by malonyl-CoA. The N-terminal domain of CPT-I is essential for malonyl-CoA inhibition. In liver CPT-I activity is also regulated by changes in the enzyme’s sensitivity to malonyl-CoA. As fluctuations in tissue malonyl-CoA content are parallel with changes in acetyl-CoA carboxylase activity, which in turn is under the control of 5′-AMP-activated protein kinase, the CPT-I/malonyl-CoA system is part of a fuel sensing gauge, turning off and on fatty acid oxidation depending on the tissue’s energy demand. Additional mechanism(s) of short-term control of CPT-I activity are emerging. One proposed mechanism involves phosphorylation/dephosphorylation dependent direct interaction of cytoskeletal components with the mitochondrial outer membrane or CPT-I. We have proposed that contact sites between the outer and inner mitochondrial membranes form a microenvironment which facilitates the carnitine transport system. In addition, this system includes the long-chain acyl-CoA synthetase and porin as components.

Introduction

Fatty acids represent a major energy source for the heart and skeletal muscle. In the liver, oxidation of fatty acids also serves an additional role. It produces ketone bodies which are important fuels for extrahepatic organs. The β-oxidation of activated fatty acids occurs within the mitochondrial matrix and is catalyzed by the sequential action of four enzyme families (acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase), each with different substrate specificity for short-, medium- and long-chain acyl-CoAs [1], [2]. Long-chain fatty acids are activated on the mitochondrial outer membrane by the long-chain acyl-CoA synthetase (LCAS) but the mitochondrial inner membrane is not permeable to these acyl-CoAs. The carnitine dependent transport of these activated fatty acids precedes their β-oxidative chain shortening. This transport system consists of three proteins, carnitine palmitoyltransferase I (CPT-I), carnitine:acylcarnitine translocase (CACT) and carnitine palmitoyltransferase II (CPT-II), each with a different submitochondrial localization. As a first step, acyl-CoAs formed by the catalytic action of LCAS in the mitochondrial outer membrane (MOM) are converted to acylcarnitines. This transesterification is catalyzed by CPT-I, also localized in the MOM. The reaction products, long-chain acylcarnitines, are then translocated into the mitochondrial matrix in an exchange reaction catalyzed by CACT, an integral inner membrane protein. Within the matrix the acylcarnitines are then reconverted to the respective acyl-CoAs by CPT-II, an enzyme associated with the inner leaflet of the mitochondrial inner membrane (MIM).

Although the importance of the mitochondrial carnitine system in overall fatty acid oxidation has long been recognized [3], [4], significant progress in our understanding of the structure, function and regulation of the mitochondrial carnitine system has only been possible with the development and application of molecular biology tools. The scope of this presentation is to provide a brief review about our current knowledge of the individual enzymes of the carnitine dependent mitochondrial fatty acid uptake mechanism, their structural and functional organization and the role of this pathway in the regulation of overall mitochondrial fatty acid oxidation.

Section snippets

Cellular uptake and activation of long-chain fatty acids

Long-chain fatty acids represent a main energy source for many organs, especially for muscle and liver. Since most tissues contain only small amounts of storage lipids, energy production depends on a continuous supply of fatty acids, mostly from adipose tissue. Fatty acids are produced by lipolysis, transported bound to albumin in blood and taken up by tissues in a process mediated by transport proteins present in the plasma membrane [5]. Once within the cell, free fatty acids are bound to

Carnitine palmitoyltransferase I

CPT-I catalyzes the formation of long-chain acylcarnitines from activated fatty acids and free carnitine, thus committing them to oxidation. By virtue of its inhibition by malonyl-CoA, CPT-I represents a key regulatory site controlling the flux through β-oxidation. Consistent with its central role in mitochondrial fatty acid oxidation, the enzyme exists in at least two isoforms with significantly different kinetic and regulatory properties [16]. The liver type (L-CPT-I) displays higher affinity

Regulation at transcriptional level

A rapid increase in CPT-I mRNA abundance and CPT-I activity was observed in liver immediately after birth, and coincided with the switch to a high-fat low-carbohydrate diet (milk). CPT-I activity remains high when rats are weaned to a high-fat diet but not when weaned to a high-carbohydrate diet [48]. Similarly, increased enzyme activities and mRNA content were found in livers of fasted and insulin dependent diabetic rats [51]. These changes in mRNA abundance coincided with changes in the

The mitochondrial carnitine system: structure-function relationships

In the foregoing, we described in some detail the properties and membrane topology of the individual components of the mitochondrial carnitine system. The reversibility of the reactions, the localization of enzymes in different membranes and the limited water solubility of intermediates would suggest an inefficient system. So how can this fatty acid transport system function as efficiently as observed in vivo? A kinetic based explanation would be that under physiological conditions the inward

Interorganellar acyl traffic and reverse operation of the mitochondrial carnitine system

The mitochondrial uptake of activated fatty acids requires all three components of the mitochondrial carnitine system. However, CACT, CPT-II and carnitine acetyltransferase (CAT) seem to have additional functions, namely shuttling chain-shortened fatty acids from peroxisomes into the mitochondria and buffering the mitochondrial free CoASH/acyl-CoA ratio and [109]. Additionally, the role of carnitine in conjunction with the respective carnitine acyltransferases in interorganellar acyl traffic

Future directions

In light of the pivotal role of CPT-I in overall mitochondrial fatty acid oxidation a greater understanding of the molecular mechanisms for malonyl-CoA and etomoxiryl-CoA inhibition, as well as isoform selectivity should provide insight for the design of drugs as inhibitors of this critical step. These drugs would be advantageous in the treatment of diabetes mellitus [118], [119] and myocardial ischemia/reperfusion injury [64], [79]. Furthermore, identification of the topography of the outer

Acknowledgements

We thank Drs. Edward Lesnefsky and John Mieyal for reviewing the manuscript and Peter Turkaly for his help in preparing the figures and manuscript. This research was supported by the Medical Research Service of the Department of Veterans Affairs and NIH grant POI AG15885-01.

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