control of mitochondrial β-oxidation flux

Review

Control of mitochondrial b-oxidation flux

Simon Eaton*

Surgery Unit and Biochemistry, Endocrinology and Metabolism Unit,Institute of Child Health (University College London), 30 Guilford Street, London WC1N 1EH, UK

Received 9 November 2001; accepted 20 November 2001

Abstract

The control of mitochondrial b-oxidation, including the delivery of acyl moieties from the plasmamembrane to the mitochondrion, is reviewed. Control of b-oxidation flux appears to be largely at the levelof entry of acyl groups to mitochondria, but is also dependent on substrate supply. CPTI has much of thecontrol of hepatic b-oxidation flux, and probably exerts high control in intact muscle because of the highconcentration of malonyl-CoA in vivo. b-Oxidation flux can also be controlled by the redox state of NAD/NADH and ETF/ETFH2. Control by [acetyl-CoA]/[CoASH] may also be significant, but it is probably viaexport of acyl groups by carnitine acylcarnitine translocase and CPT II rather than via accumulation of 3-ketoacyl-CoA esters. The sharing of control between CPTI and other enzymes allows for flexible regulation ofmetabolism and the ability to rapidly adapt b-oxidation flux to differing requirements in different tissues.# 2002 Elsevier Science Ltd. All rights reserved.

0163-7827/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.

PI I : S0163-7827(01 )00024-8

Progress in Lipid Research 41 (2002) 197–239

www.elsevier.com/locate/plipres

Contents

1. Introduction ........................................................................................................................................................... 199

2. Cellular uptake of fatty acids and cytoplasmic transport of fatty acids and acyl-CoA esters ............................... 2022.1. Uptake of fatty acids ..................................................................................................................................... 2022.2. Fatty acid binding proteins ........................................................................................................................... 2032.3. Activation of fatty acids to acyl-CoA esters.................................................................................................. 204

2.4. Acyl-CoA binding protein (ACBP) ............................................................................................................... 2052.5. Delivery of acyl-CoA esters and partitioning between oxidation and esterification...................................... 207

* Present address. Unit of Paediatric Surgery, Institute of Child Health, 30 Guilford Street, London WC1N 1EH,UK. Tel.: +44-20-7905-2158; fax: +44-20-7404-6181.

E-mail address: [email protected] (S. Eaton).

3. Entry of acyl-groups to mitochondria.................................................................................................................... 208

3.1. Carnitine palmitoyl transferase I and control of b-oxidation flux in liver .................................................... 2083.2. Carnitine palmitoyl transferase I and control of b-oxidation flux in heart and muscle ................................ 2093.3. Carnitine palmitoyl transferase I and control of b-oxidation flux in other tissues........................................ 213

3.4. Carnitine-acyl-carnitine translocase............................................................................................................... 2133.5. Carnitine palmitoyl transferase II ................................................................................................................. 214

4. Control exerted by the intramitochondrial enzymes of b-oxidation ...................................................................... 2144.1. Acyl-CoA dehydrogenases............................................................................................................................. 2144.2. 2-Enoyl-CoA hydratases................................................................................................................................ 2174.3. 3-Hydroxyacyl-CoA dehydrogenases ............................................................................................................ 217

4.4. 3-Ketoacyl-CoA thiolases.............................................................................................................................. 2184.5. Redox control of b-oxidation........................................................................................................................ 2194.6. Feedback control of b-oxidation ................................................................................................................... 2214.7. Control exerted by disposal of acetyl-CoA and the acylation state of the mitochondrial CoA pool............ 2214.8. Concentration of carnitine and CoA............................................................................................................. 2244.9. Supramolecular organisation of the intramitochondrial enzymes of b-oxidation ......................................... 225

5. Overall control of the pathway .............................................................................................................................. 2275.1. Intermediates of b-oxidation ......................................................................................................................... 2275.2. Modeling of b-oxidation................................................................................................................................ 228

6. Conclusions ............................................................................................................................................................ 228

Acknowledgements...................................................................................................................................................... 229

References ................................................................................................................................................................... 229

Nomenclature

HSL hormone-sensitive lipase.LPL lipoprotein lipase.NEFA non-esterified fatty acids.VLDL very-low density lipoprotein.CPT I mitochondrial outer membrane carnitine palmitoyl transferase (CPTo).CPT II mitochondrial inner membrane carnitine palmitoyl transferase (CPTi).SCAD short-chain acyl-CoA dehydrogenase.MCAD medium-chain acyl-CoA dehydrogenase.LCAD long-chain acyl-CoA dehydrogenase.VLCAD very-long-chain acyl-CoA dehydrogenase.ETF electron transfer flavoprotein.ETF:QO electron transfer flavoprotein:ubiquinone oxidoreductase.ACBP acyl-CoA binding protein.

198 S. Eaton / Progress in Lipid Research 41 (2002) 197–239

1. Introduction

b-Oxidation is the major process by which fatty acids are oxidised, by sequential removal oftwo-carbon units from the acyl chain. In mammals, it provides a major source of ATP for theheart and for skeletal muscle [109,270,408]. Hepatic b-oxidation serves a different role by pro-viding ketone bodies (acetoacetate and b-hydroxybutyrate) to the peripheral circulation [313,330].In addition, kidney [395], small intestine [32], white adipose tissue [396] and brain astrocytes [80]are ketogenic under some conditions. Ketone bodies are another significant fuel for extra-hepaticorgans, especially the brain, when blood glucose levels are low [272,330]. Fat is an efficient energysource and so constitutes the major fuel reserve of the body. Fat oxidation is especially importantduring fasting, sustained exercise, stress [124] and during the neonatal-suckling period [143]. Inaddition, because it is possible to infuse a high amount of calories in a small volume, lipid emulsionsare widely used in intravenous nutritional support of patients [65]. Fat accumulation in obese indi-viduals, on the other hand, is a major cause of disease, contributing to cardiovascular disease andinsulin resistance [124,125,334]. Hence, the control of the rate of fatty acid b-oxidation is of greatimportance, and an understanding of which steps in the pathway contribute significantly to the con-trol of flux is crucial for the design of pharmacological agents to increase or decrease fat breakdown.Quantitatively, most b-oxidation takes place in mitochondria. Peroxisomes are necessary for

the b-oxidation of a wide range of unusual and xenobiotic acyl groups. Initiation of the b-oxi-dation of polyunsaturated and very-long-chain fatty acids takes place in peroxisomes, but per-oxisomal b-oxidation probably accounts for a maximum of around 10% of b-oxidation flux oflong-chain fatty acids that are metabolic substrates, even in tissues where peroxisomes are abun-dant, such as liver. The consequences of deficiencies of enzymes of peroxisomal b-oxidation are,generally speaking, therefore those caused by accumulation of toxic fatty acid moieties ratherthan symptoms caused by inability to oxidise a metabolic fuel [71]. The enzymology and controlof peroxisomal b-oxidation are dealt with elsewhere [289,426].For b-oxidation of the acyl-groups of stored, ingested or infused triacylglycerol to take place, non-

esterified fatty acids must be released. This can take place distantly from the site of utilisation by theaction of hormone-sensitive lipase (HSL) [444] in the adipocyte, or locally by the action of endothe-lial lipoprotein lipase (LPL) [113,146]. Non-esterified fatty acids (NEFA) bound to albumin provide

FABPpm plasma membrane fatty acid binding protein.FATP fatty acid transfer protein.FAT fatty acid translocase.FABP fatty acid binding protein.FOAT fat oxidation activation-transport complex.TDCA top down control analysis.BUCA bottom-up control analysis.AICAR 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside.SHOAD short-chain 3-hydroxyacyl-CoA dehydrogenase.LHOAD long-chain 3-hydroxyacyl-CoA dehydrogenase.CoASH unesterified CoA.

S. Eaton / Progress in Lipid Research 41 (2002) 197–239 199

the main substrate that is taken up and b-oxidised by tissues, although there is some evidence that theheart can take up very-low density lipoprotein (VLDL) or chylomicron triacylglycerol directly[163,237]. In addition, intracellular triacylglycerol can also provide a significant source of acyl moi-eties for b-oxidation in the heart and skeletal muscle [90,339] again through the action of HSL [379].HSL and LPL are under control of the hormonal and nutritional milieu so that fatty acid oxidation ispartly controlled by the supply of NEFA to the tissue. This is true both in liver, as is evident from therelationship between circulating NEFA levels and circulating ketone body levels during fasting(Fig. 1) and in heart and skeletal muscle [186,187,221,251]. Much of the control of b-oxidation flux is,however, intracellular, as are several potentially important regulatory sites, both physiological andpharmacological. The process of intracellular fatty acid oxidation, including delivery of fatty acids tothemitochondrion, is shown in Fig. 2. For the purposes of this review, steps involved in the control of

Fig. 1. Relationship of plasma NEFA concentrations to blood ketone body concentrations during the course of fastingin controls (solid lines, no symbols), patients with proven medium-chain acyl-CoA dehydrogenase deficiency (*), and

a patient with a probable b-oxidation disorder (&). From Bartlett et al. [24] with permission from Nestec Ltd.


Fig.2.Fluxofacylgroupswithin

ageneralisedcell.


mitochondrial b-oxidation will include those intracellular steps likely to influence the delivery of acylgroups to mitochondria, but not those controlling the rate of fatty acid delivery to the cell. Similarly,the control of ketogenesis per se, i.e. the generation of ketone bodies from acetyl-CoA, is outside thescope of this review.My aim in this article is to review the current prevailing hypotheses concerning the control of

mitochondrial b-oxidation flux and to highlight potential regulatory sites, but not to reviewthe regulation of fatty acid b-oxidation. Metabolic control analysis provides a theoretical andpractical framework for delineating how a pathway may be controlled and regulated. Manypathways for which rate-limiting steps had been postulated have been shown to be subject tomulti-site control and regulation; indeed multi-site control has been suggested to be a pre-requisite for flexible regulation in metabolism [112]. Where metabolic control analyses have beenundertaken, I will consider these results together with more traditional approaches to delineatingcontrol in biochemical systems. Necessarily, most of the observations are from experimentalanimals but where possible, I will try to consider human inherited and acquired diseases andexperimental studies that address the question of the control of mitochondrial b-oxidation flux.For a more comprehensive review of inherited disorders of b-oxidation, rather than a discussionof what inherited disorders of b-oxidation can tell us about the control of mitochondrial b-oxi-dation flux, the reader is referred to other excellent recent reviews [56,425]. Similarly, a moredetailed review of the enzymology of mitochondrial b-oxidation can be found elsewhere[96,207].

2. Cellular uptake of fatty acids and cytoplasmic transport of fatty acids and acyl-CoA esters

2.1. Uptake of fatty acids

The fasting plasma concentration of NEFA is 0.2–0.6mM, mostly bound to albumin. Con-troversy still exists over whether: (1) it is the albumin-bound or the unbound fatty acid that istaken up by cells; (2) whether fatty acid cross the membrane by diffusion or by a protein-medi-ated transport [158,239]. Three main types of tissue-specific putative fatty acid transport/bindingproteins have been identified [131]: the plasma membrane fatty acid binding protein (FABPpm)[370,372]; fatty acid translocase (FAT)/CD36 [161]; and the fatty acid transport proteins [364].Antibodies against FABPpm [371], disruption of the FAT/CD36 gene in mice [73], human defi-ciency in FAT/CD36 [389] and decreases in FATP4 expression by antisense oligonucleotides [365]all cause a reduction in fatty acid uptake. Diminished tissue fatty acid uptake in these systems doesnot, however, prove that the putative transporter actually transfers fatty acid molecules across theplasma membrane. The proteins could have receptor or catalytic functions; indeed several mem-bers of the FATP family have been found to have acyl-CoA synthetase activity [74,167,406].Recently, it has been suggested that both diffusional and saturable (presumed protein mediated)pathways occur in adipocytes [373] and given the wide variety of tissue-specific isoforms ofputative fatty acid transporters, the relative importance of each pathway may vary between tis-sues, with circulating albumin and NEFA concentrations (and molar ratio) and on the intracel-lular fate of the fatty acid. As the essentiality or otherwise of protein-mediated fatty acidtransport into the cell is disputed, it is difficult to speculate on the potential role of these proteins


in the control of mitochondrial b-oxidation flux, although some workers have suggested a corre-lation between the amount of FABPpm and the oxidative capacity of muscle fibre types [49] andof trained muscle [399]. Recently, it has also been shown that electrical stimulation of cardio-myocytes increased FAT/CD36-dependent fatty acid uptake and also b-oxidation, suggestingthat the activity of FAT/CD36 may not be in excess [225].

2.2. Fatty acid binding proteins

Long-chain fatty acids have extremely low solubility in water yet regardless of the means oftransfer across the plasma membrane must be transported rapidly to sites of utilisation. Cytosolicfatty acid binding proteins (FABP) are found in high concentrations (0.5–5% of cytosolic pro-tein, �0.1–1.0 mM) [239]. They can be thought of as providing a similar role to that of plasmaalbumin i.e. transfer of hydrophobic fatty acids through an aqueous medium to a site of utilisa-tion and thus providing a degree of protection from the potentially damaging effects of free fattyacids. Their biochemistry, structure and function has been comprehensively reviewed elsewhere[38,145,168,348,369]. Theoretical and experimental approaches have suggested that FABPs increasethe transfer of fatty acids from membrane to membrane via an aqueous environment [421,422]. Insupport of an essential role for FABPs are the findings that transfection of cells with FABP isoformsincrease fatty acid flux rates [264,265] and that mice deficient in heart-type FABP have decreasedfatty acid uptake in heart, increased plasma NEFA, decreased fasting plasma glucose [43] anddecreased fatty acid uptake and oxidation in cardiac myocytes [343]. However, anomalous resultshave been obtained with intestinal fatty acid binding protein: overexpression can reduce fatty acidincorporation [81,311] andmice lacking intestinal FABP are still capable of fatty acid absorption andindeed show increased plasma triacylglycerol levels [412]. Studies in humans with polymorphisms ofthe intestinal FABP do support the hypothesis that this FABP isoform affects fatty acid traffic[20,21]. The results in heart-FABP knockout mice suggest that FABP isoforms may be importantin the pathway of fatty acid utilisation in some tissues but they do not indicate whether physio-logically relevant alterations in the amount of FABP in the cell have any effect on mitochondrialb-oxidation flux. The necessary experiments are difficult to perform, as they would require eitherwell-defined inhibitors of fatty acid binding to FABP or small alterations in the amount of FABPin the cell. It has been suggested that inhibition of fatty acid binding to FABP diminishes fattyacid flux in liver [226], but the results should be interpreted with caution as bromopalmitate is anextremely promiscuous inhibitor, inhibiting carnitine palmitoyl transferase, acyl-CoA synthetaseand 3-ketoacyl thiolases, amongst others [61,62,67,75,293,296]. One study using both peroxisomeproliferators to increase FABP content and antisense-RNA to decrease expression in HepG2 cellsshowed that there was a correlation between FABP content and fatty acid uptake (Fig. 3),although oxidation of the labelled fatty acid was not reported [437].It has been suggested that as fatty acid oxidation rates can vary extremely rapidly in muscle

with no alteration in the amount of fatty acid binding protein, diffusion of fatty acids cannot berate-limiting under normal conditions [324]. This does not exclude the possibility that flux controlof b-oxidation shifts during exercise such that diffusion of fatty acids across the cytosol becomesa limitation to b-oxidation flux under conditions of extreme exercise. FABP content does seem tovary with fatty acid flux in some conditions in different issues (muscle, heart, liver) although thisfinding is not universal [45,227,254,410,414,415,453].


FABPs mostly bind fatty acids of 14–16 carbon atoms or more [132,145]. However, in addition totheir fatty acid binding properties, many FABPs can also bind retinoids [419], bile acids [145], pros-taglandins [194] and eicosanoids [432] and, more importantly for the current discussion, acyl-CoAesters [132] and acyl-carnitine esters, although the latter is disputed [230,300]. Fournier and Richardsuggested that self-aggregation of FABPmay play a role in the control of cardiac fatty acid oxidationflux via binding of acyl-carnitine esters, although the relevance of these studies is uncertain as theirpreparation of FABP probably contained acyl-CoA binding protein (see later) [117–121].

2.3. Activation of fatty acids to acyl-CoA esters

Long-chain acyl-CoA synthetase activity is present in mitochondrial outer membrane [277],peroxisomes [355] and endoplasmic reticulum [205]. These activities were catalytically and immuno-logically indistinguishable and it was thought that there was a single long-chain acyl-CoA synthetasethat was directed to different compartments [255,388], expressed in most tissues [377] and result-ing in a single intracellular pool of acyl-CoA that was used by all acyl-CoA dependent enzymes[152]. However, it has become apparent that there several acyl-CoA synthetases with activitytowards long-chain acyl-CoA esters that vary in their subcellular localisation, tissue expressionand preference for fatty acid substrate [133–135,192,282,390,406]. As well, there are multipletranscripts of the originally described acyl-CoA synthetase [378] and several members of theFATP family have been found to have acyl-CoA synthetase activity [74,167,406]. The wide range offatty acyl-CoA synthetases active towards cytosolic fatty acids/FABP-bound fatty acids has

Fig. 3. Correlation of L-FABP content and oleic acid uptake rate. Reprinted from Wolfrum et al. [437], ‘‘Variationof liver-type fatty acid binding protein content in the human hepatoma cell line HepG2 by peroxisome proliferators

and antisense RNA affects the rate of fatty acid uptake’’ Biochim Biophys Acta 1999;1437:194–201, with permissionfrom Elsevier Science.


raised the possibility that there is functional channelling of acyl-CoA esters. Using the differentialsensitivity of isoforms of acyl-CoA synthetase to triacsin C and troglitazone, and specific anti-bodies against the different isoforms, Coleman’s group has suggested functional channelling of acyl-CoA esters to esterification or b-oxidation in liver, and that there is a further acyl-CoA synthetaseassociated with mitochondria that is not one of the isoforms already cloned and characterisedwith respect to inhibitor sensitivity [177,195,217,263]. Further evidence suggestive of functionalchannelling of acyl-CoA esters directly to mitochondrial b-oxidation is the localisation of acyl-CoA synthetase activity, and a protein recognised by a polyclonal antibody against the micro-somal protein, in contact sites between the mitochondrial outer- and inner-membranes, togetherwith carnitine palmitoyl transferase I (CPT I) and carnitine palmitoyl transferase II (CPT II)[122,174]. There may be a further component to the pathway of mitochondrial b-oxidation toallow passage of acyl-CoA between the cytosolic and the intermembrane faces of the outermitochondrial membrane; Hoppel’s group have suggested that porin may be necessary [400].Thus there may be a fat oxidation-activation transport (FOAT) complex associated with themitochondrial membranes.The activation of fatty acids to CoA esters is not usually thought to be a major site for control

and regulation of b-oxidation. However, acyl-CoA synthetase is subject to potential control bythe availability of substrate and removal of product. It has been suggested that as the cytosolicconcentration of free CoA (henceforward referred to as CoASH) is equal to or less than the Km ofthe enzyme, the availability of CoASH could influence the activity of the enzyme andthereby b-oxidation flux [288]. Similarly, the Km for ATP of the enzyme is 2–5 mM[23,303,304,388] so that lack of cytosolic ATP could impair activity and flux. Acyl-CoA synthe-tase is also inhibited by its acyl-CoA products [294] so that in principle, b-oxidation flux could begoverned by the [acyl-CoA]/[CoASH] ratio. However, this product inhibition is not expected tooccur in vivo as acyl-CoA binding protein (ACBP, Section 2.4) [325] or channelling of acyl-CoAdirectly to oxidation via contact sites [122,174] would result in a low nanomolar concentration offree acyl-CoA [239]. Some studies have compared the effect of inhibition of acyl-CoA synthetasewith inhibition of b-oxidation flux. In rat heart mitochondria, acyl-CoA synthetase activity wastitrated with enoximone and b-oxidation flux from palmitate decreased in parallel, suggestingthat acyl-CoA synthetase has much of the flux control over b-oxidation in intact mitochondria[1,2]; similar results were obtained [445] in liver mitochondria. The experiments could not, how-ever, be repeated in intact cardiomyocytes because enoximone exerted an independent stimula-tory effect on palmitate oxidation [1,2]. In hepatocytes, use of troglitazone as an inhibitor ofmitochondrial and microsomal acyl-CoA synthetase demonstrated that acyl-CoA synthetaseactivity could affect b-oxidation flux and partitioning of fatty acids between b-oxidation, tria-cylglycerol synthesis and phospholipid synthesis [137] (Section 2.5).

2.4. Acyl-CoA binding protein (ACBP)

FABPs were thought to physiologically bind acyl-CoA esters within the cytosol. However, a fur-ther protein was isolated, which had originally co-purified with FABP [256], that has �1000-foldhigher affinity for acyl-CoA esters of chain length C14–C22 (although the affinity is so high that itis difficult to measure by conventional means) and a much lower affinity for fatty acids and car-nitine esters [332]. It is ubiquitously expressed in mammalian tissues and is thought to be at a


concentration of 10–50 mm in the cytosol, compared to 100–300 mM for FABP [108,199]. Thedifferent binding constants and properties of ACBP and FABP towards CoA esters have beensuggested to act as a two-level system to buffer acyl-CoA levels within the cytosol [108] and protectenzymes, such as the adenine nucleotide translocase [356] and hexokinases [394] from the potentiallyinhibitory effects of acyl-CoA esters and to prevent product inhibition of acyl-CoA synthetase[294,325]. This has been modelled extensively by Knudsen’s group as shown in Fig. 4 [108].As ACBP has such a high affinity for acyl-CoA esters, it might be expected that acyl-CoA esters

would be unavailable to b-oxidation or triacylglycerol synthesis. Indeed, ACBP is able to protectacyl-CoA esters from the action of acyl-CoA hydrolases [150,325]. However, several studies havenow convincingly shown that ACBP-bound acyl-CoA is a substrate for CPT I [3,39,326] andmicrosomal triacylglycerol or phospholipid synthesis [149,190,325,326]. As delivery of acyl-CoAfrom ACBP to CPT I takes place in the face of a�100–1500 fold difference in apparent Kd,making it difficult for CPT I to complete directly with ACBP for acyl-CoA, it has been suggestedthat transfer of acyl-CoA is caused by a conformational change induced by a specific interactionbetween ACBP and CPT I [4].Although the ability of ACBP to bind acyl-CoA esters and deliver them to enzymes requiring

them for utilisation is now well established in vitro, the evidence for a role of ACBP in vivo andwhether ACBP binding of acyl-CoA is important in control of mitochondrial b-oxidation flux is

Fig. 4. Calculation of the free concentration of acyl-CoA esters in the presence of ACBP or FABP. Calculations arebased on the presence of 50 mM ACBP and 0.3 mM liver FABP (0.6 mM binding sites), and Kd values for the bindingof long-chain acyl-CoA esters of 1 nM (ACBP) and 1 mM (FABP). Reproduced with permission from N. J. Færgeman

and J. Knudsen. Role of long-chain fatty-acyl CoA-esters in the regulation of metabolsim and cell signalling. BiochemJ 323:1–12, 1997 [108]. # The Biochemical Society.


much less well known, again due to a lack of specific inhibitors. In a recent study in hepatoma cellsoverexpressing ACBP, it has been shown that ACBP increased flux of free fatty acids to triglyceridesonly at low substrate concentrations; however ACBP overexpression strikingly decreased flux of[U-14C]fatty acid to 14CO2 [441]. This would suggest a role for ACBP in the partitioning of acyl-CoAbetween esterification and oxidation (Section 2.5), and also imply that some control over b-oxidationflux is exerted at a level prior to the entry of acyl groups to the mitochondrion, at least under theexperimental conditions studied. However, 14CO2 is quantitatively a minor product of b-oxida-tion in liver, as in most tissues [106,361,413,429] and measurement of the effect of ACBP over-expression on flux to more major in vitro end products (acetyl-carnitine, ketone bodies, Krebscycle intermediates) should be made.

2.5. Delivery of acyl-CoA esters and partitioning between oxidation and esterification

As is clear from the earlier discussion of FATPs, FABPs, ACBP and the localisation of acyl-CoA synthetase and potential channelling of acyl-CoA esters within fat oxidation-activationtransport (FOAT) complexes, the precise route of transfer of fatty acid moieties from circulatingNEFA to mitochondrial b-oxidation is still poorly understood. Three possible pathways areshown in Fig. 5. It should be noted that these pathways are not mutually exclusive and acyl

Fig. 5. Diagram showing three possible pathways for transfer of acyl-groups from plasma to the mitochondrion. In A,CD36/FAT acts as a recognition site for fatty acids which are transferred across the plasma membrane but FATP,bound to FABPs, converted to acyl-CoA esters by acyl-CoA synthetase and transferred to the mitochondrion as an

ACBP/acyl-CoA complex. In B, FATP transfers fatty acids across the membrane but also acts as an acyl-CoA syn-thetase, thus obviating the need for FABP. ACBP/acyl-CoA delivers substrate to CPT I. In C, fatty acids diffusedirectly across the plasma membrane and are bound to FABP, which delivers fatty acid to acyl-CoA synthetase in

contact sites. Acyl-CoA esters are channelled directly to CPT I at the contact site, thus obviating any requirement forACBP for b-oxidation.


groups could be transferred by all of these mechanisms simultaneously, with the balance betweenpathways varying with (1) tissue studied (2) circulating NEFA concentration (3) downstream sinkfor b-oxidation flux (4) demand for acyl groups for triacylglycerol and phospholipid formation.There is a requirement for acyl-CoA by several of the enzymes of triacylglycerol synthesis [216]:

glycerol-3-phosphate acyltransferase, 1-acylglycerol-3-phosphate acyltransferase, diacylglycerolacyltransferase, monoacylglycerol acyltransferase, and also by the enzymes of phospholipidmetabolism. Hence, in tissues that synthesise triglyceride and/or phospholipids at significantrates, there may be competition between triacylglycerol formation, phospholipid metabolism andb-oxidation for acyl-CoA. Much of the control of the fate of acyl residues in liver seems to residein the balance between mitochondrial and microsomal glycerol-3-phosphate acyl transferaseactivities and the activity of CPT I under acute regulation by the concentration of malonyl-CoA[449,451]. In liver, this allows for rapid alterations in the fate of acyl groups during fasting-refeeding [257–259]. When isolated hepatocytes were incubated with an inhibitor of acyl-CoAsynthetase, phospholipid synthesis was able to outcompete both b-oxidation and neutral lipidformation for acyl-CoA moieties [137]. Muscle and heart both have significant triacylglycerolstores which can be utilised when circulating NEFA levels are low [90,339], but the control of thepartitioning of NEFA between triacylglycerol synthesis and b-oxidation is not very well understoodin these tissues [91,383].

3. Entry of acyl-groups to mitochondria

3.1. Carnitine palmitoyl transferase I and control of b-oxidation flux in liver

Much of the control of the rate of hepatic mitochondrial b-oxidation flux appears to reside atthe level of CPT I, such that hepatic b-oxidation flux is subject to regulation by different effectorsthrough inhibition of CPT I. Inhibition of CPT I by its major physiological inhibitor, malonyl-CoA, the substrate for hepatic de novo lipogenesis, was first demonstrated by McGarry andFoster [245]. Subsequently, the elucidation of the roles of AMP-dependent protein kinase andacetyl-CoA carboxylase have underlined the importance of the CPT I/ malonyl-CoA system incontrol and regulation of hepatic b-oxidation flux [152,240,241,449,451] such that CPT I is frequentlydescribed as the ‘‘rate-limiting step’’ of b-oxidation flux. In vivo animal studies, for example thoseof Chien et al. [69], and studies in adult humans such as those demonstrating decreases in plasmab-hydroxybutyrate in response to a hyperinsulinaemic/hyperglycaemic clamp when circulatingNEFA levels were maintained by intravenous lipid infusion [357] support a role for the CPT I/malonyl-CoA axis in the control of hepatic b-oxidation flux.In order to be a rate-limiting enzyme, the activity of hepatic CPT I must be such that inhibition

of the enzyme by 10% should inhibit b-oxidation flux by 10%. The question of whether CPT Ireally is rate-limiting for hepatic b-oxidation flux has been investigated by metabolic controlanalysis, largely by Quant and co-workers. For a discussion of the theory and practical applica-tion of metabolic control analysis, the reader is referred to other sources [53,110–112,312], butessentially, ‘‘rate-limiting’’ enzymes have a flux control coefficient close to 1.0, ‘‘rate-controllingenzymes’’ have a flux control coefficient of between 0.5 and 1.0, and enzymes which have little orno control over pathway flux have a flux control coefficient of close to 0. The various results


obtained for the flux control coefficient of CPT I over b-oxidation flux are indicated in Table 1.Lower flux control coefficients were obtained where the incubation conditions are less physiologicali.e. in uncoupled mitochondria in the presence of Krebs cycle inhibitors.In intact adult rat hepatocytes CPT I appears to have a high flux control coefficient over b-

oxidation flux, i.e. can be considered to be close to rate-limiting. This ensures that b-oxidationflux is responsive to changes in CPT I activity under a wide range of physiological conditions, butcan also be regulated at other steps in the pathway. However, in suckling animals, CPT I exerts alower flux control coefficient than in adults, so that although hepatic CPT I activity and amountof enzyme increase in parallel with the ability to use fatty acids as an energy source (reviewed[143]), some of the control of b-oxidation flux must reside elsewhere.

3.2. Carnitine palmitoyl transferase I and control of b-oxidation flux in heart and muscle

Fatty acids are important metabolic fuels for both heart and skeletal muscle, which lack denovo lipogenesis and therefore have no obvious role for malonyl-CoA. The rate of b-oxidationhas been described as demand-led, in that an increased work rate and ATP demand leads to fasteroxidative phosphorylation and tricarboxylic acid cycle activity. NADH and acetyl-CoA levelsdiminish, thus increasing b-oxidation flux [269,287]. In addition, control at the level of CPT Iactivity appears to be important in control of heart and skeletal muscle b-oxidation flux[19,222,435,450]. There is a different isoform of CPT I present in skeletal muscle (M-isoform)which has very different properties from the liver isoform (L-isoform); M-CPT I is very much

Table 1Review of experimentally determined flux control coeffiecients of CPT I over b-oxidationa

Tissue andpreparation

Numerical valueof flux control

coefficient

Comments Reference

Liver mitochondria (adult) <0.28b TDCA. Uncoupled, malonate [315]Liver mitochondria (adult) 0–0.5 TDCA. Varying NAD/NADH

and malonyl-CoA. Malonate,uncoupled, oligomycin, rotenone

[314]

Liver mitochondria (adult) 0.35–0.8 TDCA. Malonyl-CoA and state 3–4. [232]Hepatocytes (adult) 0.9–1.1 BUCA. Fed, starved, refed

and insulin treated

[86]

Hepatocytes (adult) 0.67–0.79 BUCA. 0.2 and 1 mM palmitate [361]Liver mitochondria 0.87–1.0 (adult)

0.73–0.96 (suckling)

TDCA. Malonyl-CoA and State 3–4 [206]

Hepatocytes (suckling) 0.51 BUCA. Fed. [273]Liver mitochondria 0.9–1.1 (adult)

0.8–0.95 (suckling)

BUCA. Malonyl-CoA and State 3–4. [273] (recalculated

from [206]

Astrocytes (neonatal) 0.77 BUCA [46]

Heart mitochondria (suckling) 0.08 BUCA. [99]

a TDCA, top down control analysis; BUCA, bottom-up control analysis.b Top-down block including CPT I.


more sensitive than L-CPT I to malonyl-CoA inhibition whereas M-CPT I has a much higher Km

for carnitine than L-CPT I [241,450]. Cardiac tissue has both M- and L- isoforms [60,431] andtherefore cardiac CPT I has overall kinetic properties intermediate between muscle and liver.However, the concentration of malonyl-CoA in the heart and skeletal muscle is estimated to be inthe range of 1–10 mM [19,147,247,248,338,435] which greatly exceeds the IC50 of malonyl-CoA onskeletal muscle and heart CPT I [247,340]. Hence it is difficult to see how b-oxidation proceeds incardiac and skeletal muscle if CPT I activity is rate-limiting for b-oxidation.Amongst the possibilities to account for this observation are the following:

1. there is a malonyl-CoA insensitive outer mitochondrial membrane CPT I activity in muscle;2. much of the measured malonyl-CoA is intramitochondrial or bound and therefore not

available to inhibit CPT I;3. CPT I is not rate-limiting for b-oxidation in cardiac and skeletal muscle.

There are additional factors which may alter the ability of malonyl-CoA to inhibit CPT I in theheart. The sensitivity of liver isoform of CPT I to malonyl-CoA is itself acutely regulated in theliver [87] and the activity of hepatic CPT I can be modified by the Ca2+/calmodulin system and/or cytoskeletal components [416,417]. However, whether the sensitivity to malonyl-CoA or theactivity of the liver isoform of CPT I expressed in the heart is altered by these factors is unknown.Recently, alternative muscle-CPT I (M-CPT I) splicing isoforms (CPT Ib-2 and CPT Ib-3) have

been demonstrated in both rat [448] and human [447]. Although the mRNA species for the twosplicing isoforms are expressed in cardiac and skeletal muscle, it is not yet known whether there isany steady-state expression of their respective proteins. M-CPT I migrates anomalously on SDS-PAGE (88.2 kDa predicted molecular mass, runs at �82 kDa) [241] and as the predicted mole-cular mass of the proteins of two splicing variants is close to 82 kDa and the migration of thesplicing variants is unknown, it has so far been difficult to quantify expression of these proteins.CPT Ib-2 completely lacks the second transmembrane domain so whether it can correctly insertinto the mitochondrial outer membrane is uncertain [447,448]. CPT Ib-3 lacks 34 amino acidstowards the N-terminal end of the catalytic region [102,447,448] and its biochemical propertiesare unknown. Much progress has been made in recent years into defining residues in the carnitineacyltransferases which are responsible for malonyl-CoA and etomoxir-CoA inhibitability, affinityfor carnitine and catalytic activity [79,184,185,262,382]. Both CPT Ib-2 and CPT Ib-3 maintainthe catalytic C-terminal of the enzyme and N-terminal amino acids crucial for malonyl-CoAsensitivity [354] but the catalytic properties and malonyl-CoA sensitivity of the novel isoformscannot be readily predicted, even if the proteins are expressed and targeted and inserted into theouter mitochondrial membrane correctly. It has, however, been suggested that the splicing iso-forms are insensitive to malonyl-CoA thus allowing b-oxidation to take place even in the presenceof significant [malonyl-CoA] [447,448] and that this accounts for the incomplete inhibition ofCPT I by malonyl-CoA that has been observed in cardiac myocytes [248]. The physiologicalrelevance of the novel M-CPT I splicing variants has yet to be elucidated.The major route for formation of malonyl-CoA in the heart and skeletal muscle, as in liver, is

by the activity of cytosolic acetyl-CoA carboxylase [40,393]. However, some of the malonyl-CoAmeasured in muscle may be intramitochondrial due to the action of propionyl-CoA carboxylaseon acetyl-CoA [347]. In lipogenic tissues, cytosolic malonyl-CoA is used as a substrate for fatty


acid synthase; however cardiac and skeletal muscle are not lipogenic and do not have a mal-onyl-CoA-dependent fatty acid elongation system [157], so extramitochondrial malonyl-CoAmust be disposed of by a different route. Malonyl-CoA decarboxylase is postulated to be presentinside the mitochondrial matrix to scavenge malonyl-CoA formed by the action of propionyl-CoA carboxylase on acetyl-CoA, thus preventing it from inhibiting pyruvate carboxylase[196,211,346]. Inborn-errors of malonyl-CoA decarboxylase occur rarely in humans and canpresent with cardiomyopathy, suggesting a physiological role for this enzyme in cardiac muscle[58,153,231,238]. Recently, a malonyl-CoA decarboxylase which can be targeted to multiple cel-lular locations (mitochondria, peroxisomes, cytosol) has been cloned and mutations in this genefound in patients with malonyl-CoA decarboxylase deficiency [93,94,114,140,337,420]. Malonyl-CoA decarboxylase mRNA, protein and activity can be detected in heart and skeletal muscle,although levels are very much greater in cardiac than skeletal muscle [93,420] but the subcellularlocation of this protein is unknown. Hamilton and Saggerson have provided evidence that thereis a cardiac mitochondrial-associated malonyl-CoA decarboxylase activity, which is not latent(i.e. would be active towards cytosolic malonyl-CoA) [157]. It is also possible that binding ofextra-mitochondrial malonyl-CoA to mitochondrial low-affinity sites [44] or to cytosolic bindingproteins such as that described in liver [89] would also prevent malonyl-CoA from inhibiting CPTI.As control analysis of b-oxidation in heart or skeletal muscle has not been undertaken, we set

out to determine the control coefficient of CPT I over b-oxidation flux in suckling rat heartmitochondria. The apparent flux control coefficient of CPT I over b-oxidation is 0.08 (Table 1[99,102]). However, the evidence from studies in intact tissues and cells would suggest that themalonyl-CoA-CPT I axis is crucial in control of b-oxidation flux in heart and skeletal muscle. Anumber of studies have demonstrated correlations between the rate of fatty acid oxidation in theperfused heart, isolated myocytes and the concentration of malonyl-CoA [19,92,148,157,338]. Themalonyl-CoA concentration in skeletal muscle correlates well with increases in respiratory quo-tient (measured by indirect calorimetry) during the starved-refed transition and, in addition toother studies in skeletal muscle [250,251,334,435], this provides good in vivo evidence for thecontrol of b-oxidation flux at the level of the malonyl-CoA/CPT I axis [69]. So how can CPT Ihave a low flux control coefficient over b-oxidation flux and be important in flux control in vivo?The answer may be indicated in Fig. 6a, which represents the curve used to derive the flux controlcoefficient of 0.08 [102].The physiological range of extramitochondrial [malonyl-CoA] may be such that in vivo, CPT I

is always partly inhibited and therefore has a high flux control coefficient over b-oxidation (i.e.the in vivo control is within the grey box indicated on Fig. 6a). Flux control over b-oxidationwould therefore be shared between CPT I and the other intra- and extramitochondrial controlsconsidered in this review. This, together with inaccessibility of CPT I to malonyl-CoA due to apartial intramitochondrial localisation, and/or malonyl-CoA binding proteins, may explain howCPT I appears to be important in flux control in vivo despite the high apparent malonyl-CoAconcentration in muscle and heart. Winder and Holmes performed experiments with perfusedhindlimb with insulin and AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside,which after metabolism stimulates AMP-dependent protein kinase, inhibits acetyl-CoA carbox-ylase and decreases malonyl-CoA levels) to provide a range of malonyl-CoA levels. Their resultsshowed the dependence of in vivo b-oxidation flux on malonyl-CoA levels (Fig. 6b) [436]. The lowest


malonyl-CoA content they obtained was �0.23 nmol/g, which corresponds very approximately toa concentration of 0.5–1 mM, in great excess of the IC50 of malonyl-CoA on skeletal muscleCPT I activity (of course these calculations are extremely crude and do not take account of anyintramitochondrial or bound malonyl-CoA).The role of malonyl-CoA inhibition of CPT I in control of muscle or cardiac b-oxidation flux in

humans is less clear. An elegant study in adult humans demonstrating that a hyperinsulinaemic/hyperglycaemic clamp, with circulating NEFA levels maintained by intravenous lipid infusion,caused a decreasee in whole body [1-13C]oleate oxidation, no change in [1-14C]octanoate oxida-tion (i.e. non-CPT I dependent) and a decrease in muscle long-chain acyl-carnitine (the productof the CPT I reaction) [357]. However, long-chain acyl-carnitine levels could also decrease due toother extramitochondrial controls or merely be reflective of decreased flux rather than decreasedCPT I activity. Malonyl-CoA levels are also markedly lower in human than in rat muscle[83,279,280] whereas the IC50 of malonyl-CoA on human muscle-type CPT I is similar to rat[452]. Whether this reflects a difference in availability of CPT I to malonyl-CoA or a difference incontrol between rat and human muscle remains an open question. The few studies that havecorrelated malonyl-CoA content of human muscle with fat oxidation rates during exercise suggestthat malonyl-CoA content does not inversely correlate with exercise-induced increases in fattyacid oxidation [279,280], although another study found modest decreases [83].Hence, the role of malonyl-CoA/CPT I control of b-oxidation flux in cardiac and skeletal

muscle is still incompletely understood. b-Oxidation cannot be controlled solely at the level ofCPT I as fatty acid oxidation decreases at high exercise despite a lack of increase in malonyl-CoAlevels in both rat and human muscle [83,280,323].

Fig. 6. Dependence of skeletal and cardiac muscle mitochondrial b-oxidation flux on CPT I activity in vitro and invivo. (a) Inhibition of cardiac in vitro b-oxidation flux by inhibition of CPT I activity. The curve represents the fittedcurve to the data [99]. The shaded box may represent in vivo control of b-oxidation flux by CPT I. (b) Palmitate oxi-dation in vivo by rat hindlimbs vs. malonyl-CoA content of muscle. The variable malonyl-CoA and palmitate oxida-

tion values were generated by perfusing muscles with insulin and AICAR. Part B from Winder and Holmes [436] withpermission from The American Physiological Society.


3.3. Carnitine palmitoyl transferase I and control of b-oxidation flux in other tissues

In addition to liver, heart andmuscle, CPT I is present in other tissues, both those which are usuallythough of as tissues/cell types with active b-oxidation and those not usually thought to oxidise fattyacids to any great degree. CPT I is generally thought to be rate-limiting or rate-controlling inthese tissues, but formal flux control experiments have only been reported in astrocytes, whereCPT I was found to have a flux control coefficient comparable to hepatocytes [46] (Table 1).The kidney has an active b-oxidation system but studies which report that CPT I is important

in control of renal b-oxidation flux are based on the finding that CPT I increases in parallel withb-oxidation flux during development [84]. This argument has been shown to be flawed in liverwhere flux control is actually lower in suckling than in adult rats, despite a similar relationshipbetween b-oxidation flux and CPT I activity [206,273].Brown adipose tissue also has a very active b-oxidation system to support its thermogenic

function, with acyl-CoA synthetase and CPT I (M-type isoform [59,107]) with Vmax/mg proteinexceeding heart and liver mitochondria �10–20 fold [275,276,301]. CPT I is assumed to be rate-limiting for b-oxidation, although this has not been demonstrated.Pancreatic beta cells are able to b-oxidise fatty acids [9,18,37,63,234,351] and the switch

between fatty acid and glucose as fuel is important in stimulation of beta-cells [233,352,411]. CPTI appears to have significant control over beta-cell b-oxidation flux, although this has not beenformally shown [18,37,68] and the malonyl-CoA-CPT I-long-chain acyl-CoA axis has been sug-gested to be very important in the control of insulin secretion [76,308].The intestine becomes a ketogenic organ during the suckling period [32,88,143] and CPT I is

usually assumed to be important in control of intestinal b-oxidation flux [16,17,154], althoughagain is based on the correlation of CPT I expression with ketogenesis rates during developmentrather than on formal inhibition-flux relationships.The differential expression of M- and L- isoforms of CPT I in testis suggests some differences in

regulation and control of b-oxidation during sperm development but these have not been corre-lated with measurements of b-oxidation flux [6,96]. CPT is present in other tissue types, such aslung and erythrocytes, which are not thought to oxidise fatty acids, where it appears to have arole in membrane lipid trafficking [13–15,320].

3.4. Carnitine-acyl-carnitine translocase

The carnitine-acyl-carnitine translocase has extremely high activity in most cell types active inb-oxidation [278,322], apparently greatly in excess of b-oxidation flux [201] so that fibroblastsfrom a carnitine-acylcarnitine translocase deficient patient with only 1% residual activity hadonly a 70% decrease in b-oxidation flux [178]. Hence the carnitine acyl-carnitine translocase isnot usually thought to play a role in control of b-oxidation flux, unless under equilibrium controlby the relative concentrations of carnitine and acyl-carnitine intra- and extra- mitochondrially[201,319]. This would account for export of acyl-carnitine and acetyl-carnitine esters from mito-chondria and cells [25,97,98,228,229], which has great utility in the diagnosis of inborn errors ofb-oxidation [27] and will be considered in Section 4.7.Early studies with (+)-acylcarnitines, which were then thought to be CPT Inhibitors, showed

inhibition of b-oxidation flux [85,130,242–244,246,433,434]. (+)Acylcarnitines are now known to be


inhibitors of the carnitine-acyl-carnitine translocase [22,295], so these experiments, together withothers using inhibitors of the translocase [266] and experiments with hypo- or hyperthyroid ratswhich correlated changes in translocase activity with b-oxidation flux [297,298], suggest a potentialrole for the translocase in control of b-oxidation flux. However, it is impossible to dissect out thelong-chain acyl-carnitine entry into mitochondria from the acetyl-carnitine and free carnitine exportroles of the enzyme in order to determine whether the carnitine-acyl-carnitine translocase-catalysedentry of long-chain acyl-carnitines into mitochondria has a role in control of b-oxidation flux.Interestingly, however, recent studies have showed localisation of CPT I to contact sites of mito-chondria [122,174] so that even though there is controversy as to whether there is carnitine acyl-car-nitine translocase enrichment in these contact sites [123,174], there may be a functional separation oflong-chain acyl-carnitine entry from acetyl-carnitine export, which would have implications for thecontrol of b-oxidation flux.

3.5. Carnitine palmitoyl transferase II

Unlike CPT I, CPT II is not thought to be important in the control of b-oxidation flux—it hasno known physiological allosteric effectors and any effects exerted at the level of CPT II are likelyto be mass-action effects due to the relative concentrations of its substrates and products. CPT IIdeficiency is a relatively common b-oxidation disorder that presents in two different formsdepending on the severity of the enzyme deficiency. The commoner form presents in adults withskeletal muscle symptoms triggered by exercise or fasting (i.e. conditions associated withincreased utilisation of fatty acids by muscle) [50]. There is �15–25% residual activity in fibro-blasts, and b-oxidation flux is only mildly affected (Fig. 7) [51]. When residual activity is less than10%, b-oxidation flux in fibroblasts is markedly decreased, and the disease presents in a verymuch severe form in infants, with involvement of liver together with cardiac and skeletal muscle(Fig. 7) [50,51]. Heterozygote parents of affected individuals, with �50% activity, have normal b-oxidation flux in fibroblasts and are asymptomatic (Fig. 7) [50,51]. These findings all suggest thatat least in man, there is a great excess capacity of CPT II activity, even during exercise or fasting,and that the excess capacity is greater in liver and heart than in skeletal muscle.Kunz [209], in a pioneering work, applied metabolic control analysis to rat liver mitochondria

oxidising palmitoyl-carnitine and found that CPT II exerted significant control (flux controlcoefficient of 0.35) at high flux rates. However, as pointed out by Kunz, the calculated flux con-trol coefficient is in effect a group flux control coefficient including control exerted by carnitineacylcarnitine translocase (Section 3.4) and control by the intramitochondrial concentrations ofCoASH, carnitine, acyl-CoA and acyl-carnitine (Sections 4.7 and 4.8).

4. Control exerted by the intramitochondrial enzymes of b-oxidation

4.1. Acyl-CoA dehydrogenases

The acyl-CoA dehydrogenases of b-oxidation (very-long chain, VLCAD; long-chain, LCAD;medium-chain, MCAD; and short-chain; SCAD) appear to have by far the lowest activity of theintramitochondrial enzymes of b-oxidation in rat and human tissues [182,183,249,327], although


comparison of isolated enzyme activities may be misleading since assays of the enzymes of b-oxidation are carried out under non-physiological conditions. The enzymes have a high affinityboth for their acyl-CoA substrates and for their enoyl-CoA products, resulting in product inhi-bition [82,307]; the importance of this will be considered below in feedback control of b-oxidation(Section 4.6). Electron transfer flavoprotein (ETF) is the primary acceptor for reducing equiva-lents from the acyl-CoA dehydrogenases, which are then passed to the respiratory chain at thelevel of coenzyme Q by electron transfer flavoprotein oxidoreductase (ETF:QO), both of whichwill be considered below under redox control of b-oxidation (Section 4.5).The chain length specificity of these enzymes overlaps, particularly for LCAD and VLCAD.

However, VLCAD is associated with the inner mitochondrial membrane [359] whereas theLCAD is a soluble matrix enzyme [179] suggesting that there is some differentiation of function.The finding that patients with deficiencies in VLCAD have deficient oxidation of long-chain fattyacids, accumulate long-chain acyl-CoA and carnitine esters in muscle mitochondria [281] andhave a strong correlation between genotype and phenotype [10] support the importance ofVLCAD in the b-oxidation of long-chain fatty acids in humans. However, a mouse with dis-rupted LCAD also displays a phenotype consistent with decreased b-oxidation indicating thatthis enzyme may also be important in b-oxidation [210]. Studies of the substrate specificity ofLCAD have suggested that LCAD may in fact be more important in the b-oxidation of bran-ched-chain fatty acids [236,423] and fatty acids unsaturated in the 4,5 or 5,6 position (e.g. oleicacid) [215] than in oxidation of saturated fatty acids. The recent description of a mouse model of

Fig. 7. CPT II activity and [9,10-3H]myristate oxidation in fibroblasts from CPT II-deficient patients. (*) CPT II-deficient patients with the infantile form of the disease; [&] CPT II-deficient patients with the adult form of the disease;

(*) parents of CPT II-deficient patients; and (&) controls. From Bonnefont et al. [51] with permission from the Uni-versity of Chicago. # 1996 by The American Society of Human Genetics.


VLCAD-deficiency and comparison of the LCAD- and VLCAD- deficient mouse, however,showed no accumulation of methyl-branched chain acyl-carnitines in LCAD-deficient mouse,although the LCAD deficient mouse did accumulate shorter-chain acyl-carnitine esters than theVLCAD-deficient mouse [78]. Unfortunately measurements of b-oxidation flux in different tissuesfrom the LCAD and VLCAD deficient mice were not reported, nor were circulating NEFA orketone bodies during fasting. It is possible that the precise biochemical roles of VLCAD andLCAD additionally depend on their submitochondrial localisation and association with otherenzymes rather than purely on their substrate specificities (Section 4.9).Although inhibitors based on methylenecyclopropyl fatty acids with different acyl substituents

[33,57], or related compounds [218,398], allow discrimination of the acyl-CoA dehydrogenases,they have not so far been used to correlate inhibition of enzyme activity with inhibition of b-oxidation flux. Injection of methylenecyclopropylpyruvate, a metabolite of hypoglycin [353], torats inhibits both SCAD and MCAD activity of muscle mitochondria by �80% and therebyinhibited b-oxidation flux by 30–50%, depending on the incubation conditions and substrate used[198].Evidence for the importance of the individual acyl-CoA dehydrogenases in control of b-oxida-

tion flux comes from various studies. Overexpression of VLCAD in two different hepatoma celllines with little endogenous VLCAD caused a 30% increase in b-oxidation flux whereas HepG2cells, with a higher endogenous level of VLCAD expression, showed no change in b-oxidationflux [12]. Similarly, studies in which fibroblasts from patients deficient in VLCAD were trans-fected with VLCAD showed normal b-oxidation flux with only �20% of normal levels VLCADactivity [11]. However, neither fibroblasts nor hepatoma cells are particularly physiologicallyrelevant (especially as hepatoma cells already have altered fatty acid oxidation because they lackHMG-CoA synthase [309]). There is a correlation between genotype and phenotype in VLCAD-deficient patients such that patients with complete absence of protein have the most severe disease[10]. In an adult with VLCAD-deficiency in which symptoms were induced by exercise, musclemitochondria with �11% residual palmitoyl-CoA dehydrogenase activity were able to oxidisepalmitate at �26% of control rates, although the residual activity may of course have been dueto the matrix enzyme rather than residual VLCAD activity [281].Unlike VLCAD-deficiency, MCAD deficiency is less well defined in terms of genotype-pheno-

type correlations [425]. However, it is apparent that although children with MCAD deficiency canmake limited amounts of ketone bodies during a controlled fast [115], during a fasting there is apoint at which the ability to make ketone bodies becomes inappropriate for the circulating NEFAconcentration (see Fig. 1 [24]). Presumably, this is when MCAD becomes rate-controlling forhepatic b-oxidation. As muscle and cardiac symptoms are rare in patients with MCAD [342], it isprobable that MCAD activity does not become rate-controlling in muscle, except under extremeconditions [335]; indeed some homozygotes for mutations which are normally pathogenic maynever present with symptoms [48]. In vitro studies of b-oxidation flux in fibroblasts or leukocytesfrom patients with MCAD deficiency show decreased [1-14C]octanoate [204] or [9,10-3H]myristate[235] oxidation whereas [9,10-3H]palmitate [235] or [U-14C]palmitate [306,344] oxidation is closeto normal.It is difficult to assess the importance of SCAD in control of b-oxidation flux. SCAD deficiency

is rare and displays heterogenous symptoms with apparently normal rates of ketogenesis[328,425]. Although oxidation rates of [1-14C]butyrate are decreased [328], [9,10-3H]myristate and


palmitate oxidation rates are normal [391,425]. Interestingly, acetyl-carnitine production from[U-13C]palmitate in fibroblasts is decreased [418]. Mouse models of SCAD deficiency with absentSCAD activity show no clinical signs of disease and are able to withstand fasting or medium-chain triglyceride loading well, although a severe fatty liver develops on fasting and hepaticacetyl-CoA levels are decreased [316,438,439].

4.2. 2-Enoyl-CoA hydratases

The 2-enoyl-CoA hydratases have very high activity and are not usually thought to have a rolein the control of b-oxidation flux. The short-chain 2-enoyl-CoA hydratase is only very slowlyactive towards substrates longer than dec-2-enoyl-CoA, which was suggested to be important inthe control of b-oxidation flux [428] until the purification of a long-chain enoyl-CoA hydratase[349], now known to be the long-chain trifunctional protein of b-oxidation [64,405]. This mem-brane-bound protein comprises the activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoAdehydrogenase (a-subunit) and 3-ketoacyl-CoA thiolase (b-subunit) and its importance in the con-trol of b-oxidation flux will be considered with these enzyme activities in Sections 4.3 and 4.4.

4.3. 3-Hydroxyacyl-CoA dehydrogenases

There are at least two mitochondrial 3-hydroxyacyl-CoA dehydrogenases involved in b-oxida-tion, the short-chain 3-hydroxyacyl-CoA dehydrogenase (SHOAD) and the long-chain 3-hydro-xyacyl-CoA dehydrogenase (LHOAD) activity of the a-subunit of the trifunctional protein.There have been additional 3-hydroxyacyl-CoA dehydrogenases described but their physiologicalrole is uncertain [139,200]. Although there is little known about the importance of the 3-hydro-xyacyl-CoA dehydrogenases in the control of b-oxidation flux, short-chain 3-hydroxyacyl-CoAdehydrogenase activity is frequently used as an index of fatty acid oxidation in muscle mito-chondria (e.g. Lange et al. [212]). There are no well-defined inhibitors of the 3-hydroxyacyl-CoAdehydrogenases, although salicylic acid and its metabolites inhibit both 3-hydroxyacyl-CoAdehydrogenases and b-oxidation flux in fibroblasts [144] and benzotript, which inhibits all threeactivities of the trifunctional protein, also inhibits b-oxidation flux in fibroblast homogenates [162].Inherited disorders of SHOAD and LHOAD have been described. Disorders of the trifunc-

tional protein are relatively common and fall into two groups: LCHAD deficiency and completetrifunctional protein deficiency. Complete trifunctional protein deficiency is a quite rare and verysevere, often fatal, disease, in which there is complete absence of the trifunctional protein andtherefore very little residual activity of long-chain 2-enoyl-CoA hydratase, LCHAD and long-chain 3-ketoacyl-CoA thiolase [183,424,425]; a mouse model shows a similarly severe phenotypewith hypoglycaemia and sudden infant death [176]. LCHAD deficiency is much more common,frequently caused by a single point mutation near the active site of the LCHAD domain of the a-subunit, although long-chain 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities mayalso be slightly decreased [182,397,403,425]. Infants usually present with hepatic dysfunction andhypoketotic hypoglycaemia, together with muscle and heart involvement, suggesting that hepaticand muscle b-oxidation flux are severely affected [175,425]. An infant with 30% of normalLCHAD activity in fibroblasts who underwent a fasting and fat loading test showed impairedketogenesis in vivo and �60% of normal b-oxidation flux in fibroblasts [397]. b-Oxidation flux


was also found to be decreased in fibroblasts [285,286,306,425] and peripheral blood cells [344].An interesting observation in LCHAD deficiency which contrasts with other b-oxidation defectsis the frequent finding of opthalomological abnormalities [402,404] and we have recently shownthat retinal pigmentary epithelial cells are capable of b-oxidation and normally posses LCHADactivity [401] suggesting that LCHAD activity is important in the control of b-oxidation flux inthese cells which are highly energy demanding.SCHAD deficiency on the other hand is a rare disorder that is less well defined than LCHAD

deficiency. Until our recent description of a patient with the point mutation in the SCHAD gene [72],none of the patients described as having SCHAD deficiency had mutations found [34,35,329,392].However, it is difficult to draw any inferences about the role of SCHAD in control of b-oxidationflux from the clinical findings in our patient because of the associated hyperinsulinism [72].

4.4. 3-Ketoacyl-CoA thiolases

There are three intra-mitochondrial thiolases, also called acetyl-CoA acyltransferases. Aceto-acetyl-CoA thiolase is specific for acetoacetyl-CoA and 2-methylacetoacetyl-CoA and is prob-ably involved in branched-chain amino acid metabolism, in ketogenesis per se and in ketonebody utilisation, but not b-oxidation [253]. The general acyl-CoA thiolase and the long-chain 3-ketoacyl-CoA thiolase activity of the trifunctional protein have overlapping substrate specifi-cities [363,405]. The reaction is greatly in favour of product formation, thus pulling b-oxidationto completion [252] although product inhibition may be important (see feedback control ofb-oxidation, Section 4.6).The 3-ketoacyl-CoA thiolases have been proposed as the rate-limiting step of b-oxidation in

different tissues as well as important in the feedback control of b-oxidation and control by theacetyl-CoA:CoASH ratio (Section 4.7). The low 3-ketoacyl-CoA thiolase activity has been sug-gested to limit b-oxidation flux in brain mitochondria [440] but heterogeneity between cells typesand must be considered; CPT I activity appears to be rate-controlling in astrocytes [46]. Someevidence for the importance of the 3-ketoacyl-CoA thiolases in the control of b-oxidation flux onother tissues comes from the use of inhibitors. Several inhibitors act on 3-ketoacyl-CoA thiolases,presumably binding to the sulphydryl group at the active site. The hypoglycin analogue 4-pente-noic acid was first thought to act by sequestration of intramitochondrial CoASH but was subse-quently shown to inactivate 3-ketoacyl-CoA thiolases after metabolism to 3-keto-4-pentanoyl-CoA [41,42,116,172,173]. Subsequently, various other compounds which inhibit acetoacetyl-CoA,the general 3-ketoacyl-CoA thiolase and the long-chain 3-ketoacyl-CoA thiolase of the trifunc-tional protein to different degrees have been studied. The structural homologues 4-bromocroto-nate [171,284], 4-bromo-2-octenoate [219] and 4-bromotiglate [220], which irreversibly inactivatethiolases after conversion to their 4-bromo-3-ketoacyl-CoA derivatives, are all potent inhibitorsof palmitoylcarnitine oxidation in liver mitochondria. The inhibition of palmitoylcarnitine oxi-dation parallels or is close to that of thiolase activity, suggesting that 3-ketoacyl-CoA thiolase israte-limiting, or close to rate-limiting, for b-oxidation of palmitoylcarnitine in liver mitochondria[219,220,284]. However, Kunz, using 4-pentenoic acid, estimated the flux control coefficient of3-ketoacyl-CoA thiolase over palmitoylcarnitine b-oxidation to be only 0.13, even at high fluxrates [209]. It has been difficult to determine the importance of the 3-ketoacyl-CoA thiolase in heartbecause several of the inhibitors seem to be ineffective. 4-Bromocrotonate inhibits b-oxidation in


the intact heart [260], but 4-bromo-2-octenoate is inactive in heart mitochondria [219] apparentlydue to lack of activation by acyl-CoA synthetase [442] as is the 3-ketoacyl-CoA thiolase inhibitor2-bromooctanoate [317,318,350] and 4-pentenoate is inactive in the heart due to rapid oxidation[169]. Recently, the antianginal drug trimetazidine has been demonstrated to inhibit long-chain3-ketoacyl-CoA thiolase activity and its pharmacological action suggested to be due to switchingfrom fat to carbohydrate metabolism. However, the degree of inhibition of 3-ketoacyl-CoAthiolase was not measured with inhibition of b-oxidation flux in the perfused heart [193].Only a single case of 3-ketoacyl-CoA thiolase deficiency has been described [191] in which

medium-chain 3-ketoacyl-CoA thiolase activity was decreased by 60%, [1-14C]octanoate oxid-ation decreased by 60% and [1-14C]palmitate oxidation slightly outside the normal range. Thepatient died at 13 days of age after liver and muscle dysfunction, suggesting that general 3-ketoacyl-CoA thiolase is an important enzyme in control of b-oxidation flux. Long-chain 3-ketoacyl-CoA thiolase deficiency has been found only in association with complete absence of thetrifunctional protein (Section 4.3), and acetoacetyl-CoA thiolase deficiency is an inborn error ofbranched chain amino acid and ketone body metabolism rather than of b-oxidation [253].

4.5. Redox control of b-oxidation

b-Oxidation provides ATP not just via acetyl-CoA production, but also by direct generation ofreducing equivalents. One mol ETFH2 (fully reduced ETF, from the acyl-CoA dehydrogenases)and 1 mol NADH+H+ (from the 3-hydroxyacyl-CoA dehydrogenases) are produced from eachturn of the b-oxidation spiral. In tissues for which the primary function of b-oxidation is provision ofATP for contraction, such as skeletal and cardiac muscles, there has to be a method of control of b-oxidation flux though the ATP level, and this may be accomplished via the NAD+/NADH[271,287], reduced and oxidised ETF/ETF:QO and the acetyl-CoA/CoASH couples[271,287](Fig. 8). Redox control can also be important in control of b-oxidation flux in othertissues, such as liver [106,214]. Control by acetyl-CoA/CoASH will be considered in Section 4.7.TheKm values of the acyl-CoA dehydrogenases for ETF are in the low micromolar range [180,341]

and ETF appears to be at substrate levels within mitochondria, greatly in excess of this value[128,129]. ETF-semiquinone, the partially reduced form of ETF, can accumulate when the coenzymeQ pool is reduced [128] and is a potent inhibitor of acyl-CoA dehydrogenase [31]. However, ETF-semiquinone disproportionates to the fully oxidized and fully reduced forms in a reaction catalysedby ETF:QO [30,321] so that the levels of the various ETF species occurring in intact mitochondriaare unknown. The disproportionation reaction appears to be important, as suggested by a mutationwhose major effect appeared to cause a 4-fold decrease in the rate of disproportionation and therebythe rate of the reaction acyl-CoA-MCAD-ETF-ETF:QO-coenzyme Q1 [341]. This mutation is themost common mutation in ETF causing the disease glutaric aciduria type II (which can also becaused by deficiencies in ETF:QO) [151] and causes a 75%decrease in fibroblast b-oxidation flux anda severe neonatal presentation of disease [126]. The disease glutaric aciduria type II causes hypo-ketotic hypoglycaemia, suggesting impairment of hepatic b-oxidation, and there appears to be acorrelation between residual b-oxidation flux in fibroblasts and age at presentation, with patientswith greater residual flux presenting at a later age [126]. Kunz [208,209] has suggested control ofb-oxidation at the level of ETF, and it has been suggested that the ETF-reduction state isresponsible for changes in b-oxidation flux with osmolality [106,155,156].


Early experiments in isolated liver mitochondria demonstrated that when the rate of oxidationof NADH in isolated mitochondria is limited, as it would be in vivo by increased [ATP]/[ADP] orischaemia, 3-hydroxyacyl- and 2-enoyl- CoA intermediates accumulated [55,224,366–368],whereas they did not under conditions of maximal flux (i.e. well oxygenated mitochondria with alow [ATP]/[ADP]). This was interpreted as reflecting control of b-oxidation flux by the [NADH]/[NAD+] redox state and this was supported by studies including measurements of [NADH]/[NAD+] redox state in liver mitochondria [106,189,208,209,214]. However, measurements of theintact CoA ester intermediates of b-oxidation of palmitate or palmitoyl-carnitine in rat skeletalmuscle mitochondria showed accumulation of 3-hydroxyacyl- and 2-enoyl- CoA esters even underwell-oxygenated, non-ADP limited conditions, which was not due to gross changes in intrami-tochondrial [NAD+]/[NADH] [100]. The accumulation of 3-hydroxayacyl- and 2-enoyl- CoAesters could have three possible explanations:

1. 3-Hydroxyacyl-CoA dehydrogenase activity is lower. However, the flavoprotein acyl-CoAdehydrogenases appear to have a very much lower activity than any of the other of theenzymes of b-oxidation (Section 4.1).

2. NAD+ and NADH are channelled between 3-hydroxyacyl-CoA dehydrogenase and com-plex I and it is turnover of this pool rather than gross [NAD+]/[NADH] that is responsible

Fig. 8. Redox and feedback control of intramitochondrial b-oxidation. Solid lines indicate pathways of flux of carbonor reducing equivalents, dotted lines indicate possible feedback controls. Reproduced with permission from S. Eaton etal. [96]. #The Biochemical Society.


for the accumulation of 3-hydroxyacyl- and 2-enoyl- CoA esters. Some support for thisidea comes from studies showing direct interaction of complex I with a variety of mito-chondrial dehydrogenases [136,376,407].

3. The 3-hydroxyacyl-CoA dehydrogenase activity of the trifunctional protein is very sensi-tive to [NAD+]/[NADH] so that even the small changes in [NAD+]/[NADH] observed inrat skeletal muscle mitochondria could cause accumulation of 3-hydroxyacyl- and 2-enoyl-CoA esters. However, this does not appear to be the case [103].

In experiments in rat heart mitochondria to determine whether impairment of the respiratory chain(simulating graded ischaemia or [ATP]/[ADP] increases) inhibited b-oxidation primarily via theETF-linked or primarily via the NADH-linked step, measurements of the acyl-CoA and acyl-carni-tine esters of b-oxidation simultaneously with the redox states of the NAD/NADH and oxidised/reduced coenzyme Q pools suggested that both stages were inhibited together, thus preventing theexcessive accumulation of 3-hydroxyacyl-CoA esters intramitochondrially [105]. This is similar tothe findings of Kunz [208] using two-channel fluorimetry of the ETF and NADH redox states.Interestingly, the redox control of b-oxidation may be different in humans: a comparison of

the CoA esters generated by human skeletal muscle or heart mitochondria with those from rattissues demonstrated that 2-enoyl- and 3-hydroxyacyl- CoA and carnitine esters were almostundetectable [95,183,281].

4.6. Feedback control of b-oxidation

The acyl-CoA dehydrogenases can be inhibited by their 2-enoyl-CoA products [82,307] as canthe enoyl-CoA hydratases by their 3-hydroxyacyl-CoA products [164], 3-hydroxyacyl-CoAdehydrogenases by their 3-ketoacyl-CoA products [165,345,443] and the 3-ketoacyl-CoA thio-lases by either acetyl-CoA [283,443] or an increased [acetyl-CoA]/[CoASH], more closelymimicking the situation in intact mitochondria [103]. In addition, the enoyl-CoA hydratases[428] and short- medium- and long-chain acyl-CoA dehydrogenases [82] can all be inhibited by3-ketoacyl-CoA esters so that accumulation of 3-ketoacyl-CoA esters would be likely to lead toinhibition of b-oxidation flux (Fig. 8). However, although 3-ketoacyl-CoA esters are readilyobserved as intermediates of peroxisomal b-oxidation [29], we have never observed accumulationof 3-ketoacyl-CoA esters as intermediates of mitochondrial b-oxidation in skeletal muscle, heartor liver [26,100,105,106], suggesting that their concentration is less than about 2 mM within themitochondrial matrix. As the Ki of 3-ketoacyl-CoA esters on the acyl-CoA dehydrogenases isabout 80 mM [82,307], on the 2-enoyl-CoA hydratases 20 mM [428] and that on the 3-hydroxyacyl-CoA dehydrogenases 2–7 mM [345,443], whether feedback inhibition of b-oxidation via 3-ketoa-cyl-CoA esters actually takes place is uncertain.

4.7. Control exerted by disposal of acetyl-CoA and the acylation state of the mitochondrialCoASH pool

The general [283,349,427] and long-chain [443] 3-ketoacyl-CoA thiolases are strongly inhibited bytheir product, acetyl-CoA, with a Ki of �4 mM. The inhibition of 3-ketoacyl-CoA thiolase by ace-tyl-CoA is strongly dependent on the concentration of CoASH [427] so that the rate of b-oxidation


could be controlled by the [acetyl-CoA]/[CoASH] ratio. Hence were disposal of acetyl-CoA toketogenesis, the tricarboxylic acid cycle or to acetyl-carnitine inhibited, the [acetyl-CoA]/[CoASH] ratio would increase and feedback inhibition of b-oxidation would result; it has beensuggested that this may be particularly important in control of b-oxidation flux in cardiac mito-chondria [160,287,427]. However, carnitine is usually present at millimolar concentrations in thecytoplasm, providing an adequate buffer of acetyl-CoA via acetyl-carnitine in the heart[105,228,229], skeletal muscle [100,429] and hepatocytes [54,98] so whether control of b-oxidationvia the [acetyl-CoA]/[CoASH] ratio is important in vivo is uncertain. Quant et al. provided evi-dence that the rate of b-oxidation in rat liver mitochondria, in the presence of 2 mM carnitine(but uncoupled and in the presence of a Krebs cycle inhibitor), could be altered by the [acetyl-CoA]/[CoASH] ratio [315] but the [acetyl-CoA]/[CoASH] ratio was probably artificially high dueto the inhibition of the Krebs cycle. Kunz calculated a group flux control coefficient of 0.35 athigh flux rates of palmitoylcarnitine in rat liver mitochondria, including control exerted by CPTII, carnitine acylcarnitine translocase and control by the intramitochondrial concentrations ofCoASH, carnitine, acetyl-CoA and acetyl-carnitine [209], but the control exerted by each these ofsteps was not investigated. In another study in rat liver mitochondria, it was shown that there wasa good correlation between b-oxidation flux and the [acetyl-CoA]/[CoASH] ratio, which wasinterpreted as suggesting that liver mitochondrial b-oxidation flux was relatively insensitive to[acetyl-CoA]/[CoASH] [214]. However, the data could be interpreted as suggesting that b-oxidationflux is limited by [acetyl-CoA]/[CoASH] at high flux rates.As the mitochondrial CoA pool is limited in size, depletion of free CoASH will inhibit both

CPT II and the 3-ketoacyl-CoA thiolase. Garland et al. found that 90–95% of intramitochondrialCoA was acylated during maximal b-oxidation flux, so that only a small amount of CoASH cansustain b-oxidation [141]. As intramitochondrial total CoA is about 3 mM, depending on valuesfor the intramitochondrial volume, 90–95% acylation corresponds to 150–300 mM CoASH.Control via lack of intramitochondrial CoASH with accumulation of 3-ketoacyl-CoA esters andsubsequent feedback inhibition of the previous steps (Fig. 8) would most likely lead to accumu-lation of further acyl-CoA esters, thus lowering intramitochondrial CoASH even further. Otherintramitochondrial enzymes dependent on CoASH (including pyruvate dehydrogenase, bran-ched-chain ketoacid dehydrogenase and a-ketoglutarate dehydrogenase) would also be inhibited.Hence complete acylation of the mitochondrial CoA pool would result in the breakdown ofmitochondrial oxidative metabolism; surely a very risky control method. An alternative controlon b-oxidation in the event of lack of CoASH is to prevent entry of further acyl groups. As CPTII is also CoASH dependent, and the carnitine acyl-carnitine translocase favours export of acyl-carnitine esters when the acyl-carnitine/carnitine ratio rises intramitohondrially [319], this wouldprovide an excellent way of preventing further acylation, freeing some intramitochondrialCoASH (via the reversal of CPT II activity and the export of acyl-carnitine esters) [319] withoutaccumulating further acyl-CoA esters intramitochondrially and decreasing the CoA pool still further.The apparent Michaelis Constants for various intramitochondrial CoASH dependent enzymes

are shown in Table 2. The measurements are clearly made under very different assay conditions ina variety of tissues so should be compared with caution. This is particularly true for the 3-ketoacyl-CoA thiolases, which exhibit ping-pong kinetics so that the Km for CoASH is directlyrelated to the concentration of the 3-ketoacyl-CoA substrate. However, it does appear strikingthat the Km for CoASH of those enzymes causing acylation of the intramitochondrial CoA pool


(CPT II, medium chain acyl-CoA synthase) are an order of magnitude higher than those of the 3-ketoacyl-CoA thiolases and the CoASH-dependent steps related to the tricarboxylic acid cycle.Indeed, the Km of CPT II for CoASH appears to be of the order of 120 mM [274], much higherthan the Km

app of the general 3-ketoacyl-CoA thiolase [252] and the 3-ketoacyl-CoA thiolaseactivity of the trifunctional protein [162]. In addition, the Km of CPT II for acyl-CoA in thereverse direction is low, favouring export of acyl groups when the acylation state of the mito-chondrion increases. Hence, control of entry of acyl groups to the mitochondria by CoASH levelsappears to be a possibility on kinetic grounds.Several inhibitors of b-oxidation have been proposed to act by sequestration of intramitochondrial

CoASH but have subsequently been shown to have specific inhibitory effects [41,42,172,173,290]and very high concentrations of non-metabolisable fatty acids are probably required beforeintramitochondrial CoA is sequestered to any great extent. However, maleate does appear tosequester mitochondrial CoASH, by direct sequestration of 2 moles CoASH per mole maleate,one mole reversibly as thioester and one irreversibly as thioether [291,292]. In rat heart mito-chondria, maleate inhibited b-oxidation flux inhibited by 75% but rather than accumulation of 3-ketoacyl-CoA esters and other intramitochondrial CoA esters, caused accumulation of extra-mitochondrial palmitoyl-CoA, suggesting that CPT II rather than 3-ketoacyl-CoA thiolase isinhibited by lack of intramitochondrial CoASH [104]. An additional pathogenic role for CoASHsequestration, besides metabolism of xenobiotics, has been suggested for the impairment in cardiacfunction related to the long-term oxidation of ketone bodies [336,384].As well as recycling of intramitochondrial CoASH by citrate synthase or ketogenic acetoacetyl-

CoA thiolase, there are other routes via which CoASH can be recycled. These are indicated inTable 2. Firstly, the carnitine acyltransferases, coupled with carnitine-acylcarnitine translocase,

Table 2Michaelis constants of intra-mitochondrial CoASH and acyl-CoA dependent enzymes

Enzyme Km for CoASH (mM) Reference

CPT II 121 [274]

Trifunction protein 3-ketoacyl-CoA thiolase 5 [162]General 3-ketoacyl-CoA thiolase 18 [252]Acetoacetyl-CoA thiolase (acetoacetyl-CoA to 2 acetyl-CoA) 21 [252]Pyruvate dehydrogenase 20 [302]

2-Oxoglutarate dehydrogenase 4.5 [181]Branched chain amino-acid dehydrogenase 10.5 [52]Acetyl-CoA synthetase 400 [127]

Medium-chain acyl-CoA synthase 850 [430]Succinic thiokinase 20 [66]

Km for acyl-CoA ( mM)

CPT II (reverse direction) 3.5 (decanoyl-CoA) [274]Carnitine acetyl-transferase 21 (acetyl-CoA) [47]

Short-chain acyl-CoA thioesterase 1000 (acetyl-CoA) [358]Medium-chain acyl-CoA thioesterase 500 (hexanoyl-CoA) [290]Long-chain acyl-CoA thioesterase 17 (palmitoyl-CoA) 6 (palmitoyl-CoA) [36,380]

Acyl-CoA:glycine-N-acyltransferase 210 (acetyl-CoA) 9 (benzoyl-CoA) [28]


can export acyl groups, thus recycling intramitochondrial CoASH [319]. Secondly, acyl-CoAthioesterases can hydrolyse the thioester link directly. They have been shown to be active inrecycling CoASH from medium- and short-chain acyl-CoAs during inhibition by hypoglycinmetabolites [7,41,42,290]. As well as having a high Km

app for acyl-CoA esters, medium acyl-CoAthioesterase is inhibited by CoASH, thus further limiting its action. Long-chain acyl-CoAthioesterases are present in mitochondria but their physiological role is uncertain [36,305,380,381].Recently, it has been suggested that the novel ‘‘uncoupling protein’’ UCP3, which is found inskeletal muscle and is upregulated in correlation with increased fatty acid oxidation, may have afunction in export of acyl groups hydrolysed by long-chain acyl-CoA thioesterases but directevidence for this is lacking [70,170,261]. Another important route both of detoxification andrecycling of CoASH is glycine conjugation by the matrix enzyme acyl-CoA:glycine-N-acyl-transferase, which has maximal activity towards aromatic acyl-CoA esters and is the route ofappearance of hippuric acid [8,28,142]. Finally, intramitochondrial acyl-CoA synthases arereversible so that acylation of the CoA pool can be reversed in the presence of pyrophosphate andAMP [333].

4.8. Concentration of carnitine and CoA

Carnitine and CoA are clearly both essential for mitochondrial b-oxidation, but their distribu-tion within the cell is different and reflects their function. CoA is mostly found in the mitochon-drion and carnitine mostly within the cytosol, although this varies between tissues and withnutritional state [319]. Supplementation with carnitine as a health food has been proposed tostimulate exercise capacity although there is little evidence that this is effective, as muscle carni-tine pools are not increased by supplementation [188]. Rats with moderate carnitine depletionhave a normal exercise capacity and hepatic b-oxidation flux [166,267] and it appears from studiesin animal models of severe carnitine deficiency and studies in patients with inherited defects incellular carnitine uptake that much more severe carnitine depletion is necessary before b-oxida-tion flux is affected (Fig. 9) [5,56,268,286,360]. However, although these studies all suggest thatthe concentration of carnitine has little effect on b-oxidation flux under the normal range ofphysiological conditions, a recent study in man suggested that during high intensity exercise, freecarnitine in skeletal muscle falls to a level approaching the Km of muscle CPT I for carnitine, sothat the drop in fatty acid oxidation at high exercise rates could have been caused by a slowerentry of fatty acids into the mitochondrion [409].The effect of the total size of the CoA pool on mitochondrial b-oxidation flux is less well

understood. In liver, rats that had the total CoA pool depleted by 23% by feeding with a pan-tothenic acid-deficient diet, mitochondrial b-oxidation flux appeared unaltered whereas perox-isomal b-oxidation was decreased [446]. However, the distribution of hepatic CoA betweenmitochondria, cytosol and peroxisomes was not measured in this study. In a study on heartsperfused with CoA precursors that had �20% increase in total CoA, 14CO2 production from[14C]palmitate was decreased concomitantly with an increase in triglycerides, although whetherthis represented decreased b-oxidation flux or decreased oxidation of [14C]palmitate-derived[14C]acetyl-CoA is uncertain [223]. Again, the distribution of CoA between compartments wasnot reported. CoA can be taken up by mitochondria [385,385–387], and the recent identificationof a mitochondrial CoA carrier protein [310] and cloning of mammalian pantothenate kinase, an


important step in the control of CoA synthesis [331], should help in an understanding of thecontrol of mitochondrial CoA levels and their effect on b-oxidation flux.

4.9. Supramolecular organisation of the intramitochondrial enzymes of b-oxidation

The pathway of long-chain fatty acid oxidation to acetyl-CoA is one of the longest unbranchedpathways in metabolism, containing 27 intermediates between palmitoyl-CoA and acetyl-CoA,and it has long been suggested that the enzymes of b-oxidation are organised into a multienzymecomplex. This was initially based on the detection of low concentrations of intermediates [141]and later the observation that the intermediates of b-oxidation that did accumulate behavedmore like products than intermediates [97,100,105,106,366–368]. This led to the ‘‘leaky hose-pipe’’ model for the control of b-oxidation flux [366–368] in which channelling of a small, quicklyturning-over pool of intermediates is implied. In addition, the measured concentrations of acyl-CoA esters are close to the concentrations of the enzymes of b-oxidation themselves [362]. Ascarnitine acyl-carnitine translocase, CPT II, VLCAD, the trifunctional protein, ETF:QO andcomplex I are bound to the inner membrane and could be associated with CPT I and acyl-CoAsynthetase in contact sites, all the enzymes required for b-oxidation of long-chain acyl-CoAesters, serviced by NAD and ETF (which is present at substrate levels in mitochondria) could beassociated in a metabolon [96,101] (Fig. 10).However, direct evidence is lacking, both for such a complex and for channelling within such a

metabolon. The only direct evidence for channelling of long-chain acyl-CoA esters is between thealpha- and beta- subunits of the trifunctional protein [443] and it cannot be assumed that simplybecause it would appear to make sense that b-oxidation is a channelled process undertaken by a

Fig. 9. Relationship between carnitine concentration and ketogenesis. Livers from mice with juvenile visceral steatosis,which are profoundly carnitine deficient, were perfused with different concentrations of carnitine and the rate of keto-

genesis measured. The concentration of carnitine in control mice was 211 nmol/g wet liver. From Nakajima et al. [268].


metabolon that it does take place by in that way [77]. Another explanation for the low con-centrations of intermediates observed could simply be that most of the control within the path-way of b-oxidation is exerted at or before the level of CPT I. In addition, as pointed out by thelate Paul Srere, the teleological argument that channelling of b-oxidation would have evolvedbecause it is more efficient does not hold because evolution appears to have led from a multi-functional system in procaryotes to multiple enzymes rather than the other way round [362].Although direct evidence for a b-oxidation metabolon is lacking, there is much evidence for

associations of various types between b-oxidation enzymes and between b-oxidation enzymes andtheir associated proteins. Complex I binds several dehydrogenases, including SHOAD [376], andNADH can be channelled between them [136,407]. General 3-ketoacyl-CoA thiolase binds tocitrate synthase [374], and there is at least one SHOAD binding protein in the inner mitochon-drial membrane [138,197] which could provide an anchor for the soluble enzymes of b-oxidationto bind to. The finding that gently sonicated mitochondria oxidised short-chain substrates morerapidly than disrupted mitochondria was also interpreted as providing evidence for association ofthe soluble enzymes of b-oxidation with their redox partners (i.e. ETF, ETF:QO and complex I)[375]. Recently, Parker and Engel showed that functional assemblies consisting of MCAD orsarcosine dehydrogenase (an enzyme of one-carbon metabolism which is also dehydrogenated byETF) together with ETF, ETF:QO, CoQ and complex III could be isolated from sonicated por-cine liver mitochondria [299]. Hence, a true b-oxidation metabolon appears tantalisingly close,but has not been conclusively demonstrated. A possible arrangement of enzymes in such a com-plex I is shown in Fig. 10.

Fig. 10. Supramolecular organisation of mitochondrial b-oxidation. The diagram indicates established and potentialinteractions between b-oxidation enzymes.


5. Overall control of the pathway

5.1. Intermediates of b-oxidation

With the advent of sensitive methods for the analysis of intact acyl-CoA esters and acyl-carni-tine esters, it is now possible to get a picture of how the pathway as a whole might operate. Inisolated rat liver, muscle and heart mitochondria, saturated, 2-enoyl- and 3-hydroxyacyl-CoAesters and their corresponding carnitine esters can be detected [97,100,106] (Fig. 11), suggestingthat at least some control may be exerted at these steps.In human skeletal muscle and heart, 2-enoyl- and 3-hydroxyacyl-CoA esters are at much lower

concentration compared to the corresponding saturated acyl-CoA esters, suggesting that morecontrol is exerted at the acyl-CoA dehydrogenase step [95,183,281]. Measurement of the fullrange of acyl-CoA and carnitine esters has not been undertaken in intact cells or tissues becausethe acyl-CoA esters are at extremely low amounts compared to the amount of cellular protein,although isolated hepatocytes accumulate a similar range of acyl-carnitine esters to the acyl-CoAesters accumulated in intact liver mitochondria [98]. Newer, more sensitive methods for the

Fig. 11. Radio-HPLC chromatogram showing the accumulation of CoA esters from [U-14C]hexadecanoyl-CoA by rat

heart mitochondria. Rat heart mitochondria were incubated with 90 mM [U-14C]hexadecanoyl-CoA and CoA estersextracted and analysed by radio HPLC. Peak identification: 1, Krebs cycle intermediates; 2, acetyl-; 3, octanoyl-; 4, 3-hydroxydodecanoyl-; 5, decanoyl-; 6, dodec-2-enoyl-; 7, 3-hydroxytetradecanoyl-; 8, dodecanoyl-; 9, tetradec-2-enoyl-;

10, 3-hydroxyhexadecanoyl-; 11, tetradecanoyl-; 12, hexadec-3- enoyl-; 13, hexadec-2-enoyl-; 14, hexadecanoyl-. FromEaton et al. [97]. Intermediates of myocardial mitochondrial beta-oxidation: possible channelling of NADH and ofCoA esters. Biochim Biophys Acta 1437:402–408, 1999, with permission from Elsevier Science.


measurement of acyl-CoA esters based on fluorescent derivatives [213] or mass-spectrometry [159]should allow the measurement of the full range of CoA esters in the intact tissue.

5.2. Modelling of b-oxidation

One full model of b-oxidation flux was reported in a series of papers by Kohn and Garfinkel[201–203]. They modelled b-oxidation in the intact perfused heart and included models fortriacylglycerol metabolism, creatine kinase, glycolysis, Krebs cycle, and glutamine oxidation.Their results suggested that b-oxidation itself did not appear to limit fatty acid utilisation undernormoxic conditions and that control was shared between several enzymes. However, manyadvances in the enzymyology and the control of b-oxidation flux have been made since the dataon which this model is based was collected. In particular, the model does not include any con-trol of CPTI by malonyl-CoA via cytosolic citrate because although the control exterted bymalonyl-CoA over b-oxidation flux in the liver was known [245], the presence of malonyl-CoA inheart and the exquisite sensivity of M-CPTI to malonyl-CoA were not [247]. The model could beuseful to combine with more refined and up-to-date models of cardiac carbohydrate metabolism.Apart from the information gained from an examination of the accumulating intermediates of

b-oxidation mentioned earlier, the only other work to generate any overall view of the control ofb-oxidation flux was that of Kunz [209], who undertook control analysis of b-oxidation flux in ratliver mitochondria. He showed that control was shared between different enzymes and that thiecontrol was shifted depending on flux rates and the NAD/NADH redox state. However, theexperiments were conducted using palmitoyl carnitine as the substrate, so the control exerted byCPTI was again missing from his model.

6. Conclusions

b-Oxidation of fatty acids is clearly a long and complex pathway. Control of b-oxidation fluxdoes appear to be largely at the level of entry of acyl groups to mitochondria, but is also depen-dent on substrate supply. CPTI has much of the control of hepatic b-oxidation flux, and althoughit has low control over b-oxidation in isolated heart mitochondria, probably exerts much controlin the intact organ because of the high concentration of malonyl-CoA in vivo. In addition, thecontrol of flux by the redox state of NAD/NADH and ETF/ETFH2 is probably importantunder some conditions. Control by [acetyl-CoA]/[CoASH] may also be significant, but it isuncertain whether this is exerted via accumulation of 3-ketoacyl-CoA esters or via export of acylgroups by carnitine acylcarnitine translocase and CPT II. The likely sharing of control betweenCPTI and other enzymes allows for flexible regulation of metabolism and the ability to rapidlyadapt b-oxidation flux to differing requirements in different tissues.

Acknowledgements

The British Heart Foundation and the Child Health Research Appeal Trust are thanked fortheir support.


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