Eur. J. Biochem. 244, 1-14 (1997)
0 FEBS 1997
The mitochondrial carnitine palmitoyltransferase system
From concept to molecular analysis
J. Denis McGARRY and Nicholas F. BROWN
Departments of Internal Medicine and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas
(Received 9 October 1996) - EJB 96 150810
First conceptualized as a mechanism for the mitochondrial transport of long-chain fatty acids in the
early 1960s, the carnitine palmitoyltransferase (CPT) system has since come to be recognized as a pivotal
component of fuel homeostasis. This is by virtue of the unique sensitivity of the outer membrane CPT I
to the simple molecule, malonyl-CoA. In addition, both CPT I and the inner membrane enzyme, CPT 11,
have proved to be loci of inherited defects, some with disastrous consequences. Early efforts using classical approaches to characterize the CPT proteins in terms of structure/function/regulatoryrelationships
gave rise to confusion and protracted debate. By contrast, recent application of molecular biological tools
has brought major enlightenment at an exponential pace. Here we review some key developments of the
last 20 years that have led to our current understanding of the physiology of the CPT system, the structure
of the CPT isofornis, the chromosomal localization of their respective genes, and the identification of
mutations in the human population.
Keywords: carnitine palmitoyltransferase ; isoform; tissue distribution ; chromosome mapping ; mutation ;
malonyl-CoA; Boxidation ; pancreatic p-cell ; fuel homeostasis ; insulin secretion ; mitochondrial protein.
The essential role of carnitine in the oxidation of long-chain
fatty acids by mammalian tissues first emerged from the pioneering work of the late Irving Fritz in the mid-1950s [I]. Soon
thereafter, independent studies from the laboratories of Bremer
[2] and Fritz [3] gave rise to a conceptual framework to account
for how carnitine enables long-chain fatty acids esterified to
CoA in the extramitochondrial compartment of the cell to gain
access to the enzymes of P-oxidation in the mitochondrial matrix, thus overcoming the permeability barrier of the inner membrane to acyl-CoA esters. Implicit in their prophetic formulation
was that the acyl-CoA molecule first reacts with carnitine under
the influence of a camitine palmitoyltransferase on the outer aspect of the mitochondrial inner membrane (now referred to as
CPT I), generating free CoA and acylcarnitine. The latter would
then permeate the inner membrane (possibly via a specific carrier mechanism) and react with a matrix pool of CoA in a reaction catalyzed by CPT I1 on the inner face of the inner membrane. The re-formed acyl-CoA then enters the pathway of poxidation, while the released carnitine would return to the extramitochondrial compartment.
Correspondence to J. D. McGarry, Professor of Internal Medicine
and Biochemistry, U. T. Southwestern Medical Center at Dallas, 5323
Harry Hines Boulevard, Dallas, Texas 75235-9135, USA
Abbreviations. ACC, acetyl-CoA carboxylase; CPT, carnitine palmitoyltransferase; L-CPT I, liver CPT I; M-CPT I, muscle CPTI; COT,
carnitine octanoyltransferase; FFA, free fatty acids ; LCFA, long-chain
fatty acids; LCFA-CoA, long-chain fatty acyl-CoA; VLDL, very-lowdensity lipoprotein; TG-CoA, tetradecylglycidyl-CoA; Bt,-CAMP, dibutyryl CAMP; BAT, brown adipose tissue; WAT, white adipose tissue.
Dedication. We dedicate this article to the memory of the late Irving
Fritz (1927-1969) whose pioneering work was a major impetus to the
field of study reviewed.
Despite the elegance and unique biochemical features of this
proposed transport mechanism, it seems fair to say that it received relatively little attention during the subsequent decade.
However, all of this changed in the mid-1970s following two
important developments. First, it became clear that inherited defects at the level of the mitochondrial CPT system form the basis
of serious human disease [4]. Second, it was recognized that, at
least in liver, CPT I plays a pivotal role in the regulation of fatty
acid oxidation [ S ] . Together, these observations sparked intense
interest in the structure/function/regulatory relationships surrounding the CPT enzymes. Fortunately, this renewed enthusiasm coincided with the explosion of the ‘new biology’, the tools
of which proved indispensable in bringing us to our current understanding of this intriguing system. In the sections that follow
we shall attempt to summarize some of the key developments in
the CPT arena over the past two decades. Although emphasis
will be on more recent advances, earlier studies will be touched
upon in order to provide an appropriate historical context for
some of the debate that arose along the way.
Emergence of the regulatory role of CPT I in liver
Twenty years ago a major focus of our laboratory was on
the question of how liver accelerates its production of ketone
bodies in ketotic states such as starvation and uncontrolled diabetes. Available evidence at the time indicated that the ketogenic
process is triggered by a fall in the absolute level of circulating
insulin, coupled with elevation of the glucagonlinsulin ratio.
This bi-hormonal perturbation has two important consequences :
first, it allows mobilization of free fatty acids (FFA) from peripheral fat depots; second, it alters the metabolic set of the liver
such that the balance of fatty acid traffic through the esterifica-
2
McGany and Brown ( E m J . Biochem. 244)
VLDL
4
I
TRIGLYCERIDE
I
1
I
GLUFOSE
CARNITINE
I
I
I
I
I
C INSULIN
I
I
I
ACETYL-coll
I
I
I
PYRUVATE
t
KETONE BODIES
Fig. 1. Relationship between fatty acid synthesis and oxidation in
liver. Dashed and solid arrows, pathways of LCFA synthesis and oxidation, respectively; ACC, acetyl-CoA carboxylase; shaded arrow and box,
inhibition of CPT I by malonyl-CoA.
tion and oxidative pathways is shifted towards the latter. It
turned out that control over this key metabolic branch point is
vested largely in the concentration of a simple molecule, malonyl-CoA, the first committed intermediate in the pathway of
fatty acid synthesis and which proved to be a potent inhibitor of
CPT I, the first step specific to the opposing process of fatty
acid oxidation [6, 71. Thus, with carbohydrate feeding (high insulin ; low glucagon/insulin ratio) hepatic lipogenesis is active,
the concentration of malonyl-CoA rises, CPT I is suppressed
and newly formed long-chain fatty acyl-CoAs (LCFA-CoA)are
directed away from oxidation (thus avoiding a futile cycle of
fatty acid carbon) and into esterification products, principally
triacylglycerols. These leave the liver in the form of very-lowdensity lipoproteins (VLDL) and are transported to adipose tissue for storage. Conversely, in ketotic states (low insulin; high
glucagon/insulin ratio) carbon flow through glycolysis and acetyl-CoA carboxylase diminishes, the malonyl-CoA level falls,
fatty acid synthesis comes to a halt, and, for reasons still unclear,
the carnitine content of the liver rises. In this setting, CPT I
becomes derepressed and incoming FFA readily undergo p-oxidation with accelerated production of ketone bodies 16, 71. The
model (schematically illustrated in Fig. I ) has subsequently been
refined to include an additional level of control, namely, that
most conditions of enhanced hepatic fatty acid oxidation are
characterized not only by a fall in the tissue content of malonylCoA, but also by a reduced sensitivity of CPT 1 to the CoA
ester. For a discussion of the relevance of this phenomenon to
the regulation of hepatic substrate flux in different physiological
and pathophysiological states the reader is referred to [8].
Role of the malonyl-CoNCPT I partnership
in non-hepatic tissues
The inhibitory effect of malonyl-CoA on mitochondria1 CPT
I in liver (Fig. 1) made teleological sense in that it provided a
simple mechanism for the reciprocal control of two opposing
pathways, fatty acid synthesis and fatty acid oxidation. What
came as something of a surprise was the finding that in the rat,
non-lipogenic tissues such as heart and skeletal muscle also contain malonyl-CoA and that its concentration in these sites fluctu-
ates with feeding and fasting, just as in liver [9]. In addition,
CPT I of heart and skeletal muscle mitochondria proved to be
far more sensitive to malonyl-CoA and to have a much higher
K,, for carnitine than the liver enzyme [9, 101. These observations raised the possibility that a malonyl-CoA/CPT I interaction
might be important in the function of some non-hepatic tissues.
They also provided the first hint that there might be more than
one isoform of CPT I (see below).
The notion that malonyl-CoA and CPT I constitute an important element of fuel cross-talk in heart and skeletal muscle was
bolstered by the finding that both tissues do contain acetyl-CoA
carboxylase (ACC), albeit an isoform of the enzyme (280 kDa)
distinct from that present in liver and fat (265 kDa) [I1-141.
Studies with the perfused rat heart [lS] and isolated cardiac myocytes [I61 established that substrates such as glucose and lactate, particularly in the presence of insulin, cause elevation in
the tissue content of malonyl-CoA with concomitant suppression
of palrnitate oxidation. Dichloroacetate, an activator of pyruvate
dehydrogenase, also raised the tissue malonyl-CoA (and acetylCoA) level ; here again, fatty acid oxidation was suppressed and
this was accompanied by stimulation of glucose oxidation [15,
171. Imposition of global ischemia on the perfused working rat
heart resulted in a fall in the malonyl-CoA content (attributed
to AMP-induced inhibition of ACC) and accelerated fatty acid
oxidation during reperfusion of the organ [18]. The implications
are that in the heart the synthesis of malonyl-CoA is determined
primarily by the availability of cytosolic acetyl-CoA and that its
role is restricted to the regulation of CPT I (rather than acting
simultaneously as a precursor of fatty acids as in lipogenic tissues) [18].
The situation in skeletal muscle is less clear. Thus, in the rat
red quadriceps and gastrocnemius muscles, a reduction in malonyl-CoA content was observed in response to exercise or electrcal stimulation, both conditions being associated with enhanced
rates of fatty acid oxidation [19]. Notably, the soieus muscle of
the hyperinsulinemic, insulin resistant KKAY mouse exhibited an
elevated malonyl-CoA content [20]. This fell after pioglitazone
treatment of the animals which also improved their insulin sensitivity, suggesting a possible link between cytosolic LCFA-CoA
levels and insulin action [20]. However, no change in malonylCoA content was seen in human vastus lateralis muscle during
submaximal exercise [21]. Whether this represents a species difference or a difference in experimental design between the rat
and human studies remains to be established. An additional uncertainty, which also applies to studies in the heart, has to do
with how much of the malonyl-CoA measured in these tissues
is accessible to CPT I. As noted previously, this must be only a
small fraction of the total malonyl-CoA present or else fatty acid
oxidation would be suppressed at all times [22]. The paradox
might be explained if the cytosol contains a binding protein that
sequesters malonyl-CoA when the tissue has a need for fatty
acid oxidation and releases the inhibitor when suppression of
the process is required. Alternatively, it is conceivable that a
significant fraction of the malonyl-CoA measured in heart and
skeletal muscle is present within the mitochondria (possibly produced there by the action of propionyl-CoA carboxylase on acetyl-CoA), in which case it would be unavailable for interaction
with CPT I. Clearly, the issue requires further study.
A third extrahepatic site in which CPT I regulation by malonyl-CoA appears to play a crucially important physiological role
is the pancreatic p-cell. This new development stems from work
aimed at answering a question that has defied 75 years of intensive investigation : which aspects of glucose metabolism render
this fuel the preeminent insulin secretagogue? The picture
emerging from studies with insulinoma cell lines, the perfused
rat pancreas and isolated rat islets [23-321 can be summarized
McGarry and Brown (Eur J . Biachem. 244)
3
former containing both inhibitor-binding sites and catalytic activity within the same polypeptide. In this model, exposure of
mitochondria to strong detergents solubilizes CPT I1 in active
form, but causes complete loss of CPT I activity through disruption of its essential membrane environment.
The pros and cons of each of these arguments are detailed
in [7, 361. Suffice it to say here that resolution of the debate
required application of the tools of molecular biology in order
to isolate the cDNAs encoding the individual CPT proteins and
thus gain insight into their primary structures. The results proved
highly informative, as summarized in the sections below.
Primary structure of CPT I1
Fig. 2. The mitochondria1 CPT system.
as follows. When the glucose concentration is raised from a nonstimulatory to a stimulatory level, the sugar enters the p-cell via
the high-K,,, GLUT-2 transporter and is phosphorylated by the
high-K, glucokinase; the glucose 6-phosphate formed then enters the glycolytic pathway. The p-cell, like liver (Fig. l), has
all of the enzymes necessary to convert a portion of the glycolytically derived pyruvate into malonyl-CoA which, in turn, suppresses the activity of CPT I and causes elevation of the cytosolic concentration of LCFA-CoA. Current thinking is that
LCFA-CoAs act to stimulate exocytosis of insulin granules by a
mechanism(s) still to be elucidated and discussed in [31, 321. If,
as we suspect, this concept has validity, it brings to light a hitherto unrecognized feature of fatty acid metabolism in the workings of the pancreatic p-cell (see also [33]) and confers on the
malonyl-CoNCPT I partnership a central role in the regulation
of insulin secretion. The exciting possibility is now raised that
derangements in this coupling mechanism might be instrumental
in the p-cell dysfunction characteristic of insulin-resistanthype
2 diabetes syndromes.
Molecular characterization of the CPT proteins
By 1987, understanding of the mitochondria1 transport of
fatty acids in broad operational terms had evolved to the point
depicted in Fig. 2. The model incorporated two refinements into
the original formulation: (a) recognition by Pande and colleagues of a carnitine-acylcarnitine translocase component present on the inner membrane [34]; and (b) reassignment by the
same group of CPT I from the outer aspect of the inner membrane to the outer membrane itself [35]. At a structural level,
however, three interrelated questions remained the subject of
major controversy. First, within a given tissue are CPT I and
CPT I1 the same or different proteins? Second, how do CPT I
and CPT I1 compare in different tissues? Third, how do malonyl-CoA (reversible inhibitor of CPT I activity) and agents such
as tetradecylglycidyl-CoA (TG-CoA) and etomoxir-CoA
(irreversible inhibitors) interact with the enzyme? Briefly, there
were two schools of thought. One held that CPT I and CPT I1
are the same protein (at least in a given tissue if not body-wide),
but that the former has an associated regulatory subunit to which
inhibitors bind and somehow suppress activity of the catalytic
component. A corollary of this argument was that the wellknown loss of inhibitor sensitivity of detergent-solubilized CPT
results from disruption of the link between the regulatory and
catalytic components of CPT I. An alternative view, which we
favored, saw CPT I and CPT I1 as distinct proteins with the
Our initial focus at the molecular level was on the detergentsoluble, active and malonyl-CoA-insensitive CPT enzyme from
rat liver mitochondria which we considered to represent CPT 11.
The protein was readily purified and a specific antibody was
raised against it [37]. Partial peptide sequencing allowed the
generation of oligonucleotides with which to screen a rat liver
cDNA library. The full-length cDNA clone ultimately isolated
predicted a protein of 658 amino acids containing a 25 amino
acid NH,-terminal leader sequence that is cleaved upon mitochondrial import to yield a mature protein with molecular size
of approximately 71 kDa [38, 391. Expression of the full-length
cDNA in COS cells resulted in marked overexpression of malonyl-CoA-insensitive CPT activity that was measurable only after detergent solubilization of the mitochondria and was detectable by an antibody specific to the original purified protein [38],
i.e., the product had all of the characteristics expected of CPT
11. In a wide variety of rat tissues ([37, 401 and unpublished
studies) the CPT I1 mRNA was found to be identical in size
( ~ 2 . kb)
5 and the product to be immunologically indistinguishable, implying the operation of a single gene and expression of
the same protein body-wide.
The above described studies with rat CPT I1 were soon followed by isolation of the cDNA for its human counterpart [41,
421. As in the case of the rat enzyme, the human cDNA also
predicted a nascent product of 658 amino acids. Not surprisingly, the two proteins showed strong similarity (85% and 82%
identity at the nucleotide and amino acid level, respectively).
However, the human CPT I1 mRNA (=3 kb) is approximately
0.5 kb larger than its rat equivalent [41, 421. Curiously, although
the rat and human mature proteins differ in molecular mass by
only about 100 Da (the former being larger), their mobilities on
SDS/polyacrylamide gels suggested a much greater difference.
This was true both for the proteins extracted from liver mitochondria [37] and those generated by in vitro transcription and
translation of the respective cDNAs [39, 431. It seems likely that
the apparently larger size of the rat compared with the human
enzyme reflects an intrinsic difference in their primary amino
acid sequences. It is also probable that the rat protein behaves
anomalously, in light of the fact that human, mouse and monkey
CPT I1 all comigrate on SDS/polyacrylamide gels [37].
Primary structure of CPT I
Liver. Characterization of the CPT I molecule proved to be
much more difficult because of two properties of the protein
predicted by our earlier studies [7]: its tight membrane association and its loss of catalytic activity when removed from its
natural environment. The strategy ultimately employed entailed
exposure of rat liver mitochondria to ['H]etomoxir-CoA to label
CPT I covalently, followed by treatment of the membranes with
a mixture of proteases. This resulted in truncation of the CPT I
protein such that the shorter version (still carrying the label)
4
McGarry and Brown ( E m 1.Biochent. 244)
could be isolated in pure form from SDS gels in amounts sufficient to obtain peptide sequences [44]. With this information,
oligonucleotides were synthesized and used to screen our rat
liver cDNA library [45]. The full-length clone isolated predicted
a protein of 773 amino acids (88 kDa) ind, when expressed in
COS cells, resulted in a 10-20-fold induction of CPT activity
that was both inhibitable by malonyl-CoA (and etomoxir-CoA)
and detergent labile [45]. Similar results were obtained when the
cDNA was expressed i n yeast cells which are totally devoid of
endogenous CPT activity [46]. These findings established unequivocally two key points: (a) that the CPT I molecule consists
of a single polypeptide containing both the inhibitor binding and
catalytic domains; and (b) that CPT I and CPT I1 are distinct
entities (and are derived from transcripts of very different size,
approximately 4.7 and 2.5 kb, respectively, due largely to a particularly long 2 kb 3’-untranslated region in the CPT I mRNA).
A longstanding debate (see above) was thus finally put to rest.
Unlike CPT 11, and characteristic of outer-membrane proteins, no NH,-terminal signal peptide is removed from the nascent L-CPT I during its mitochondria1 import [47].
With the rat liver CPT I cDNA in hand, it was possible to
isolate its human counterpart from a human liver cDNA library
[48]. As was true for CPT 11, both the nucleotide sequence of
the human liver CPT I cDNA and its predicted primary structure
(773 amino acids) proved to be very similar to those of the rat
enzyme (82% and 88% identity, respectively).
quences and predicted primary structures of the rat and human
proteins turned out to be highly similar (=85% and 86% identity, respectively). In both cases, the transcript size (”3 kb) is
much smaller than that for rat and human L-CPT I ( ~ 4 . kb),
7
the difference reflecting mainly the much shorter 3’-untranslated
region of the muscle mRNA [52, 531.
A comparison of the predicted primary structures of the rat
and human CPT proteins is shown in Fig. 3.
Tissue expression of CPT I isoforms
Designation of the two known mitochondrial CPT I isoenzymes as liver-type (L-CPT I) and skeletal muscle-type (M-CPT
I) is based upon the tissues originally identified as expressing
these isoforms. However, from northern blot analysis and [’HIetomoxir-CoA labeling of CPT I in mitochondria prepared from
a variety of tissues it has become apparent that the expression
of both proteins is widespread (Table 1).
Particular interest has surrounded CPT I in rat heart mitochondria, which has long been noted to display kinetics intermediate between those of liver and skeletal muscle (in terms of
IC,,, for nialonyl-CoA and K,,, for carnitine [9]). The explanation
came to light with the finding that although this tissue expresses
M-CPT I predominantly, it also contains sufficient L-CPT I to
account for the kinetic data [49,54]. The level of heart L-CPT I
is particularly high in the late fetalheonatal rat, declining during
development [%I. This decrease in cardiac L-CPT I expression
Skeletal muscle. That skeletal muscle (M) and liver (L) CPT occurs simultaneously with the accumulation of carnitine in the
I must be different proteins was suspected from two sets of ob- growing heart and it is inferred that the low K, liver isoform
servations. First, when assayed in intact mitochondria under makes a critical contribution to LCFA oxidation in the neonatal
standard conditions [9] the rat liver and muscle enzymes exhib- organ [55].That L-CPT I and M-CPT I are present in the myoited a 100-fold difference i n sensitivity to malonyl-CoA (ICso cyte itself has been confirmed in cells obtained from adult and
values of ~ 2 . pM
7 and 0.03 yM, respectively). They also dis- neonatal rats [49, 55, 561. Both isoforms are also seen in the
played very different K,,, values for carnitine (-30 pM and human heart (Fig. 4).
As described above, the cDNA encoding rat M-CPT I was
500 yM, respectively). Second, when labeled covalently with radioactive TG-CoA [37] or etomoxir-CoA [49], the two proteins originally isolated from BAT [SO]. In the same study it was also
migrated on SDS/polyacrylamide gels with apparent monomeric found that rat epididymal WAT expressed predominantly L-CPT
sizes of approximately 88 kDa (L) and 82 kDa (M). Formal proof I. This observation is potentially misleading, however, since it
of the distinction came with the isolation of a cDNA for the rat has now been shown that when rat white adipocytes are purified
muscle enzyme. This occurred in a rather circuitous way. Using away from the stromal tissue in the fat pad, they too express M a subtraction cloning strategy, Yamazaki et al. [50] sought to CPT I as the major isoform [51].
In studies to be published elsewhere, northern blots with
identify gene products (other than the uncoupling protein) that
are expressed i n rat brown adipose tissue (BAT) but not in white RNA from a variety of rat tissues indicate that L-CPT I is the
adipose tissue (WAT). A cDNA thought to represent such a pro- primary (or sole) isoform expressed in kidney, lung, spleen, intein was isolated and found to share approximately 63% identity testine, ovary and pancreas (both islets and acinar tissue),
with rat L-CPT I, leading the authors to speculate that it might whereas M-CPT I predominates in the testis (Table 1). Human
encode the muscle variant of CPT I [50].This was shown to be fibroblasts express only the liver-type CPT I [48].
the case when Esser et al. [51] isolated the identical clone from
a rat heart library (known to be rich in M-CPT I), expressed it
Structural comparison of the CPT enzymes
in COS cells, and found the product to have physical and kinetic
No CPT structure has yet been determined crystallographicharacteristics identical to those o f CPT I in skeletal muscle
mitochondria. Still somewhat puzzling is why L and M-CPT 1, cally. However, information gleaned from conventional biopredicted to contain 773 and 772 amino acids with molecular chemical studies, expression of mutant forms in Escherichia coli
masses of 88 150 Da and 88 227 Da, respectively, should migrate and Saccharomyces cerevisiae, and direct comparison of the preso differently on SDS/polyacrylamide gels (see above). Because dicted primary structures has yielded substantial insight into the
this also applies to the in vitro transcribed and translated cDNAs topology of the enzymes. They also provide pointers to poten1.511, it may be inferred that the different electrophoretic mobil- tially important catalytic domains.
ity of the two proteins is an intrinsic property of their primary
Membrane topology. CPT I1 is loosely associated with the
structures, (as opposed to selective post-translational modification) reminiscent of the situation with rat and human CPT I1 inside of the inner mitochondrial membrane and can easily be
noted above. That rat L-CPT I migrates on SDS/polyacrylamide solubilized in active form by mild detergent treatment, such as
gels to a position expected for a protein of 88 kDa suggests that 1 % Tween-20 [57]. In keeping with this observation is the absence of any amino acid sequence indicative of a menibranethe rat muscle enzyme behaves anomalously [51].
Using the rat M-CPT I cDNA as a probe both we [52] and spanning domain [ 381. The matrix location of CPT 11’s catalytic
Yamazaki et a]. [53] have recently isolated its equivalent from center is confirmed by its insensitivity to protease treatment of
human heart cDNA libraries. As expected, the nucleotide se- intact mitochondria [even under conditions where adenylate ki-
5
McGarry and Brown (EUK1. Biochem. 244)
CPTII
human
rat
..................................................
..................................................
+
+++
A
+
human . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
L-CPTI human I T
K T D P S L G I I A K I N R T L E l . . A N C M S 98
rat I T
K V D P S L G H I A K I S R T L D T . . T G R M S 98
M-CPT I human L R
" J D I S L G L V S C l Q R C L P Q G C G P Y Q T 1 W
rat L R
K V V l S M G L V H C l Q R C L P T R Y G S Y G T l O O
CPTII
(+)
CPT II
human
rat
H1
........................
CPT It
human
rat
L-CPT I human
rat
M-CPT I human
rat
human
rat
L-CPT I human
rat
M-CPT I human
rat
M
vP
000 000
-
R L
(+I(+)
+
4
~ A
w RP
R GP A
v
G P
~
PA
~ L s P26
....
H2
CPT II
(+) (+)
A
A
125
125
246
246
248
248
N P D P K S E Y N D
N P D P K S E Y N D
..........
..........
..........
..........
175
175
246
246
248
248
Fig.3. Amino acid sequence alignment of rat and human mitochondria1 CPT proteins. Sequences were obtained from the following sources;
human CPT I1 [41, 421, rat CPT I1 [38], human L-CPT I [48], rat L-CPT I [45], human M-CPT I 152, 531, rat M-CPT I [51]. Residue numbering
is shown at the right of each line. Boxed amino acids are those conserved in at least four sequences. Shaded areas labeled H1 and H2, hydrophobic
regions in CPT 1; arrow, cleavage site of nascent CPT 11; open triangles, histidine residues conserved in the CPT I NH,-terminal region; filled
triangles and asterisk, aspartate and histidine residues, respectively, conserved throughout the carnitinekholine acyltransferase family of enzymes.
Charged amino acid residues are indicated only in the region of HI : + and -, positively and negatively charged residues, with parentheses denoting
lack of conservation. See text for details.
nase (inter-membrane space) and P-hydroxybutyrate dehydrogenase (inner membrane) are destroyed], whereas after exposure
of the matrix face by freezehhawing, the enzyme is readily proteolysed [58].
The situation with the outer membrane CPT I is markedly
different. The enzyme is not extracted from the membrane by
mild detergents but can be solubilized by more powerful agents
such as Triton X-100 or octyl glucoside [57]. However, under
these conditions it loses catalytic activity and, presumably, threedimensional structure, consistent with its being an integral mernbrane protein. The deduced sequences of both L-CPT I and MCPT I contain two largely hydrophobic domains encompassing
amino acids 53-75 (HI) and 103-122 (H2) in the liver se-
quences (shaded areas in Fig. 3) While H2 is almost certainly
membrane spanning, it is not clear whether H1 also has this
property, given the presence of prolines at positions 56 and 59
which would be atypical for a transmembrane domain. However,
Zammit and coworkers have suggested that H1 acts as a stoptransfer signal and does indeed span the membrane [47]. Moreover, a cluster of five completely conserved positively charged
residues is located close to the NH,-terminus of H1 as opposed
to conservation of one negative and one positive charge in similar proximity to the carboxy-terminal end. This charge distribution is typical for proteins where the NH,-terminus faces the
cytosol, the orientation of the remaining membrane spans being
dependent on the most NH,-terminal one [47]. Thus, it might be
6
McGarry and Brown ( E m J. Biochem. 244)
human
rat
L-CPT I human
rat
M-CPT I human
rat
CPT II
T I T F K RL I R F V P S S L S W Y G A YL V N A Y P L D M
tP
iS
.L S W Y G A YL V N A Y P L D M
T D A F K R L. Il R[ FS VT P
.........
......
.......IRLLGSTIPLCSA. .........
.. . V M A L G . I V P M C S Y . . . . . . . . . .
.....
...VMALG.MVPMCSY.
human
rat
L-CPT I human
rat
M-CPT I human
rat
CPT II
L V L R K G N F Y I F D V L D Q D G N I V S P S E I Q A H L K Y 2 7 5
LVLRKGHFYVFDVLDQDGNIVNPLEIQAHLKY275
V V Yp H RQ G RVY F K V ..W fL iY ~
H DR G[ RR L~L~K LP ~R QE [
M EM Q~ QQ M Q R 3 6365
5
A V Y H K G R F F K L - W L Y E G A R L L K P Q D L E M Q F Q R m
AVYHKGRFFKV.WLYEGSCLLKPRDLEMQFQR366
human
rat
L-CPT I human
rat
M-CPT I human
rat
CPT II
L M S
........
A
CPT II
human
rat
L-CPT I human
rat
M-CPT I human
rat
T T D S T V T V 412
A T N S S A S V 412
%
A
Fig. 3. Continuation
predicted that almost all of the CPT I molecule resides on the
cytosolic face of the outer membrane with a stretch of less than
30 amino acids protruding into the inter-membrane space. Such
a topology would be entirely compatible with a study in which
malonyl-CoA and octanoyl-CoA, both rendered impermeable to
the outer membrane by coupling to agarose beads, were still
capable of acting as inhibitor and substrate, respectively, demonstrating that domains necessary for regulation and catalysis are
accessible to the cytosol [59]. This model, taken in conjunction
with the evidence presented below regarding the locations of the
inhibitor and substrate binding sites within the CPT I polypeptide, is extremely persuasive.
Substrate-binding and inhibitor-binding sites on CPT I.
Expression of rat L-CPT I in COS cells [45] and yeast 1461
established unequivocally that the catalytic center of the enzyme
and the binding sites for both the physiological, reversible, inhibitor malonyl-CoA, and the synthetic, covalent inhibitor, etomoxir-CoA, reside on a single polypeptide. However, the princi-
pal sites of action of these two agents may be distinct within the
enzyme molecule.
A critical question has been whether malonyl-CoA acts as a
simple, competitive inhibitor by binding at the active site or
whether it acts at a separate allosteric locus. Several authors
have reported competition between malonyl-CoA and the palmitoyl-CoA substrate [60-631. However, detailed kinetic analysis
of the inhibition of CPT I by malonyl-CoA in rat liver mitochondria [64] or permeabilized cultured neonatal myocytes [65]
suggests that the phenomenon is not purely competitive, but also
contains an allosteric component.
Some light has been shed on the problem by experiments
with proteases. Treatment of rat liver mitochondria with Nagarse, chymotrypsin, papain or proteinase K results in a marked
loss of sensitivity to malonyl-CoA before catalytic activity is
entirely destroyed 158, 59, 66, 671. The clear inference is that
under the conditions used these proteases are able to digest a
region of the protein essential for allosteric inhibition while the
catalytic center remains intact. Trypsin is capable of destroying
7
McGarry and Brown ( E M J. Biochem. 244)
A
CPT II
human
rat
L-CPT I human
rat
M-CPT I human
rat
CPT II
human
rat
L-CPT I human
rat
M-CPT I human
rat
P S
QMMV
P S
DPAQTVEQRL
DP KSTAEQRL
EG
R H L F A L R 560
ig
R H L Y A L R 560
MIY
TG
Y
LTK
AV
v .. HnD Mj WGI G[ c VN vh s . .
R LA M T
R H L F C L . 660
R H L F C L . 660
GJAIG
R H L F C L . 660
I D R H LF
c
LI.
660
1
. s y
I L V G E N L I N F H I S S K F S C
I I V G E N F I H F H I S S K F S S
M I A G E N T I F F H I S S K F S S
M I A G E N T M F F H V S S K L S S
658
658
773
773
772
772
Fig. 3. Continuation
CPT I catalytic activity while having little [58] or no [67] effect
on malonyl-CoA sensitivity when the mitochondria remain undamaged. This indicates that the desensitization phenomenon is
probably the result of protease specificity and the availability of
cleavage sites and is not due simply to the active site being
located on the inner side of the membrane and so being protected, as has been suggested [66, 681.
Proteases have a similar desensitizing effect on L-CPT I with
regard to inhibition by succinyl-CoA, methylmalonyl-CoA and
Ro-25-0187 (a malonyl-CoA analog), whereas the potency of
CoASH, acetyl-CoA and propionyl-CoA is unaffected [68]. This
suggests that the dicarboxylic acid esters are capable of binding
at the same allosteric site, which is the primary site of inhibition
by malonyl-CoA. If malonyl-CoA is included in the incubation
during protease treatment both desensitization and inactivation
of the enzyme are curtailed [58, 691, and the same is true for
those other inhibitors believed to bind at the allosteric site [68].
Proteolytic treatment does not appear to diminish sensitivity to
the presumed active-site-directed inhibitors, D~-2-bromopalmitoyl-CoA [67], (+)-hemipalmitoylcarnitinium [68] or etomoxirCoA [58]. Prior treatment of rat liver mitochondria with etomoxir-CoA prevents total destruction of imniunoreactive L-CPT
I by a mixture of trypsin and chymotrypsin, an observation that
was exploited in the initial purification of the enzyme [44, 451.
However, due to the covalent nature of the inhibitor-protein interaction, this cannot be related to protection of enzyme activity.
In addition to protecting against the action of proteases, the
presence of malonyl-CoA also prevents the well-established desensitization to the inhibitor normally seen when rat liver mitochondria are allowed to warm from 0" to 22°C [58, 70, 711.
Furthermore, addition of malonyl-CoA at 22°C to liver mitochondria that have been allowed to desensitize in this way can
restore full sensitivity [58, 70, 711. Predictably, malonyl-CoA
cannot reverse the desensitizing effect of proteases [58]. One
can speculate that binding of the physiological inhibitor causes
a substantial change in the conformation of CPT I such that the
enzyme is rendered more accommodating to subsequent binding
of a malonyl-CoA molecule and (perhaps coincidentally) less
susceptible to proteolysis (discussed in [42, 581). It is uncertain
how these temperaturehalonyl-CoA effects on the affinity of
CPT I for malonyl-CoA in vitro may be related to the decrease
in sensitivity of the rat liver enzyme that accompanies starvation
and diabetes.
Unlike the CPT I of liver, the enzyme in adult rat heart mitochondria ( ~ 9 8 %
M-CPT I [54]) displays little loss of malonylCoA sensitivity on warming. Protease-induced desensitization,
however, is observed with the muscle-type enzyme [58].
As seen in Fig. 3, the greater size of CPT I relative to CPT
I1 is due largely to an extended NH,-terminal region in the former that bears no significant similarity to CPT I1 for approximately the first 170 amino acids. Naturally, one might suspect
that this region of marked departure from the otherwise close
8
McGarry and Brown (Euc J. Biochem. 244)
Table 1. Overview of mitochondrial CPT enzymes. Relative levels of
CPT I isoform expression for each organ are based on northern blot
analysis (and in some cases ['Hletomoxir-CoA labeling), but do not indi-
cate precise ratios. (+), trace expression compared with the alternative
isoform; -, undetected. Tissue expression data refer to the rat, except
in the case of fibroblasts. The K,,, for carnitine of CPT I1 was measured
after solubilization of mitochodria in 1% octyl glucoside and therefore
should not be compared directly with the values for CPT I.
Feature
~_____
L-CPTI
M-CPTI
CPTII
=88 kDa
=2.5 pM
=30 pM
=X8 kDa
=70 kDa
llq13
22q13.3
_____
Mass
Malonyl-CoA IC,,,
Carnitine K,,,
Human chromosome locus
Tissue expression
Liver
Skeletal muscle
Heart
Kidney
Lung
Spleen
Intestine
Pancreas (islets and acinar)
Brown adipose tissue
White adipocytes
Ovary
Testis
Human fibroblasts
Human deficiency described
++++
+
++++
++++
++++
++++
++++
(+I
+
++++
(+I
++++
(+)
yes
~ 0 . 0 3pM
=500 pM
-
++++
+++
(+I
(+I
~
++++
+++
(+I
++++
~
no
-
=I20 pM
1p32
+
+
+
+
+
+
+
+
+
+
+
+
+
yes
Fig.4. ['H]Etomoxir-CoA labeling of CPT I in human heart mitochondria. Patient age is indicated in months (mo.) or years (yr.). Heart
tissue was obtained during surgery for correction of a congenital defect
(15 mo.) or at transplant (others). Mitochondria1 preparation and labeling
of CPT I isoforms was as described for rat heart [49]. L and M, migration positions of L-CPT I and M-CPT I, respectively.
similarity between the outer and inner membrane enzymes may
relate to the most salient distinction between them: the unique
inhibitability of CPT I by malonyl-CoA. Evidence for a critical
role of the NH,-terminus in malonyl-CoA inhibition of L-CPT I
was obtained by expression of the full-length rat enzyme and a
truncated version of the protein in yeast [46]. The shorter product, lacking the first 82 amino acids of the wild-type protein
showed a greatly decreased sensitivity to malonyl-CoA (ICso
=80 pM vs 5 pM). This i s consistent with a model in which the
NH,-terminus is essential for forming the high-affinity, allosteric
malonyl-CoA binding site, with residual inhibition exerted at the
level of competition at the active site located within the remainder of the polypeptide.
The active site. Due to its relative ease of solubilization,
purification and expression, CPT TI has received more attention
than CPT I as regards reaction mechanism, but given the close
similarity of the two proteins it seems likely that both active
sites share some important features. By studying the kinetics
of purified bovine CPT IT, Nic A'Bhaird and colleagues found
evidence for an ordered mechanism in which the binding of
acyl-CoA or CoA binding must precede that of carnitine [72].
An acyl-enzyme intermediate does not appear to be formed. The
authors propose that an ordered mechanism also exists for CPT
I in intact mitochondria. (This is not true for COT, which seemingly employs a random-order mechanism [72]). Transition-state
theory has led to the synthesis of several transition-state analogs
that have proved to be effective inhibitors of both CPT I1 and
CPT I [72, 731.
It was observed early on that lowering the pH from 8 to 6
decreases the affinity of CPT I for carnitine while increasing
that for malonyl-CoA [74, 751. This pH change is associated
with the protonation of histidine residue imidazole groups and
it has been inferred that one or more of these may be involved in
the CPT reaction. Furthermore, treatment of the closely related
carnitine acetyltransferase with the inhibitor bromo-acetyl-L-carnitine, believed to bind at the active site, results in the specific
alkylation of a histidine residue [76]. In addition, both CPT 11
[77, 781 and COT [78] are rapidly inactivated by the histidinemodifying reagent, diethylpyrocarbonate.
The use of site-directed mutagenesis has recently allowed
candidate catalytic residues to be investigated individually. Examination of the primary structures of the CPT proteins (Fig. 3)
and of closely related enzymes such as COT, carnitine acetyltransferase and choline acetyltransferase from species as widely
divergent as humans, yeast and insects [77] reveals the perfect
conservation of a single histidine residue (marked * in Fig. 3).
By analogy with the highly cliaracterized histidinelaspartatelserine catalytic triad of the serine proteases, we have suggested that
a similar mechanism may operate in the case of CPT [77]. In
the case of the serine proteases, the serine is deprotonated by
histidine, the aspartate acting in a charge relay system 1791. For
acyltransferases, the hydroxyl group of the carnitine substrate
might correspond to that of the serine residue [77]. In line with
this theory, three aspartate residues are also seen to be conserved
throughout the carnitinelcholine acyltransferase family (marked
A in Fig. 3). To examine the roles of these residues, wild-type
rat CPT 11 and a series of mutants in which the putative catalytic
histidines and aspartates were replaced by alanine were expressed in S. cerevisiae [77]. Mutation of His372 or Asp376 or
Asp464 (residue numbering for CPT 11) resulted in complete
loss of function although levels of imniunoreactive protein were
comparable in each case. Collectively, these data provide overwhelming evidence for the critical role of a histidine in catalysis
(probably His372 in CPT 11), though direct proof of the reaction
mechanism is not yet available.
The five- to ten-fold increase in the IC,, for malonyl-CoA
of CPT I in mitochondria from rat liver, heart or skeletal muscle
with a rise in pH from 6.8 to 7.6 has also been proposed to result
from titration of a histidine residue [75]. The magnitude of the
effect indicates that it is being exerted largely at the allosteric
site and is not connected with the catalytic histidine. Intriguingly, the NH,-terminal regions of the CPT I enzymes, so critical
for malonyl-CoA sensitivity (see above), do contain three conserved histidine residues (marked A in Fig. 3).
The carnitine-acylcarnitinetranslocase. Less well characterized than the CPT enzymes, but just as important for the overall function of the CPT system, is the specific carnitine-acylcarnitine transporter located in the inner mitochondrial membrane.
The protein, with an apparent molecular mass of 32.5 kDa on
SDSIPAGE, has been purified to homogeneity from rat liver
[go]. Reconstitution of the carrier into liposomes has been used
to analyze the transport mechanism kinetically. Transport of
['Hlcarnitine into the liposomal lumen was shown to occur readily when the vesicles were loaded with acylcarnitines of various
McGarry and Brown (Eul: J. Biochem. 244)
chain lengths or with carnitine itself, but not when such countersubstrates were absent [80]. The mechanism appears to be of the
ping-pong type, which makes the carnitine carrier unique among
all the mitochondrial metabolite transporters characterized [8l,
821. Clones encoding the entire carnitine carrier have reportedly
been isolated from a rat liver cDNA library [82]. The sequence
is said to predict a protein of 301 amino acids consisting of three
homologous tandem repeats, features common to other mitochondrial transporters [82].
Insight into the CPT genes
Chromosomal mapping. The first of the human CPT genes
to be assigned a chromosomal residence was that for CPT 11; it
was localized by Gellera et al. [83] to chromosome lp32. More
recently, we have been able to assign the genes for liver and
muscle CPT I to chromosomes 11q13 and 22q13.3, respectively
[48, 521. These advances both reinforce and allow refinement of
three key points made earlier (a) that in any given tissue CPT I
and CPT I1 are indeed distinct entities and are now seen to be
encoded by different genes; (b) that CPT I1 is almost certainly
the product of a single gene body-wide; and (c) that CPT I exists
in at least two isoforms, the liver and muscle variants, and these
also arise from separate genes.
Structure. To date, only the CPT I1 gene has been analyzed
i n detail. In the human it is approximately 20 kb in size and is
composed of five exons, ranging from 81 bp to 1305 bp, separated by four introns varying from 1.5 kb to 8 kb in length [84].
The gene has some very unusual features. For example, exon 4
(1305 bp) is exceptionally long and accounts for 66% of the
translated portion of the mRNA. It has been reported that in
vertebrates only approximately 0.5 % of internal exons are more
than 550 bp in length, the average size being 137 bp [85]. Intron
3 of the CPT I1 gene (=8 kb) is also particularly long, given the
fact that less than 8% of vertebrate introns are said to be more
than 3 kb in size [85]. Similarly, the 5’-untranslated region
(516 bp) greatly exceeds the average length (77 bp) for vertebrate genes, placing it in the second size percentile for such
domains [84]. The murine CPT I1 gene is very similar to its
human counterpart in terms of its exon-intron organization [86].
Putative regulatory elements have been identified in the promoter regions of the two genes, including SP-1 and AP-1 sites,
an insulin-responsive sequence and a DNA-recognition sequence
for steroidkhyroid hormone receptors, but whether all of these
sites are present in both constructs is unclear at present [84, 861.
However, the CIEBPa and CREB binding sites claimed to be
present in the promoter region of the rat CPT 11 gene by Brady
et al. [87] were not evident in the murine or human genes [84,
861, substantiating our belief [88] that the rat CPT I1 genomic
clone reported in [87] was an artifact.
Regulation. Studies on the regulation of the CPT genes are
still in their infancy and have been restricted for the most part
to understanding how changes in nutritional and/or hormonal
status, as well as the administration of pharmacological agents,
affect expression of the CPT proteins in rat liver. Manipulations
that enhance the capacity for hepatic fatty acid oxidation, such
as fasting, fat feeding, induction of diabetes or treatment of rats
with peroxisomal/mitochondrial proliferating agents (e.g., clofibrate or diethylhexylphthalate), all cause an increase in the
mRNA and activity levels of CPT I and CPT 11. The effects are
variable and sometimes profound. For example, whereas in 48 h
fasted and streptozotocin diabetic rats the specific activiy of
CPT I in liver mitochondria was found to increase by 2-3-fold,
the CPT I mRNA level rose by 7.5-fold and 15-fold, respectively
9
[89]. No information was provided on CPT I1 gene expression
in that study. Compared with hypothyroid rats, hyperthyroid animals exhibited a 40-fold elevation in liver CPT l mRNA abundance, and this was paralleled by changes in CPT I activity [90] ;
again, CPT I1 expression was not examined.
A highly selective effect on transcriptional activity of the
CPT I and CPT I1 genes has been found to occur during the
perinatal period in the rat. Thus, in the late fetal liver both the
mRNA level and activity of CPT I are very low, but both rise
dramatically during the first day of extra-uterine life and remain
elevated throughout the suckling period [91-931. Whereas transcription of the CPT I gene is markedly attenuated if the pups
are weaned onto a high-carbohydrate diet, it remains robust if
the solid diet is rich in fat [92]. By contrast, the level of CPT I1
mRNA and the activity of the enzyme are already high prior to
birth and remain constant during suckling and weaning regardless of the fat content of the solid diet [92, 931. All of these
findings are consistent with earlier reports that during the fetalneonatal transition, not only is the activity of CPT I increased
but both the liver content of malonyl-CoA and sensitivity of
CPT I to malonyl-CoA inhibition are markedly decreased, allowing for efficient fatty acid oxidation and ketone body production (reviewed in [94]). Conversely, with weaning onto a high
carbohydrate diet all of these parameters ar reversed [92-941.
In the rat intestine the pattern of CPT I and CPT I1 gene expression during the perinatal period, as well as that for mitochondrial
hydroxymethylglutaryl-CoA synthase (which mirrors the CPT I
profile), are very similar to those found in liver [93], consistent
with a ketogenic role for the intestine at this time [93, 951.
The factors responsible for turning on the liver CPT I gene
at birth have recently been examined using cultured hepatocytes
from 20-day-old fetal rats [96]. The level of CPT I mRNA,
which was initially very low, rose dramatically upon exposure
of the cells to dibutyryl cAMP (Bt,-CAMP). Similar results were
obtained when LCFA were added to the culture medium,
whereas medium-chain fatty acids were ineffective. The effects
of Bt,-CAMP and LCFA were both offset by insulin. That Bt,cAMP stimulated CPT I gene transcription under conditions
where intracellular triacylglycerol hydrolysis was chemically
suppressed suggests that the cyclic nucleotide and LCFA act
through separate mechanisms. In addition, while linoleate and
Bt,-CAMP enhanced CPT I gene transcription by twofold and
fourfold, respectively, the former also increased the half-life of
the CPT I mRNA by approximately 50%. It was also noted that
2-bromopalmitate, which is readily converted into the non-metabolizable CPT I inhibitor, 2-bromopalmitoyl-CoA, was more
affective than oleate or linoleate in raising the concentration of
CPT I mRNA. Moreover, another CPT I inhibitor, tetradecylglycidate, when used alone at a concentration that did not alter
the level of the CPT I transcript, enhanced the stimulatory effect
of linoleate [96]. It might be inferred, therefore, that induction
of the CPT 1 message by LCFA does not require the oxidation
of these substrates but does require their conversion into CoA
esters.
Collectively, the studies cited above suggest that the sharp
rise in hepatic CPT I mRNA abundance that accompanies the
fetal to neonatal transition in the rat is triggered by the fall in
circulating insulin levels which then provides two key signals :
an elevated plasma LCFA concentration and an increase in the
cAMP content of the liver.
In contrast to their powerful inductive effect towards CPT I
mRNA, LCFA had no influence on the level of the CPT I1 transcript, which was already high in the late fetal hepatocyte [92,
961. However, the peroxisome proliferator, clofibrate, increased
both CPT I and CPT TI mRNA levels, and neither effect was
antagonized by insulin [96]. It thus appears that the fibrate
10
McGarry and Brown (Eul: J. Biochern. 244)
works through the peroxisome-proliferator-activated receptor
while LCFA operate via a different mechanism, possibly involving a distinct fatty acid-activated receptor [96]. Undoubtedly,
this point will be clarified when the structure of the liver CPT I
gene promoter becomes available. Also of interest will be how
the promoter region of the muscle CPT 1 gene compares with
its liver counterpart and whether this is also subject to dietary
and/or hormonal regulation.
Mutations. Recessively inherited defects have been described both at the level of CPT I and CPT 11. CPT I deficiency
appears to be very rare, or possibly to have been under-diagnosed because of its potentially lethal character. In the small
number of cases reported to date the affected enzyme is clearly
the liver isoform. This is evidenced by the fact that the defect is
easily identifiable in homogenates of fibroblasts (which we now
know to express the liver-type enzyme essentially exclusively
[48]) when appropriate assays are employed to discriminate between the activities of CPT I and CPT 11 [97]. Moreover, the
clinical symptomatology, which is usually seen in infancy, displays classical liver involvement ; the hallmark feature is hypoketotic hypoglycemia which, if not treated, can be fatal [98].
Typically, symptoms are precipitated by fasting, a situation
where euglycemia is normally maintained by accelerated hepatic
gluconeogenesis. However, because this process is dependent
upon fatty acid oxidation for maximal efficiency it becomes inefficient in the patient lacking hepatic CPT I activity, such that
the blood glucose level falls precipitously [99]. The low capacity
for hepatic fatty acid oxidation also explains the concomitant
hypoketonemia [99]. The condition is readily treatable either by
administration of medium-chain triacylglycerols, the fatty acids
of which do not require the CPT system for oxidation, or by
maintaining the affected patient on a high carbohydrate/low-fat
diet 1991.
The molecular basis of hepatic CPT I deficiency has yet to
be determined, but with the recent emergence of the relevant
cDNA structure and partial characterization of the gene [48, 521
such information will no doubt soon be forthcoming. Now that
muscle and liver CPT I are known to be different proteins encoded by separate genes (see above) the question is raised as to
whether there exists a syndrome of muscle CPT I deficiency. To
date, no such cases have been reported. If the condition does
exist, it might be expected to show a clinical phenotype similar
to that seen with severe CPT I1 deficiency (see below). However,
loss of muscle CPT I might be incompatible with life, given the
importance of the enzyme for heart function.
In contrast to the limited number of patients with documented CPT I insufficiency, derangements at the CPT I1 locus
are now being reported with ever increasing frequency. It is now
believed that inherited CPT I1 deficiency is the single most common cause of abnormal lipid metabolism in skeletal muscle.
Classically, the disease presents in young adults with recurrent
bouts of muscle pain and myoglobinuria, usually brought on by
episodes of fasting, heavy exercise or infection ; renal failure is
common. Although the enzyme defect is presumably present
body-wide (assuming there is only one CPT I1 gene), in most of
the earlier described patients only muscle symptomatology was
evident. Recently, however, there have been a number of reports
of infantile CPT I1 deficiency with more serious clinical sequelae including hypoketotic hypoglycemia, liver failure,
cardiac involvement and even sudden death [loo- 1031.
A number of different mutations in the CPT 11 gene have
now been established as disease causing. Particularly common
among European subjects with the classical adult muscular form
of the disorder is a C-T transition at nucleotide 439 which
changes amino acid 113 from serine to leucine. In 25 such pa-
tients, all unrelated, the Serll3Leu mutation was found on 28
of the 50 mutant alleles, but in none of 31 unaffected control
subjects [104]. Curiously, in all cases the defective allele also
contained two polymorphic variants, Va1368Ile and Met647Va1,
which occur in the general population with allele frequencies of
0.51 and 0.25, respectively [104]. Taroni et al. [lo41 designate
this allele ILV. Whereas the presence of the two polymorphisms
on the mutant allele appeared not to affect the low residual activity of the protein when expressed in COS cells, it exacerbated
the deleterious impact of a second disease-causing mutation
identified in an infantile case of CPT I1 deficiency as Arg631Cys
[104]; the authors refer to the latter defective allele as ICV. One
of the 25 adult patients studied in [lo41 proved to be a compound heterozygote carrying both ILV and ICV mutant alleles.
According to Verderio et al. [84], other mutations responsible
for the adult form of CPT I1 deficiency include the Pro5OHis
transition (which occurs in a Leu-Pro motif that is highly conserved among acyltransferases) and an Asp553Asn substitution.
Additional causes of neonatal CPT I1 deficiency have been
found to involve an 11-nucleotide duplication (which produces
a frameshift giving rise to a truncated protein) as well as Pro227Leu, Tyr628Ser, Glu174Lys and Phe383Tyr substitutions [84].
The first three of these proved fatal. Precisely how these various
alterations translate into loss of CPT I1 activity is not yet clear.
In most of the cases studied the mutations were associated with
low tissue levels of a protein with normal kinetic features, indicating decreased stability of the mutant enzymes. Also to be
clarified is why different mutations in the CPT I1 gene present
with such disparate clinical symptomatology. One possibility
suggested by Bonnefont et al. [102], is that the severity of symptoms is dictated by the level of residual enzyme activity. According to this view, a value of 15-25% of control is associated
only with muscle symptoms, but below this threshold abnormal
function extends to the liver and heart. Others have proposed
that CPT I1 may form a physical association with distal components of the Boxidation machinery such that mutations at different sites in the protein result in variable efficiency of the overall system [104].
Extramitochondrial CPT proteins
The preceding sections have dealt exclusively with the CPT
proteins associated with mitochondria. It has been known for
some time, however, that other subcellular organelles, in particular microsomes and peroxisomes, also contain CPT-like enzyme
activities. Although details of these two extramitochondrial CPT
systems are still scanty, the picture emerging is that both may
operate in a functional sense in a manner analogous to that present in mitochondria. Thus, rat liver microsomes have been
shown to contain a membrane-bound and soluble (luminal) form
of CPT activity [105-1071. The former displays properties remarkably similar to those of mitochondrial CPT I in that it is
inhibited reversibly by malonyl-CoA and irreversibly by etomoxir-CoA. None of these properties were shared by the luminal
enzyme [105-1071, as is true for mitochondrial CPT I1 [7]. The
microsomal CPT I-like enzyme is also completely inactivated
when the membranes are solubilized with octyl glucoside, but
apparently retains considerable activity and malonyl-CoA sensitivity when deoxycholate is used as the detergent [106].
Despite its functional similarity to mitochondrial CPT I
(88 kDa), the microsomal membrane-bound enzyme is clearly a
distinct entity, having a much smaller monomeric mass of only
about 47 kDa (as judged by migration of the [3H]etomoxirCoA-labeled protein on SDS/polyacrylamide gels [ 1051). Similarly, the microsomal soluble CPT (54 kDa) differs from mitochondrial CPT I1 (71 m a ) ; indeed, the former now appears to
McGarry and Brown ( E m J. Biockern. 244)
be identical to a stress-related protein, GRP.58, for which the
cDNA bad been previously cloned [log].
Liver peroxisomes have also been found to contain a membrane-associated, malonyl-CoA - inhibitable and detergent-labile
form of CPT [109, 1101, but how this protein relates to its counterparts in mitochondria and microsomes is not yet known. It
has, however, been established that the readily solubilized, detergent-stable and malonyl-CoA insensitive CPT activity of peroxisomes, previously referred to as carnitine octanoyltranferase
(COT) differs in its monomeric size (=63 kDa), primary structure and immunologically from the internal CPT proteins of the
other two organelles [110, 1111. It seems likely, therefore, that a
similar fatty-acid-transport mechanism operates in mitochondria,
microsomes and peroxisomes, but that the intra-organellar and
inter-organellar proteins involved are all different.
While the role of the CPT system in mitochondria is clear
(see above) its function in the other organelles has not yet been
elucidated. Although it is generally believed that isolated peroxisomes do not require carnitine for the oxidation of fatty acylCoAs [112], the possibility exists that in situ these structures are
less permeable to CoA esters and thus require a CPT shuttle for
the inward transport of the fatty acyl moiety. By the same token,
a CPT system may be needed for the export of medium-chain
acyl groups, which are major products of liver peroxisomal fatty
acid oxidation [112, 1131. With regard to the CPT enzymes in
liver microsomes, two potential roles have been suggested [106].
One is in the process of fatty acylation of certain secreted proteins, an event thought to occur at some point in the secretory
pathway. A second relates to the possibility that some portion of
VLDL triacylglycerol assembly occurs in the secretory compartment of the endoplasmic reticulum. In either case, a requirement
for fatty acyl-CoA might necessitate a CPT shuttle for transmembrane movement of fatty acyl groups from the cytosolic
compartment. Whatever the precise functions of these extramitochondrial fatty-acid-transport systems may be, the intriguing
question remains as to whether they too are subject to control
by malonyl-CoA in the intact cell.
Epi1ogue
For years after its original formulation by bright minds the
mitochondria1 CPT system remained an elegant but still largely
conceptual framework to explain how fatty acyl groups gain access to the /I-oxidation machinery of the cell. As the poet might
have said, the situation has now changed utterly. Not only has
this transport mechanism come to enjoy an ever-expanding role
in fuel homeostasis, but thanks to the powerful tools of molecular biology it has finally begun to divulge its closely guarded
secrets. Particularly exciting has been the recent emergence of
the primary structures of CPT I (liver and muscle forms) and
CPT 11, together with chromosomal mapping of their genes and
insights into some of their structural features. While these advances have put to rest a number of controversial issues, some
burning questions pose major challenges for the future. To name
just a few : Precisely how are CPT I and CPT I1 arranged relative
to each other on their respective membranes and how does each
relate to the carnitine-acylcarnitine translocase ? Just how do
malonyl-CoA and other inhibitors interact with CPT I and which
features of the liver and muscle enzymes account for their very
different kinetic characteristics and inhibitor sensitivities ? Are
there more than two isoforms of CPT I? Why, in the rat at least,
do brown and white fat cells express the muscle type of CPT I?
In addition to their known pathogenicity, might partial defects in
CPT I andlor CPT II activity underly some other disease states
characterized by abnormal lipid dynamics, such as obesitylinsulin-resistance syndromes ? Might selective CPT inhibitors have
11
therapeutic potential in conditions where fatty acid oxidation is
excessive ?
Clearly, the past decade has seen explosive growth in the
CPT arena and has generated a number of surprises. The coming
years promise to be even more rewarding.
Studies from the authors’ laboratory were supported by grants from
the National Institutes of Health (NIH; DK18573), the NIHlJuvenile Diabetes Foundation Diabetes Interdisciplinary Research Program, Sandoz
Pharmaceuticals, the Chilton Foundation and the Forrest C. Lattner
Foundation.
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