(Received for publication, December 13, 1995; and in revised form, January 18, 1996)
From the
We set out to determine if the cDNA encoding a carnitine
palmitoyltransferase (CPT)-like protein recently isolated from rat
brown adipose tissue (BAT) by Yamazaki et al. (Yamazaki, N.,
Shinohara, Y., Shima, A., and Terada, H.(1995) FEBS Lett. 363,
41-45) actually encodes the muscle isoform of mitochondrial CPT I
(M-CPT I). To this end, a cDNA essentially identical to the original
BAT clone was isolated from a rat heart library. When expressed in COS
cells, the novel cDNA and our previously described cDNA for rat liver
CPT I (L-CPT I) gave rise to products with the same kinetic
characteristics (sensitivity to malonyl-CoA and K for carnitine) as CPT I in skeletal muscle and liver
mitochondria, respectively. When labeled with
[
H]etomoxir, recombinant L-CPT I and putative
M-CPT I, although having approximately the same predicated masses (88.2
kDa), migrated differently on SDS gels, as did CPT I from liver and
muscle mitochondria. The same was true for the products of in vitro transcription and translation of the L-CPT I and putative M-CPT I
cDNAs. We conclude that the BAT cDNA does in fact encode M-CPT I.
Northern blots using L- and M-CPT I cDNA probes revealed the presence of L-CPT I mRNA in liver and heart and its absence from skeletal muscle and BAT. M-CPT I mRNA, which was absent from liver, was readily detected in skeletal muscle and was particularly strong in heart and BAT. Whereas the signal for L-CPT I was more abundant than that for M-CPT I in RNA isolated from whole epididymal fat pad, this was reversed in purified adipocytes from this source. These findings, coupled with the kinetic properties and migration profiles on SDS gels of CPT I in brown and white adipocytes, indicate that the muscle form of the enzyme is the dominant, if not exclusive, species in both cell types.
Transport of long chain fatty acyl groups into the mitochondrial
matrix to undergo -oxidation is effected by the mitochondrial
carnitine palmitoyltransferase (CPT) (
)enzyme system. CPT I,
an integral outer membrane protein, catalyzes the transfer of an acyl
group from coenzyme A to carnitine, the acylcarnitine product
traversing the inner membrane by means of a specific translocase. The
transesterification is then reversed by CPT II, associated with the
matrix face of the inner membrane. CPT I has been the focus of
particular attention due to its unique inhibition by malonyl-CoA, a
property of the enzyme that is central to the physiological regulation
of the
-oxidation pathway(1, 2) . As a
consequence, CPT I has aroused interest as a potential site for
pharmacological inhibition of fatty acid oxidation in the liver in
states where this process occurs at excessive rates (such as poorly
controlled diabetes(3, 4) ) or in the ischemic heart,
where elevated levels of acylcarnitines have been associated with
arrhythmias(5) .
Whereas CPT II appears to be the same
protein in all tissues(6) , CPT I exists as at least two
isoforms(6, 7) . ()These have been
designated L-CPT I (expressed in liver and fibroblasts (9, 10) ) and M-CPT I (expressed in skeletal
muscle(6) ). The heart expresses both forms, the muscle variant
becoming increasingly predominant during neonatal development in the
rat(7, 11, 12) . A fuller understanding of
the tissue distribution and properties of CPT I isoforms has been
impeded by the fact that although cDNAs for L-CPT I and CPT II have
been cloned from both rats (9, 13) and
humans(10, 14) , the same goal has not been achieved
with certainty in the case of M-CPT I.
Recently, a cDNA encoding a
CPT I-like protein was isolated from rat brown adipose tissue
(BAT)(15) . This was obtained by a subtractive cloning strategy
aimed at identifying proteins expressed in BAT, but not in white
adipose tissue (WAT). The derived product was predicted to be a protein
of 772 amino acids having 62.6% identity to rat L-CPT I (773 amino
acids). Northern blot analysis indicated high levels of expression in
BAT, skeletal muscle, and heart(15) . However, the study cited
left unanswered three important questions. First, does this cDNA in
fact correspond to M-CPT I or to an additional member of the growing
family of carnitine acyltransferases that have been characterized in
recent years(16, 17, 18, 19) ?
Second, if the new cDNA does encode M-CPT I with a predicted mass of
88,227 Da, which is almost identical to the value of 88,150 Da
predicted for L-CPT I(9) , why do the two proteins (when
labeled with [H]etomoxir) migrate so differently
on SDS gels(6, 7) ? Third, why should brown and white
fat express different CPT I isoforms?
The studies outlined below leave little doubt that the CPT I expressed in rat BAT is the muscle isoform of the enzyme. They also address the question of why the liver and muscle forms of CPT I behave differently on SDS gels. Finally, the new findings allow refinement of the pattern of CPT I isoform expression in brown and white fat.
pCMV6-rL-CPT I encoding rat liver CPT I is the same plasmid referred to as pCMV6-CPT I in (9) . pB-rL-CPT I was constructed by inserting into pBluescript SK(+) the coding sequence excised from pY-CPTI-MetI (24) with the restriction enzyme EcoRI.
Whole mitochondria were used
directly for assay of CPT I. To allow for variation in the extent of
malonyl-CoA-insensitive CPT II exposed during mitochondrial
preparation, the CPT I values given refer to enzyme activity inhibited
by 100 µM malonyl-CoA (see ``Results''). For
measurement of CPT II, mitochondria were made 1% (w/v) with octyl
glucoside and kept on ice for 30 min before assay, with frequent
vortexing. This procedure solubilizes CPT II in active form while
inactivating CPT I(31) . Treatment of mitochondrial fractions
with the covalent inhibitors [H]etomoxir and
DNP-etomoxir were as described(11) .
COS-M6 cells were
transfected with plasmid pCMV6-rM-CPT I or pCMV6-rL-CPT I (encoding rat
L-CPT I). Mock transfections contained no plasmid. Table 1shows
the CPT activity measured in crude homogenates of the cells 48 h after
transfection in two independent experiments. pCMV6-rL-CPT I caused a
6-7-fold induction of CPT I activity (i.e. activity
measured in the absence of octyl glucoside), consistent with previous
results(9) . A 5-fold induction was observed with pCMV6-rM-CPT
I. Both activities were substantially inhibited in the presence of 100
µM malonyl-CoA. Putative M-CPT I was the more sensitive,
residual activity being similar to that in untransfected cells. For
assay of CPT II, the membranes were solubilized in 1% octyl glucoside.
Under these conditions, no change was observed in pCMV6-rM-CPT
I-transfected relative to untransfected cells, but after transfection
with pCMV6-rL-CPT I, enzyme activity in the presence of octyl glucoside
did rise 1.5-fold. This may represent incomplete inactivation of
the induced L-CPT I by the detergent or up-regulation of endogenous CPT
II.
A more detailed analysis of the kinetic properties of the two
expressed CPTs is presented in Fig. 1. A gross difference in
sensitivity to malonyl-CoA is apparent, with I values
(concentration needed for 50% inhibition) of
8 and 0.15
µM, respectively, for the recombinant L-CPT I and putative
M-CPT I variants (Fig. 1A). The response of each enzyme
to increasing concentrations of carnitine is shown in Fig. 1B. Expressed L-CPT I saturated rapidly (K
25 µM), whereas the other
enzyme displayed a much more gradual response (higher K
). In the case of expressed M-CPT I, the presence
of endogenous COS cell CPT I (probably the liver isoform) rendered the
corresponding Eadie-Hofstee plot markedly nonlinear (more than one
component), preventing simple calculation of an accurate K
. It is clear, however, that the exogenous CPT in
this case contributed a high K
M-CPT I-like
component.
Figure 1: Kinetics of rat CPT I isoform expressed in COS cells. A, effect of malonyl-CoA. Results are expressed relative to values in the absence of malonyl-CoA. B, response to carnitine. Data have been normalized to unity at 500 µM carnitine. Results are from two independent and closely agreeing experiments.
Transfected and untransfected COS cells were incubated
for 6 h in medium containing 3 µM [H]etomoxir to covalently label the
expressed CPT I isoforms. Total cell membrane extracts were then
analyzed by SDS-PAGE and subsequent fluorography (Fig. 2A). Both types of transfected cells contained
highly induced labeled bands. Labeled L-CPT I migrated more slowly than
its presumed muscle-type counterpart (
88 and
82 kDa,
respectively), mirroring the behavior of
[
H]etomoxir-labeled CPT I isoforms in
mitochondria from rat liver and muscle (see below). Only a faint
labeled band, of approximately the size of the L-CPT I expression
product, was visible in untransfected cells.
Figure 2:
SDS-PAGE analysis of CPT I isoforms
expressed in vitro. A,
[H]etomoxir labeling of rat CPT I isoforms
expressed in COS cells. Cells were transfected with the indicated
plasmid and subsequently exposed to [
H]etomoxir.
Membranes were then subjected to SDS-PAGE followed by fluorography. B, mobilities of mitochondrial and in vitro synthesized CPT I isoforms.
S,
S-labeled product of in vitro transcription and
translation;
H,
[
H]etomoxir-labeled liver or muscle mitochondrial
membranes; MIX, combined
S- and
H-labeled samples.
To address the question
of the differential mobility of the two enzymes during SDS-PAGE, we
used an in vitro transcription and translation system to
synthesize S-labeled protein products from the two CPT I
clones. These would not be subject to post-translational modification,
as might occur in the whole cell (e.g. upon mitochondrial
import). In this system also, the radioactive proteins migrated
differently (Fig. 2B). Furthermore, the apparent sizes
of the in vitro synthesized
S-labeled proteins
were indistinguishable from those of the corresponding
[
H]etomoxir-labeled enzymes from rat liver and
muscle mitochondria.
Figure 3:
[H]etomoxir labeling
of mitochondria from rat tissues. A, whole rat tissues; B, purified white adipocytes; C, effect of
DNP-etomoxir preincubation on [
H]etomoxir
labeling of white adipocyte mitochondria. Adipocyte mitochondria were
incubated in the absence (lane 1) or presence (lane
2) of 10 µM DNP-etomoxir before exposure to 3
µM [
H]etomoxir (see
``Experimental Procedures''). L and M indicate migration positions of L-CPT I and M-CPT I,
respectively.
The different
CPT I profiles indicated by [H]etomoxir labeling
of mitochondria prepared from BAT, whole epididymal WAT, and purified
white adipocytes were paralleled by differences in the kinetic
properties of the enzyme from those sources. When expressed relative to
mitochondrial protein (Table 2), the activity of CPT I measured
in liver, heart, and skeletal muscle varied over only a 2-fold range,
with a proportional change in the level of CPT II, so that the CPT
II/CPT I ratio remained close to unity. (It is important to note that
since the K
values for the substrates of L-CPT I,
M-CPT I, and CPT II are different, the relative activities measured for
the different isoenzymes will depend upon experimental conditions. The
present data are intended to show that wide variation exists between
tissues; however, they do not necessarily reflect the molar ratios in vivo.) In BAT mitochondria, the ratio was doubled, and in
WAT and purified adipocytes, it rose to 9 and 14, respectively. This
observation explains the apparent resistance to complete inhibition of
CPT I activity in mitochondria from whole epididymal fat pads and
adipocytes isolated from them (Table 2, sixth column). Whereas
inhibition of
90% is routine with 100 µM malonyl-CoA
in ``intact'' mitochondria from liver, heart, and skeletal
muscle, the value dropped to
83% in BAT and to only 40-50%
in white adipose-derived preparations. It is likely that in the case of
each tissue or cell type, a similar small fraction of the mitochondria
becomes damaged, but that the amount of malonyl-CoA-insensitive CPT II
rendered overt is exaggerated in those tissues where the CPT II/CPT I
ratio is highest. Accordingly, values for CPT I shown here have been
assessed as the malonyl-CoA-sensitive component of overt CPT activity.
The potency of malonyl-CoA as an inhibitor of CPT I in each
mitochondrial type is shown in Fig. 4. Skeletal muscle was the
most sensitive (I = 0.04 µM) and liver
the least, with an I
100-fold greater (Table 2,
fourth column). Heart and BAT mitochondria exhibited a sensitivity
close to that of skeletal muscle. CPT I from whole WAT behaved more
like the liver enzyme, displaying an I
of 1.5
µM. However, the malonyl-CoA response curve of CPT I in
mitochondria from purified adipocytes was clearly shifted to the left,
the I
of 0.23 µM being consistent with a
predominance of M-CPT I.
Figure 4: Effect of malonyl-CoA on CPT I in rat tissue mitochondria. Results are expressed relative to values in the absence of malonyl-CoA (mean of three independent determinations; error bars omitted for clarity). L, liver; Ad, purified adipocytes; H, heart; SM, skeletal muscle.
The K values for
carnitine of L-CPT I and M-CPT I are
30 and 500 µM,
respectively(29) . In BAT mitochondria, the K
was found to be >400 µM, close to that of the
muscle isoform of CPT I (Table 2, fifth column). Unfortunately,
the substantial contamination of CPT I by exposed CPT II in
mitochondria from whole WAT and white adipocytes precluded accurate
determination of a K
for carnitine in either case.
CPT I isoform expression was also studied at the level of mRNA for
each tissue or cell type (Fig. 5). Fig. 5(A and B) shows a Northern blot analysis of poly(A)
RNA isolated from liver, heart, skeletal muscle, and BAT. The L-CPT I
probe generated strong signals in liver and heart, as expected, and a
weak signal in BAT, while no band was detected in skeletal muscle (Fig. 5A). The M-CPT I probe revealed expression of
this isoform in heart, skeletal muscle, and BAT, but not in liver (Fig. 5B). Due to the extremely low RNA yields from
purified adipocytes, total RNA was used to perform Northern analysis on
both adipocytes and whole WAT to allow for a direct comparison. The
band representing the liver form was stronger in WAT than in adipocytes (Fig. 5C), whereas the mRNA for the muscle isoform was
found to be the more abundant species in the purified cells (Fig. 5D).
Figure 5:
Northern blot analysis of RNA from rat
tissues. A, 5 µg of poly(A) RNA from the
indicated rat tissues were analyzed as described under
``Experimental Procedures'' using a single-stranded liver
cDNA probe. B, 8 µg of poly(A
) RNA were
analyzed using a single-stranded muscle cDNA probe. Twenty micrograms
of total RNA from whole WAT and purified adipocytes were analyzed using
a single-stranded liver (C) or muscle (D) cDNA probe. kb, kilobases.
The recent cloning and expression of cDNAs encoding rat and human L-CPT I and CPT II have provided considerable insight into the structure/function relationships between the CPT isoenzymes(9, 13, 24, 28, 33, 34) . There are, however, a number of important but unresolved issues. Particularly intriguing is the question of why CPT I should exist in at least two isoforms with distinct kinetic properties and tissue distribution, and how these relate to whole body fuel homeostasis. Progress on this front has been hampered by the unavailability of a cDNA corresponding to the muscle enzyme. Our initial goal here, therefore, was to determine whether a candidate cDNA isolated from rat BAT (15) did in fact represent muscle CPT I. To this end, we used sequence information from the BAT cDNA to screen a rat heart cDNA library, and the CPT I-like sequence was confirmed in that tissue.
As with L-CPT I(9) , COS cells transfected with the putative
M-CPT I cDNA generated a CPT activity with characteristics typical of
mitochondrial CPT I, i.e. it was membrane-bound and
malonyl-CoA-sensitive and lost activity upon solubilization of the
membranes with the detergent octyl glucoside. Malonyl-CoA response
curves established that expressed putative M-CPT I was far more
sensitive to the inhibitor than was the liver enzyme. Although the
I value observed (0.15 µM) was somewhat
higher than that for CPT I from skeletal muscle mitochondria (
0.04
µM), the difference was likely due to the presence of
background endogenous CPT I activity in the COS cells, which is of the
less sensitive liver type. The I
for the expressed L-CPT I
was
8 µM, consistent with the values of 2-10
µM reported for CPT I from liver mitochondria in different
physiological states(29, 35, 36) .
Furthermore, [
H]etomoxir labeling experiments
established that the recombinant L-CPT I and putative M-CPT I enzymes
exhibited the same differential migration on SDS-PAGE as is seen with
the native proteins. Thus, when expressed in COS cells, the putative
M-CPT I clone matched in every respect the characteristics of the
native muscle enzyme. These findings, coupled with the tissue
expression data presented by Yamazaki et al.(15) and
below, provide compelling evidence that the enzyme encoded by the clone
originally isolated from BAT is identical to M-CPT I.
That being so,
the question arises as to why two highly homologous proteins with
almost identical predicted molecular masses should display such
distinct electrophoretic mobilities. To investigate this question, we
generated S-labeled protein from the L-CPT I and M-CPT I
clones using in vitro transcription and translation. The
products were found not only to run differently, but to migrate exactly
with their mitochondrial equivalents. This suggests that the phenomenon
is not the result of post-translational modification, but stems from an
intrinsic difference in the primary sequence of the two proteins. A
comparison of the cDNA-derived polypeptide molecular masses (both
88 kDa) with those estimated from SDS-PAGE analysis (
88 and
82 kDa for L- and M-CPT I, respectively) suggests that the muscle
isoform behaves anomalously.
Our final aim was to investigate the
implication of the work of Yamazaki et al.(15) , that
whereas M-CPT I is expressed in BAT, L-CPT I is the primary isoform in
WAT. An additional consideration here was the earlier work by Saggerson
and Carpenter showing that CPT I in BAT mitochondria is highly
malonyl-CoA-sensitive, as is CPT I in muscle(37) , but that in
mitochondria from purified adipocytes, CPT I exhibits a sensitivity
intermediate between that of the muscle and liver enzymes(38) .
A more detailed interpretation of these observations is now possible.
The [H]etomoxir labeling patterns of CPT I
obtained using mitochondria prepared from whole rat epididymal fat pad
and BAT appeared to conform to a model in which L- and M-CPT I are the
primary isoforms in those tissues, respectively, and Northern blots
appeared to support this notion. (
)However, when the white
adipocytes were separated from the bulk of the stromal and other cell
types, the M-CPT I band labeled with [
H]etomoxir
was seen to dominate. Moreover, kinetic analysis of CPT I in
mitochondria isolated from whole fat pad and adipocytes corroborated
the predominance of M-CPT I-like activity in the purified cells, and
this was also reflected at the level of the mRNAs for L- and M-CPT I.
Therefore, all of the above evidence pointed to a scenario in which
the muscle-type enzyme not only represents the sole species of CPT I in
BAT, but is also the dominant isoform in isolated white adipocytes from
the epididymal fat pat. Furthermore, when expressed relative to
mitochondrial protein, the CPT I activity of white adipocyte
mitochondria is seen to be 20-40-fold lower than that of
preparations from any other tissue examined (liver, heart, skeletal
muscle, or BAT). This suggests that a small contamination of the
adipocytes with any other cell type containing CPT I activity at a
similar level to those listed and that happens to express primarily
L-CPT I would result in a disproportionate effect on the overall
isoform profile. Hence, the minor quantity of L-CPT I found in the
white adipocyte mitochondrial preparations could well have resulted
from a slight impurity and might not have been of adipocyte origin. For
this reason, we cannot exclude the possibility that M-CPT I is the sole
CPT I isoform expressed both in brown and white fat cells.
Therefore, at this stage, the differential cloning exercise that led to
the original isolation of the M-CPT I cDNA (15) should not be
taken to imply that white and brown adipocytes differ fundamentally in
terms of their CPT I isoform expression. ()
A picture of tissue CPT I distribution is now emerging in which both isoenzymes are to be found in a variety of tissues (L-CPT I in liver, heart, and fibroblasts; M-CPT I in skeletal muscle, heart, and white and brown adipocytes). This will be a key consideration in the design of CPT I inhibitors as potential pharmaceutical agents. The diverse tissue expression of each isoform may also help to explain the multitissue symptoms exhibited by L-CPT I-deficient patients(39, 40, 41) . No M-CPT I deficiencies have yet been described. Obviously, further studies will be needed to establish a teleological basis for the presence of M-CPT I in adipocytes, indeed, to explain the tissue-specific expression of L- and M-CPT I generally. Identification of the rat M-CPT I cDNA represents an important step in this direction.