Báo cáo khóa học: Membrane transport of fatty acylcarnitine and free L-carnitine by rat liver microsomes pot

Membrane transport of fatty acylcarnitine and free
L
-carnitine
by rat liver microsomes
Jason M. Gooding, Majid Shayeghi and E. David Saggerson
Department of Biochemistry and Molecular Biology, University College London, UK
Recent studies have suggested that parts of the hepatic
activities of diacylglycerol acyltransferase and acyl choles-
terol acyltransferase are expressed in the lumen of the
endoplasmic reticulum (ER). However the ER membrane is
impermeable to the long-chain fatty acyl-CoA substrates of
these enzymes. Liver microsomal vesicles that were shown to
be at least 95% impermeable to palmitoyl-CoA were used to
demonstrate the membrane transport of palmitoylcarnitine
and free
L
-carnitine – processes that are necessary for an
indirect route of provision of ER luminal fatty acyl-CoA
through a luminal carnitine acyltransferase (CAT). Experi-
mental conditions and precautions were established to per-
mit measurement of the transport of [
14
C]palmitoylcarnitine
into microsomes through the use of the luminal CAT and
acyl-CoA:ethanol acyltransferase as a reporter system to
detect formation of luminal [
14
C]palmitoyl-CoA. Rapid,
unidirectional transport of free L-[
3
H]carnitine by micro-

somes was measured directly. This process, mediated either
by a channel or a carrier, was inhibited by mersalyl but
not by N-ethylmaleimide or sulfobetaine – properties that
differentiate it from the mitochondrial inner membrane
carnitine/acylcarnitine exchange carrier. These findings are
relevant to the understanding of processes for the reassembly
of triacylglycerols that lipidate very low density lipoprotein
particles as part of a hepatic triacylglycerol lipolysis/
re-esterification cycle.
Keywords: acylcarnitine; carnitine; microsomes; liver; trans-
port.
Although mammalian intracellular membranes are imper-
meable to the Coenzyme A thioesters of long-chain fatty
acids, these activated derivatives, which are synthesized
from nonesterified fatty acids by fatty acyl-CoA synthetase
on the cytosolic aspect of organelle membranes, are the
substrates for metabolic processes within at least three
cellular organelles. In mitochondria, it is well established
that CPT
1
, a carnitine acyltransferase associated with the
outer membrane, generates fatty acylcarnitine derivatives
which can then traverse the inner membrane via a
carnitine/acylcarnitine exchange carrier (CAC). Within
the mitochondrial interior, the latent carnitine acyltrans-
ferase CPT
2
then facilitates the re-formation of fatty acyl-
CoAs which are then substrates for b-oxidation within the
mitochondrial matrix [1]. Fatty acyl-CoAs are also sub-

strates for chain-shortening by b-oxidation within the
matrix of peroxisomes. As peroxisomes also contain overt
and latent carnitine acyltransferase activities [2,3] and
express the CAC protein [4], it has been concluded that
activated fatty acids access the peroxisomal matrix through
a system that is closely analogous to the mitochondrial one.
Enzymes within the lumen of the endoplasmic reticulum
(ER) also require fatty acyl-CoA thioesters as substrate.
A latent form of diacylglycerol acyltransferase (DGAT),
assigned to the luminal surface of the ER membrane and
which can be differentiated from a cytosolically oriented
DGAT, has been described [5–7]. This latent DGAT may
be involved in the reassembly of triacylglycerols which
lipidate very low density lipoprotein (VLDL) particles as
part of a hepatic triacylglycerol lipolysis/re-esterification
cycle [2,3,8–12]. Two forms of acyl cholesterol acyltrans-
ferase (ACAT) are also known and one of these is suggested
to be oriented from the ER membrane towards the lumen
and to contribute to provision of cholesteryl esters for
lipidation of VLDL particles [13–16]. Finally, acyl-CoA:eth-
anol acyltransferase (AEAT) is an enzyme activity
1
that
appears to be exclusively localized to the ER lumen [17].
As they do not readily penetrate the ER membrane
[17], it has been proposed that the fatty acyl-CoA
substrates for luminal enzymes such as DGAT, ACAT
and AEAT are generated by a malonyl-CoA-insensitive
carnitine acyltransferase (CAT) that is localized in the ER
lumen [18,19]. It has been envisaged that the substrate for

this luminal CAT is fatty acylcarnitine, which is trans-
ported from the cytosol to the ER lumen [2,3,12] (Fig. 1)
and which is generated by CPT
1
located at sites distinct
from the ER [20] or also by an ER-targeted form of
CPT
1
[12,21–23]. For the luminal CAT to function in the
Correspondence to E. D. Saggerson, Department of Biochemistry
and Molecular Biology, University College London,
Gower Street, London, WC1E 6BT, UK.
Fax: + 44 20 76797193, Tel.: + 44 20 76797320,
E-mail:
Abbreviations: ACAT, acyl cholesterol acyltransferase; AEAT, acyl-
CoA:ethanol acyltransferase; CAC, carnitine/acylcarnitine exchange
carrier; CAT, carnitine acyltransferase; CPT
1
, the overt carnitine
palmitoyltransferase of mitochondria; DGAT, diacylglycerol
acyltransferase; ER, endoplasmic reticulum; etomoxir,
2-[6-(4-chlorophenoxy)hexyl]oxirane carboxylic acid; [(Np-O)
2
P
i
],
bis-(4-nitrophenyl)phosphate; VLDL, very low density lipoprotein.
(Received 20 October 2003, revised 8 January 2004,
accepted 16 January 2004)
Eur. J. Biochem. 271, 954–961 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03997.x

way envisaged in Fig. 1 there must additionally be a
means whereby the
L
-carnitine product can escape from
the ER and there must be a supply of ER luminal free
CoASH. The CAC protein is not expressed at detectable
levels in liver microsomes [4], so at present it is not clear
how inward and outward transport of fatty acylcarnitine
and
L
-carnitine, respectively, across the ER membrane are
facilitated. In the present study, we report direct evidence
for the passage of radiolabelled
L
-carnitine across micro-
somal membranes. We also report on studies of the
uptake of [
14
C]palmitoylcarnitine into the lumen of
ÔsealedÕ microsomal vesicles. Direct measurement of this
is technically very difficult or impossible as these lipid
substrates bind nonspecifically to membrane proteins and
lipids. Therefore indirect approaches using Ôreporter
systemsÕ must be used. Broadway et al. [12] used the
ER luminal coupled system of CAT and AEAT as a
ÔreporterÕ in preliminary studies to show that ÔsealedÕ liver
microsomes could generate ethyl palmitate from palmi-
toylcarnitine provided exogenous free CoASH was pre-
sent, and concluded that these findings were consistent
with a trans-ER membrane transport of palmitoylcarni-

tine together with an ER transport process for CoASH,
the cosubstrate for microsomal CAT [12]. In support of
the notion of fatty acylcarnitine transport in the ER,
Abo-Hashema et al. [24] used the latent DGAT activity as
a Ôreporter systemÕ to demonstrate a carnitine-dependent
conversion of oleoyl-CoA into luminal triacylglycerol.
In those experiments, microsomes were fused with
liposomes encapsulating a supply of CoASH for the
luminal CAT. In the present study, we have re-evaluated
and further developed the experimental approach of
Broadway et al. [12] through the use of bis-(4-nitrophe-
nyl)phosphate [(Np-O)
2
P
i
] which is used to decrease
interference from carboxyesterase and thioesterase activ-
ities in microsomes [25]. From these studies, we confirm
the presence of a system that facilitates the entry of
palmitoylcarnitine into ÔsealedÕ microsomes but discount
the previous notion of a concomitant transport system for
CoASH [12].
Materials and methods
Chemicals
Routinely used chemicals were from BDH Ltd. (Poole,
Dorset, UK) or from Sigma Chemical Co. Ltd. (Poole,
Dorset, UK). The sodium salt of etomoxir{2-[6-(4-chloro-
phenoxy)hexyl]oxirane carboxylic acid} was from
H. P. O. Wolf, (Projekt-Entwicklung GmbH, Allensbach,
Germany). [

3
H]Carnitine, [
14
C]inulin, [
14
C]palmitoylcarni-
tine and [
14
C]palmitoyl-CoA were from Amersham Inter-
national (Little Chalfont, Bucks, UK). The radiolabelled
palmitoylcarnitine was supplied in a 50 : 50 (v/v) water/
ethanol solution which was removed by evaporation in
a stream of nitrogen before use. This ensured that no
ethanol was present in ethanol-independent acylation
assays. Ethyl[
14
C]palmitate was synthesized as described
by Diczfalusy et al. [25].
Isolation of microsomes from rat liver
2,3
Fed male Sprague–Dawley rats (180–220 g) were killed
2,3
by
an approved schedule/method [schedule/method approved
by the UK Home Office under the Act of Parliament ÔAnimals
(Scientific Procedures) Act 1986Õ and the local UCL Animal
Experimentation Ethics Committee]. Livers were immedi-
ately removed and washed in ice-cold isolation medium
(5 m
M

Tris/HCl buffer, pH 7.4, containing 220 m
M
mann-
itol, 80 m
M
sucrose, 1 m
M
EDTA, 5 lgÆmL
)1
bestatin,
5 lgÆmL
)1
leupeptin and 1 m
M
phenylmethanesulfonyl
fluoride) and then homogenized in 4 vols of the same
buffer with four strokes of a motor-driven Potter-type
homogeniser. The homogenate was centrifuged at 500 g for
10 min; the supernatant was then centrifuged at 10 000 g
for 20 min followed by centrifugation of the 10 000 g super-
natant at 20 000 g for 20 min. The resulting 20 000 g
supernatant was then ultra-centrifuged at 100 000 g for
60 min. The pellet was resuspended in fresh isolation
medium and recentrifuged at 100 000 g for 60 min. The
microsomal pellet was then resuspended (80–100 mgÆmL
)1
)
and stored at )70 °Cas100 lL aliquots in HS buffer (5 m
M
Hepes buffer, pH 7.4 containing 250 m

M
sucrose). The
specific activity of cytocrome c oxidase (a mitochondrial
inner membrane marker) was less than 1% of that found in
mitochondrial fractions (results not shown). Similar micro-
somal fractions have previously been shown to contain very
little activity of monoamine oxidase (a mitochondrial outer
membrane marker) [19].
In some instances 4 m
M
CoASH with 4 m
M
dithiothre-
itol were included in isolation medium and in HS buffer.
Microsomal vesicles described as ÔsealedÕ were obtained by
thawing stored aliquots and suspending at the required
concentrationinHSbuffer.
The intactness of microsomal preparations was routinely
assessed by the latency of mannose 6-phosphatase [26] which
exceeded 90%. Further evidence of the intactness of micro-
somal vesicles was obtained from measurements of AEAT
activity (see below). Microsomal vesicles described as
ÔpermeabilisedÕ were similarly suspended in HS buffer to
which the pore-forming antibiotic alamethicin [27–29] had
been added from a stock solution of 20 mgÆmL
)1
dissolved in
dimethyl sulfoxide. At the concentration used (15 lgÆmL
)1
)

Fig. 1. A scheme summarizing processes to provide the fatty acyl-CoA
substrate for AEAT, DGAT and ACAT in the ER lumen.
Ó FEBS 2004 Transport of carnitine and acylcarnitine (Eur. J. Biochem. 271) 955
we found that alamethicin abolished latency of mannose
6-phosphatase essentially immediately (results not shown).
Treatment of microsomes with etomoxir
Microsomes were diluted to approximately 10 mgÆmL
)1
in
HS buffer containing 5 m
M
ATP, 10 m
M
MgCl
2
,2m
M
CoASH, 50 l
M
sodium etomoxir and BSA (1 mgÆmL
)1
)
and incubated at 25 °C for 30 min followed by addition of
4 vols of ice-cold HS buffer. The diluted microsomes were
then centrifuged at 200 000 g for 30 min, resuspended in HS
buffer (approximately 50 mgÆmL
)1
) and stored at )70 °C.
Assay of ethanol-dependent and -independent
acyltransferase activities

Assays were performed either with sealed microsomes or in
the permeabilized state (with alamethicin [15 lgÆmL
)1
]) at
30 °C in a final volume of 1.4 mL in 10 m
M
Tris/HCl buffer
(pH 7.4) containing 300 m
M
sucrose, 10 m
M
MgCl
2
,
0.8 m
M
EDTA, bovine albumin (1 mgÆmL
)1
)and40l
M
[
14
C]palmitoyl-CoA (1.8 lCiÆlmol
)1
) with or without 10 lL
of ethanol (123 m
M
final concentration). Where indicated,
250 l
M

(Np-O)
2
P
i
[25] was included in assays. Assays were
initiated by the addition of 700 lg of microsomal protein.
At zero, 1, 2, 5, 10, 15 and 20 min 200 lL samples of
the assay mixture were removed and mixed with 1.5 mL
propan-2-ol/heptane/water (80 : 20 : 2; v/v/v). After mixing
with a further 1 mL of heptane and 0.5 mL of water the
upper (heptane) layer was removed and washed with 2 mL
of 50 m
M
NaOH dissolved in 50% (v/v) ethanol. Washed
heptane layer (650 lL) was taken for liquid scintillation
counting. AEAT (ethanol-dependent) activity was calcula-
ted by subtracting
14
C-labelled product formation in the
absence of ethanol from that in its presence. Measurement
of AEAT activity in this way was found in preliminary
experiments to agree with the formation of ethyl[
14
C]pal-
mitate as detected by TLC [25]. In experiments in which
AEAT was used as a reporter enzyme to detect entry of
palmitoylcarnitine into sealed microsomal vesicles,
[
14
C]palmitoyl-CoA was replaced by 40 l

M
[
14
C]palmitoyl-
carnitine (1.8 lCiÆlmol
)1
) with or without 0.5 m
M
CoASH
and 1 m
M
dithiothreitol.
Measurement of uptake of
L
-[
3
H]carnitine into
microsomal vesicles
Assays were performed at 10 °C and were commenced by
the addition of 500 lL of microsomes in HS buffer
(60–80 mgÆmL
)1
protein, pre-equilibrated at 10 °C) to
500 lL of pre-equilibrated HS buffer containing various
concentrations of unlabelled carnitine, 5 lCi [
3
H]carnitine
and 2 lCi [
14
C]inulin. At each time-point, two 50 lL

samples were removed quickly and transferred to tubes
containing 2 mL of ice-cold HS buffer containing polyethy-
lene glycol 8000 (5%, w/v). One of each pair of tubes
additionally contained 200 lg of alamethicin to permeablise
the microsomal vesicles. Both tubes were immediately
centrifuged for 60 s at 6000 g to sediment the microsomal
material. The supernatants were removed by aspiration and
the walls of the tubes wiped with tissue to remove adhering
fluid. The pellets were dissolved in 500 lL of Triton X-100
(10%, v/v), transferred to scintillation fluid and
3
Hand
14
C
measured by liquid scintillation counting. The amount of
[
3
H]carnitine within microsomal vesicles at each time-point
was calculated by subtracting the
3
H-associated with the
alamethicin-treated pellet from the
3
H-associated with the
untreated pellet after correcting for adhering medium, which
was determined from the amount of associated [
14
C]inulin.
Measurement of protein content
This was by a bicinchoninic acid assay kit (Sigma).

Statistical methods
Values are shown in figures as means of the number of
separate measurements (n) ± SD. Where SD bars are not
shown in figures, these are within the symbol.
Results
Transport of palmitoylcarnitine into sealed microsomal
vesicles
TheexperimentsshowninFig.2wereperformedinthe
absence of (Np-O)
2
P
i
. Figure 2A confirms the previously
reported high degree of latency of AEAT [5,17] in that
minimal formation of ethanol-dependent product (ethyl
palmitate) from [
14
C]palmitoyl-CoA was seen with sealed
microsomes whereas permeabilization by alamethicin
allowed ethyl palmitate formation at an initial rate of
1020 ± 30 pmolÆmin
)1
Æmg
)1
.When[
14
C]palmitoylcarnitine
was provided alone, there was no appreciable formation of
ethyl palmitate by sealed microsomes (Fig. 2B). A similar
lack of ethyl palmitate formation from palmitoylcarnitine

was seen with alamethicin-permeabilized microsomes, con-
firming that palmitoylcarnitine is not a substrate for AEAT
(data not shown). However, when CoASH was also present
(together with dithiothreitol to keep coenzyme A in the
reduced form) ethyl palmitate formation from [
14
C]palmi-
toylcarnitine by sealed microsomes was observed at a steady
Fig. 2. Radiolabelled product formation by microsomes in the absence of
(Np-O)
2
P
i
. All values are means ± SD of four independent meas-
urements. (A) Ethanol-dependent product formation from 40 l
M
[
14
C]palmitoyl-CoA. h, sealed microsomes; j, microsomes permea-
bilized by alamethicin. (B) Product formation from 40 l
M
[
14
C]palmitoylcarnitine by sealed microsomes. s, ethanol-dependent
without CoASH and dithiothreitol; j, ethanol-dependent with
0.5 m
M
CoASH + 1 m
M
dithiothreitol.

956 J. M. Gooding et al. (Eur. J. Biochem. 271) Ó FEBS 2004
rate of 180 ± 20 pmolÆmin
)1
Æmg
)1
over 10 min (Fig. 2B).
From experiments such as these, Broadway et al. [12]
arrived at the conclusion that although palmitoyl-CoA
could not gain access to the interior of sealed microsomes,
both palmitoylcarnitine and CoASH could do so, thereby
becoming substrates for the coupled microsomal CAT/
AEAT system – it is reasonable to expect that diffusion
across the microsomal membrane of ethanol, the cosub-
strate for AEAT, is not rate-limiting [30].
Figure 2A also shows that ethyl palmitate formation
from [
14
C]palmitoyl-CoA by alamethicin-permeabilized
microsomes plateaued and then declined after 10 min. As
only  10% of the available
14
C was in ethyl palmitate or in
ethanol-independent products (not shown) at this 10 min
time interval, extensive destruction of the product(s) and/or
of the substrate must have occurred. To a lesser extent, the
same is suggested by Fig. 2B where the timecourse of
product formation from palmitoylcarnitine became nonlin-
ear after only  5% of the available
14
C could be detected

in ethanol-dependent or -independent products. Diczfalusy
et al. [25] have shown that the serine esterase inhibitor
(Np-O)
2
P
i
at 250 l
M
enhanced AEAT activity in rat liver
microsomes by approximately fivefold. This effect was
primarily explained through inhibition of carboxyesterase
ES-4, which has both ethyl esterase and thioesterase
activities. We therefore reinvestigated the formation of
ethyl palmitate from [
14
C]palmitoyl-CoA and [
14
C]palmi-
toylcarnitine in the presence of 250 l
M
(Np-O)
2
P
i
(Figs 3
and 4). Ethyl palmitate formation from [
14
C]palmitoyl-CoA
was considerably enhanced by (Np-O)
2

P
i
in alamethicin-
permeabilized microsomes (compare Fig. 2A and 3A) and
also facilitated the detection of a small but significant
amount of this conversion by sealed microsomes (Fig. 3A).
However, even though  40% of the available [
14
C]palmi-
toyl-group now appeared in ethyl palmitate after 5 min with
permeabilized microsomes, (Np-O)
2
P
i
did not totally abol-
ish the destruction of this product (Fig. 3A). Reasonable
estimates of initial rates of ethyl palmitate formation
(AEAT activity) can be made from Fig. 3A. These were
480 ± 52 pmolÆmin
)1
Æmg
)1
for sealed microsomes and
18.4 ± 1.2 nmolÆmin
)1
Æmg
)1
for those permeabilized by
alamethicin, i.e. 97.5% of AEAT activity was latent,
indicating that these experiments were conducted with

vesicles that had a high degree of ÔintactnessÕ.Itwas
important to establish this because palmitoyl-CoA, albeit at
higher concentrations in the absence of BSA, permeabilizes
rat liver microsomes [31]. AEAT is inhibited by high
concentrations of its fatty acyl-CoA substrate [25].
Figure 3B shows that our experimental conditions
employed a palmitoyl-CoA concentration (40 l
M
)that
was not inhibitory. Also in general (not shown), we
observed that ethanol-independent product formation from
[
14
C]palmitoyl-CoA or [
14
C]palmitoylcarnitine was hardly
increased by permeabilization of the microsomes, suggesting
that these unspecified products are largely made by enzymes
oriented towards the cytosolic face of the ER.
In the presence of 250 l
M
(Np-O)
2
P
i
,ratesofformation
of ethanol-dependent and -independent products from
40 l
M
[

14
C]palmitoyl-CoA or [
14
C]palmitoylcarnitine by
sealed microsomes were consistently linear from 1 to 5 min
and usually were linear from 1 to 10 min. Figure 4 shows
values obtained within this linear range. Under these
experimental conditions, rates of ethanol-dependent and
-independent product formation of 548 ± 34 and
148 ± 25 pmolÆmin
)1
Æmg
)1
, respectively, were seen when
thesubstratewas40l
M
[
14
C]palmitoylcarnitine with
CoASH/dithiothreitol (essentially zero product formation
was seen in the absence of CoASH/dithiothreitol – results
not shown). CoASH/dithiothreitol had minimal effect on
ethanol-dependent product formation from [
14
C]palmitoyl-
CoA by sealed (Fig. 4) or permeabilized (not shown)
microsomes. When microsomes were pretreated with etom-
oxir, ATP and CoASH in order to generate etomoxiryl-
CoA (an irreversible inhibitor of CPT
1

in microsomal
fractions [19]) AEAT activity measured in alamethicin-
permeabilized microsomes was not inactivated (results not
Fig. 3. Radiolabelled product formation from palmitoyl-CoA by
microsomes in the presence of 250 l
M
(Np-O)
2
P
i
. All values are
means ± SD of four independent measurements. (A) shows ethanol-
dependent product formation from 40 l
M
[
14
C]palmitoyl-CoA.
h, Sealed microsomes; j, microsomes permeabilized by alamethicin.
(B) Dependence of product formation by alamethicin-permeabilized
microsomes on the concentration of [
14
C]palmitoyl-CoA. Values are
calculated from the difference between zero and 2 min time-points. j,
Ethanol-dependent products; h, ethanol-independent products.
Fig. 4. Radiolabelled product formation by sealed microsomes in the
presence of 250 l
M
(Np-O)
2
P

i
. All values are means ± SD of four
independent measurements calculated from the difference between 1
and 5 min time-points. Product formation was measured from 40 l
M
[
14
C]palmitoyl-CoA or [
14
C]palmitoylcarnitine with additions of
0.5 m
M
CoASH + 1 m
M
dithiothreitol (DTT) as indicated. In some
instances, microsomes were pretreated with etomoxir to inactivate
CPT
1
(Materials and methods). Open bars, ethanol-independent
product formation; solid bars, ethanol-dependent product formation;
cross-hatched bar, ethanol-dependent product formation by CoASH-
loaded microsomes (Materials and methods).
Ó FEBS 2004 Transport of carnitine and acylcarnitine (Eur. J. Biochem. 271) 957
shown) and there was only a small increase in overt AEAT
activity, suggesting that the pretreatment caused little
increase in leakiness of the microsomes (Fig. 4). Inactivation
of CPT
1
by etomoxiryl-CoA totally inhibited the formation
of ethanol-dependent and -independent products from

[
14
C]palmitoylcarnitine. This suggested that the effect of
CoASH/dithiothreitol to facilitate formation of ethanol-
dependent product from [
14
C]palmitoylcarnitine, in contra-
diction of [12], cannot be explained by CoASH being
transported into the ER lumen to provide a cosubstrate for
the ER luminal CAT. Rather, it strongly suggested that this
product formation was due to conversion of [
14
C]palmi-
toylcarnitine by CPT
1
(either an integral component of the
ER membrane [21] or a contaminant arising from mito-
chondrial contact sites [20]) to external [
14
C]palmitoyl-CoA,
which is then converted into ethyl palmitate by the small
amount of AEAT activity that is overt because of incom-
plete sealing of the vesicles.
Free CoASH is not detectable in rat liver microsomal
fractions [32] implying that any luminal pool of CoASH is
lost during tissue extraction and/or fractionation. In order
to attempt to make CoASH available to the interior of the
microsomal vesicles, some preparations were isolated with
CoASH present (Materials and methods). After pretreat-
ment with etomoxiryl-CoA, these microsomes were still

relatively sealed, as evidenced by an overt AEAT activity of
only 972 ± 88 pmolÆmin
)1
Æmg
)1
(indicating 95% latency of
AEAT) and had complete inactivation of CPT
1
,as
evidenced by the lack of any ethanol-independent product
formation from [
14
C]palmitoylcarnitine (Fig. 4). However,
[
14
C]palmitoylcarnitine was converted into ethanol-depend-
ent product at a rate of 939 ± 188 pmolÆmin
)1
Æmg
)1
(Fig. 4). Overall, these results support the notion that
palmitoylcarnitine can be transported to the ER lumen
where it can become a substrate for the microsomal CAT/
AEAT. However, externally derived CoASH cannot play
any significant role in the CAT reaction, which must rely on
an internal, luminal pool of CoASH.
Transport of
L
-carnitine into sealed microsomal vesicles
Transport of

L
-[
3
H]carnitine was studied by measurement
of uptake into microsomal vesicles at 10 °C. Figure 5
shows initial experiments in which 2 m
M
carnitine was
used. Because of the time needed to separate microsomes
from the incubation medium, it was not feasible to
measure uptake at times earlier than 2 min. With 2 m
M
carnitine at 10 °C the uptake reached  60% of the
equilibrium value at 2 min allowing only a crude minimal
estimate that the initial rate of unidirectional uptake was
at least 0.62 nmolÆmin
)1
Æmg
)1
.MersalylandN-ethyl
maleimide are known to inactivate the mitochondrial
CAC [33,34]. Mersalyl at 0.5 m
M
caused  40% inhibition
of carnitine uptake at 2 min and 5 m
M
mersalyl almost
abolished uptake (Fig. 5). However, we found that neither
N-ethyl maleimide nor sulfobetaine (which is a competitive
inhibitor of the mitochondrial CAC [35]) at concentrations

up to 5 m
M
had any effect on carnitine uptake by
microsomes (results not shown).
We attempted to study the concentration dependence of
L
-[
3
H]carnitine uptake (Fig. 6). These experiments appeared
to show that the rate of uptake increased linearly with
carnitine up to 10 m
M
, i.e. there was no indication of
saturation of the process. However, as indicated above,
initial rates determined from the first time-point can only be
regarded as crude minimum estimates of the true initial rates
of unidirectional uptake. These were 0.034 ± 0.003 and
2.9 ± 0.6 nmolÆmin
)1
Æmg
)1
at 0.1 and 10 m
M
carnitine,
respectively. In other experiments (results not shown),
microsomes were preloaded with 2 m
M
unlabelled
L
-carni-

tine. This had no effect on the time profile of subsequently
measured uptake of [
3
H]carnitine, suggesting that an
exchange carrier identical or similar to the mitochondrial
CAC was not involved.
Data showing unidirectional import of
L
-carnitine by
the purified mitochondrial CAC in a reconstituted system
suggest a V
max
forthisprocessat10°Cof 2nmolÆ
min
)1
Æmg
)1
with a K
m
of 0.53 m
M
(the rate of exchange
transport was much faster) [36]. Even relative to total
Fig. 5. Effect of mersalyl on uptake of
L
-[
3
H]carnitine by sealed
microsomes. Sealed microsomes were incubated with 2 m
M

L-[
3
H]carnitine and uptake measured as described under Materials and
methods. Values are means ± SD of four independent experiments.
h, No mersalyl; j,0.5m
M
mersalyl; s,5m
M
mersalyl.
Fig. 6. Effect of
L
-carnitine concentration on
L
-[
3
H]carnitine uptake
by sealed microsomes. Sealed microsomes were incubated with
L
-[
3
H]carnitine and uptake measured as described under Materials and
methods. Values are means ± SD of four independent observations.
L
-carnitine concentrations were: h,0.1m
M
; j,0.25m
M
; s,0.5m
M
;

d,1.0m
M
; n,2.0m
M
; m,10m
M
.
958 J. M. Gooding et al. (Eur. J. Biochem. 271) Ó FEBS 2004
microsomal protein we have observed comparable or higher
rates of unidirectional transport than 2 nmolÆmin
)1
Æmg
)1
.
This further differentiates the microsomal process from the
mitochondrial CAC.
Figure 7 shows a linear plot of [
3
H]carnitine uptake at
equilibrium (60 min values) vs. the
L
-carnitine concentra-
tion. The slope of this line, which represents the Ôcarnitine
spaceÕ of the microsomal vesicle preparation, was
1.03 lLÆmg
)1
. In other experiments (not shown), we found
the internal H
2
O space to be 2.4 ± 0.2 lLÆmg

)1
[3].
4
Therefore, only 43% of the vesicles present in the micro-
somal fraction appeared to contain the capability of
L
-carnitine transport.
Discussion
The findings of this study provide a framework whereby,
acting in concert with the ER CAT, enzymes in the ER
lumen (e.g. DGAT, ACAT or AEAT) are supplied with
their fatty acyl-CoA substrate via a fatty acylcarnitine
intermediate and the resulting free
L
-carnitine product can
be disposed of (Fig. 1). As AEAT activity appears to be
totally localized to the interior of the ER in cells, it,
together with the luminal CAT, provides an easily
quantifiable (via assay) and unambiguous
5
reporter of the
entry of fatty acylcarnitine into the ER lumen provided
certain experimental conditions are met, i.e., inactivation
of CPT
1
, minimization of interference from esterases and
provision of a source of luminal CoASH either as
described here or by delivery from liposomes [24]. As far
as we are aware, this study presents the first direct
demonstration of fatty acylcarnitine transport across

microsomal membranes.
After taking precautions to minimize the ambiguity
inherent in using DGAT as the reporter enzyme, Abo-
Hashema et al. observed carnitine-dependent incorporation
of [
14
C]oleoyl-CoA into liver microsomal luminal triacyl-
glycerol to the extent of 11.58 nmolÆmg
)1
over an
incubation period of 40 min at 37 °C (290 pmolÆmin
)1
Æ
mg
)1
) [24]. Initial rates were not determined [24] and so it is
not known to what extent this product formation was limited
by esterase activity or by the necessity to involve CPT
1
as an
additional enzyme in the system prior to the membrane
transport of the fatty acylcarnitine. With 40 l
M
[
14
C]palmi-
toylcarnitine, we observed formation of ethyl palmitate that
was linear with time at a rate of 939 pmolÆmin
)1
Æmg

)1
at
30 °C, provided microsomes were isolated previously with
CoASH present (Fig. 4). Activities of rat liver microsomal
luminal CAT (2.7–3.6 nmolÆmin
)1
Æmg
)1
at 25 °C[19])and
AEAT (18.4 nmolÆmin
)1
Æmg
)1
at 30 °C; see Fig. 3A) exceed
this value of 939 pmolÆmin
)1
Æmg
)1
suggesting that it is likely
to be a reasonable estimate of the rate of palmitoylcarnitine
transport. It is of note that measurements of rat liver
microsomal latent DGAT activity (0.47–1.9 nmolÆmin
)1
Æ
mg
)1
at 37 °C [5,7]) are not dissimilar from our measure-
ment of the rate of palmitoylcarnitine transport. As we
discuss below, it is highly unlikely that the CAC protein
plays any role in the ER membrane. At present, this

microsomal transport process for fatty acylcarnitine awaits
characterization in future studies which should also investi-
gate whether it makes a significant contribution to the
control of metabolic flux to luminal enzymes such as DGAT
and ACAT.
Our demonstration that
L
-carnitine can move across the
membrane of microsomal vesicles that have a high degree of
ÔintactnessÕ as judged by enzyme latency is a significant
finding as a key feature of the scheme in Fig. 1 is that free
carnitine should be able to escape from the ER after its
generation by the luminal CAT. The observation that
microsomal carnitine transport is sensitive to mersalyl
(Fig. 5) suggests that the process is protein-mediated rather
than being a simple diffusion process (the extreme hydro-
philicity of carnitine also makes this highly unlikely).
The lack of sensitivity to N-ethyl maleimide and sulf-
obetaine and the apparent lack of requirement for a
counter-transport partner for carnitine discriminates this
microsomal process from carnitine/acylcarnitine transport
in mitochondria – findings that are not at variance with the
report of Fraser & Zammit that the CAC protein is not
detectable in liver microsomes [4]. Our attempts at a kinetic
analysis (Fig. 6) could not differentiate between a carrier or
a channel that facilitates the rapid equilibrium of carnitine
across the microsomal membrane. In this regard, it is of
note that there have been reports [37–39] of a microsomal
membrane channel that permits the passage of certain small
molecules, some similar to carnitine (e.g. choline [39]). The

microsomal isotope space for choline of 1.05 lLÆmg
)1
reported by Meissner & Allen [39] is remarkably similar to
the space of 1.03 lLÆmg
)1
that we found for
L
-carnitine
(Fig. 7). Further studies are needed to characterize the
microsomal
L
-carnitine process.
Finally, the need for an ER luminal pool of CoASH
(Fig. 1) is demonstrated by this study. However, questions
regarding the source of this pool and how it is maintained
in vivo remain totally unanswered.
Acknowledgements
We are grateful to the Medical Research Council and to the Wellcome
Trust for support.
Fig. 7. Content of
L
-[
3
H]carnitine by sealed microsomes at equilibrium.
Equilibrium was reached by incubation with
L
-[
3
H]carnitine for
60 min (Fig. 6). Values are means ± SD of four independent experi-

ments. Symbols indicating
L
-carnitine concentrations are the same as
those in Fig. 6 except that values with 10 m
ML
-carnitine, which also lie
on the line, are omitted for reasons of scale. An intramicrosomal space
for
L
-carnitine (1.03 lLÆmg
)1
) is obtained from the slope of the line.
Ó FEBS 2004 Transport of carnitine and acylcarnitine (Eur. J. Biochem. 271) 959
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