Membrane transport of fatty acylcarnitine and free L -carnitineby rat liver microsomes Jason M.. Liver microsomal vesicles that were shown to be at least 95% impermeable to palmitoyl-CoA
Trang 1Membrane 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 freeL-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 [14C]palmitoylcarnitine
into microsomes through the use of the luminal CAT and
acyl-CoA:ethanol acyltransferase as a reporter system to
detect formation of luminal [14C]palmitoyl-CoA Rapid, unidirectional transport of free L-[3H]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 CPT1, 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 CPT2then 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
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 CPT1 located at sites distinct from the ER [20] or also by an ER-targeted form of CPT [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: Saggerson@biochem.ucl.ac.uk
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)
Trang 2way 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
andL-carnitine, respectively, across the ER membrane are
facilitated In the present study, we report direct evidence
for the passage of radiolabelledL-carnitine across
micro-somal membranes We also report on studies of the
uptake of [14C]palmitoylcarnitine into the lumen of
sealed 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 reporter
systems must be used Broadway et al [12] used the
ER luminal coupled system of CAT and AEAT as a
reporter in preliminary studies to show that sealed 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 reporter system 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)2Pi] 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 sealed 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) [3H]Carnitine, [14C]inulin, [14 C]palmitoylcarni-tine and [14C]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[14C]palmitate was synthesized as described
by Diczfalusy et al [25]
Isolation of microsomes from rat liver 2,3Fed male Sprague–Dawley rats (180–220 g) were killed
an approved schedule/method [schedule/method approved
by the UK Home Office under the Act of Parliament Animals (Scientific Procedures) Act 1986 and the local UCL Animal Experimentation Ethics Committee] Livers were immedi-ately removed and washed in ice-cold isolation medium (5 mMTris/HCl buffer, pH 7.4, containing 220 mM mann-itol, 80 mM sucrose, 1 mM EDTA, 5 lgÆmL)1 bestatin,
5 lgÆmL)1 leupeptin and 1 mM 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 °C as 100 lL aliquots in HS buffer (5 mM
Hepes buffer, pH 7.4 containing 250 mM 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 mMCoASH with 4 mM dithiothre-itol were included in isolation medium and in HS buffer Microsomal vesicles described as sealed were obtained by thawing stored aliquots and suspending at the required concentration in HS buffer
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
permeabilised 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)1dissolved 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.
Trang 3we 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)1in
HS buffer containing 5 mM ATP, 10 mM MgCl2, 2 mM
CoASH, 50 lM 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 mMTris/HCl buffer
(pH 7.4) containing 300 mM sucrose, 10 mM MgCl2,
0.8 mM EDTA, bovine albumin (1 mgÆmL)1) and 40 lM
[14C]palmitoyl-CoA (1.8 lCiÆlmol)1) with or without 10 lL
of ethanol (123 mMfinal concentration) Where indicated,
250 lM(Np-O)2Pi[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 mMNaOH 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 14C-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,
[14C]palmitoyl-CoA was replaced by 40 lM[14
C]palmitoyl-carnitine (1.8 lCiÆlmol)1) with or without 0.5 mMCoASH
and 1 mMdithiothreitol
Measurement of uptake ofL-[3H]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 [3H]carnitine
and 2 lCi [14C]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 and3H and14C measured by liquid scintillation counting The amount of [3H]carnitine within microsomal vesicles at each time-point was calculated by subtracting the 3H-associated with the alamethicin-treated pellet from the3H-associated with the untreated pellet after correcting for adhering medium, which was determined from the amount of associated [14C]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
The experiments shown in Fig 2 were performed in the absence of (Np-O)2Pi 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 [14C]palmitoyl-CoA was seen with sealed microsomes whereas permeabilization by alamethicin allowed ethyl palmitate formation at an initial rate of
1020 ± 30 pmolÆmin)1Æmg)1 When [14C]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 [14C]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 CoASH + 1 m dithiothreitol.
Trang 4rate of 180 ± 20 pmolÆmin)1Æmg)1over 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 [14C]palmitoyl-CoA by alamethicin-permeabilized
microsomes plateaued and then declined after 10 min As
only 10% of the available14C 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 available14C could be detected
in ethanol-dependent or -independent products Diczfalusy
et al [25] have shown that the serine esterase inhibitor
(Np-O)2Piat 250 lMenhanced 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 [14C]palmitoyl-CoA and [14
C]palmi-toylcarnitine in the presence of 250 lM (Np-O)2Pi(Figs 3
and 4) Ethyl palmitate formation from [14C]palmitoyl-CoA
was considerably enhanced by (Np-O)2Pi 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)2Pidid 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)1Æmg)1 for sealed microsomes and 18.4 ± 1.2 nmolÆmin)1Æmg)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 intactness It was 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 lM) that was not inhibitory Also in general (not shown), we observed that ethanol-independent product formation from [14C]palmitoyl-CoA or [14C]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 lM(Np-O)2Pi, rates of formation
of ethanol-dependent and -independent products from
40 lM [14C]palmitoyl-CoA or [14C]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)1Æmg)1, respectively, were seen when the substrate was 40 lM [14C]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 CPT1 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 [14C]palmitoyl-CoA or [14C]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).
Trang 5shown) 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 CPT1by etomoxiryl-CoA totally inhibited the formation
of ethanol-dependent and -independent products from
[14C]palmitoylcarnitine This suggested that the effect of
CoASH/dithiothreitol to facilitate formation of
ethanol-dependent product from [14C]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 CPT1(either an integral component of the
ER membrane [21] or a contaminant arising from
mito-chondrial contact sites [20]) to external [14C]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)1Æmg)1(indicating 95% latency of
AEAT) and had complete inactivation of CPT1, as
evidenced by the lack of any ethanol-independent product
formation from [14C]palmitoylcarnitine (Fig 4) However,
[14C]palmitoylcarnitine was converted into
ethanol-depend-ent product at a rate of 939 ± 188 pmolÆmin)1Æmg)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 ofL-carnitine into sealed microsomal vesicles
Transport ofL-[3H]carnitine was studied by measurement
of uptake into microsomal vesicles at 10°C Figure 5
shows initial experiments in which 2 mM 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 mM
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)1Æmg)1 Mersalyl and N-ethyl
maleimide are known to inactivate the mitochondrial
CAC [33,34] Mersalyl at 0.5 mMcaused 40% inhibition
of carnitine uptake at 2 min and 5 mM 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 mM had any effect on carnitine uptake by
microsomes (results not shown)
We attempted to study the concentration dependence of
L-[3H]carnitine uptake (Fig 6) These experiments appeared
to show that the rate of uptake increased linearly with
carnitine up to 10 mM, 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)1Æmg)1 at 0.1 and 10 mM carnitine, respectively In other experiments (results not shown), microsomes were preloaded with 2 mMunlabelledL -carni-tine This had no effect on the time profile of subsequently measured uptake of [3H]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 Vmax for this process at 10°C of 2 nmolÆ min)1Æmg)1with a Km of 0.53 mM (the rate of exchange transport was much faster) [36] Even relative to total
Fig 5 Effect of mersalyl on uptake of L -[3H]carnitine by sealed microsomes Sealed microsomes were incubated with 2 m M L-[3H]carnitine and uptake measured as described under Materials and methods Values are means ± SD of four independent experiments.
h, No mersalyl; j, 0.5 m M mersalyl; s, 5 m M mersalyl.
Fig 6 Effect of L -carnitine concentration on L -[3H]carnitine uptake
by sealed microsomes Sealed microsomes were incubated with
L -[3H]carnitine and uptake measured as described under Materials and methods Values are means ± SD of four independent observations.
L -carnitine concentrations were: h, 0.1 m M ; j, 0.25 m M ; s, 0.5 m M ;
d, 1.0 m M ; n, 2.0 m M ; m, 10 m M
Trang 6microsomal protein we have observed comparable or higher
rates of unidirectional transport than 2 nmolÆmin)1Æmg)1
This further differentiates the microsomal process from the
mitochondrial CAC
Figure 7 shows a linear plot of [3H]carnitine uptake at
equilibrium (60 min values) vs theL-carnitine
concentra-tion The slope of this line, which represents the carnitine
space of the microsomal vesicle preparation, was
1.03 lLÆmg)1 In other experiments (not shown), we found
the internal H2O space to be 2.4 ± 0.2 lLÆmg)1 [3]
4Therefore, 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 freeL-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
entry of fatty acylcarnitine into the ER lumen provided
certain experimental conditions are met, i.e., inactivation
of CPT1, 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 [14C]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)1Æ
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 CPT1as an additional enzyme in the system prior to the membrane transport of the fatty acylcarnitine With 40 lM[14 C]palmi-toylcarnitine, we observed formation of ethyl palmitate that was linear with time at a rate of 939 pmolÆmin)1Æmg)1at
30°C, provided microsomes were isolated previously with CoASH present (Fig 4) Activities of rat liver microsomal luminal CAT (2.7–3.6 nmolÆmin)1Æmg)1at 25°C [19]) and AEAT (18.4 nmolÆmin)1Æmg)1at 30°C; see Fig 3A) exceed this value of 939 pmolÆmin)1Æmg)1suggesting 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)1Æ
mg)1at 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 thatL-carnitine can move across the membrane of microsomal vesicles that have a high degree of
intactness 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 microsomalL-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 vivoremain totally unanswered
Acknowledgements
We are grateful to the Medical Research Council and to the Wellcome Trust for support.
Fig 7 Content of L -[3H]carnitine by sealed microsomes at equilibrium.
Equilibrium was reached by incubation with L -[3H]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 M L -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.
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