From the Departments of Nutrition and Pediatrics,
University of North Carolina, Chapel Hill, North Carolina 27599 and the
§ Department of Medicine and Canadian Institutes for Health
Research Group on Molecular and Cell Biology of Lipids, University of
Alberta, Edmonton, Alberta T6G 2S2, Canada
Received for publication, March 6, 2001, and in revised form, April 17, 2001
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ABSTRACT |
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Inhibition studies have suggested that acyl-CoA
synthetase (ACS, EC 6.2.1.3) isoforms might regulate the use of
acyl-CoAs by different metabolic pathways. In order to determine
whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated
independently, non-cross-reacting peptide antibodies were raised
against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic
reticulum, mitochondria-associated membrane (MAM), and cytosol, but not
in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5
protein was located in mitochondrial membrane. Consistent with these
locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in
microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive
inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in
endoplasmic reticulum, MAM, and mitochondria, suggesting that a
hitherto unidentified triacsin-sensitive ACS is present in
mitochondria. ACS1, ACS4, and ACS5 were regulated independently by
fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding normal
chow or a high sucrose diet for 24 h after a 48-h fast increased
both ACS1 and ACS4 protein expression 1.5-2.0-fold, consistent with
inhibition studies. These results suggest that ACS1 and ACS4 may be
linked to triacylglycerol synthesis. Taken together, the data suggest
that acyl-CoAs may be functionally channeled to specific metabolic
pathways through different ACS isoforms in unique subcellular locations.
The first step in long chain fatty acid use in mammals requires
the ligation of fatty acid with coenzyme A (CoA). This reaction, catalyzed by acyl-CoA synthetase
(ACS,1 EC 6.2.1.3), produces
acyl-CoAs, which are primary substrates for energy use via
Currently, five different rat ACS cDNAs have been cloned, each the
product of a different gene (4-8). Rat ACS1-5 share a common
structural architecture and are further classified into two subfamilies
based on amino acid identity and fatty acid preference (4-8). ACS1,
ACS2, and ACS5 make up one subfamily with about 60% homology to one
another, and ACS3 and ACS4 make up a second subfamily with about 70%
homology to each other and 30% similarity to ACS1. Within each
subfamily, the ACS isoforms differ in their mRNA size, tissue
distribution, and transcriptional regulation. ACS1, ACS4, and ACS5 are
all expressed in liver.
Studies with the ACS inhibitors triacsin and troglitazone suggested
that long chain acyl-CoAs are functionally channeled toward specific
metabolic fates. In most of these studies, de novo
glycerolipid synthesis was severely inhibited, whereas phospholipid
reacylation and ketone bodies formation was less impaired. Inhibition
of fatty acid incorporation into cholesterol esters varied with cell
type, being completely blocked in human fibroblasts and only moderately decreased in hepatocytes. For example, in hepatocytes, triacsin C did
not alter oxidation of pre-labeled intracellular lipid, but did inhibit
triacylglycerol synthesis 40% and 70% in hepatocytes isolated from
starved and fed rats, respectively (9). Additionally, in hepatocytes
isolated from fasted rats, troglitazone blocked incorporation of oleate
into triacylglycerol, but not into phospholipid (10). In addition,
troglitazone inhibited ketone body production. In human fibroblasts,
triacsin C blocked the incorporation of [3H]glycerol
into phospholipid by 80% and the incorporation into triacylglycerol by
99%, indicating severely impaired acylation of glycerol 3-phosphate,
lysophosphatidic acid, and diacylglycerol via the de novo
synthetic pathway from glycerol 3-phosphate (11). Incorporation of
[14C]oleate into triacylglycerol was also blocked 95%,
consistent with impaired acylation via the de novo pathway;
however, incorporation into phospholipids was not impaired, suggesting
that separate pools of acyl-CoAs exist and that the reacylation pathway
is functionally separate from de novo glycerolipid
synthesis. Taken as a whole, these studies suggest that there are
functionally independent acyl-CoA pools within cells, and that
acyl-CoAs might be channeled toward specific fates rather than being
freely available for all possible enzymatic reactions.
Yeast provide clear evidence for functionally different ACS-linked
pathways. In Candida lipolytica, studies using ACS mutants indicate that ACS I activates exogenous fatty acids for glycerolipid synthesis and ACS II activates them for We examined ACS1, ACS4, and ACS5 in rat liver, which contains a variety
of pathways that use acyl-CoAs, in order to determine whether the
subcellular locations, inhibition by specific inhibitors, and
nutritional regulation might link the different ACS isoforms with
different metabolic pathways. Our data indicate that ACS1, ACS4, and
ACS5 are present in different subcellular membranes; that ACS activity
is inhibited by triacsin C, troglitazone, and NEM to varying degrees in
these subcellular fractions; and that nutritional changes regulate each
ACS isoform independently.
Materials--
[2-3H]Glycerol and
[9,10-3H]palmitate were from Amersham Pharmacia Biotech.
Glycerol, palmitoyl-CoA, ATP, and bovine serum albumin (essentially
fatty acid-free) were from Sigma. Triacsin C (>95% pure) was from
Biomol. Troglitazone was the gift of Dr. Steven Jacobs,
GlaxoSmithKline. A polyclonal antibody to rat ACS1 was the gift of Dr.
Paul Watkins, Kennedy Krieger Institute.
Animals--
Animal protocols were approved by the
University of North Carolina (UNC), Chapel Hill, NC and University of
Alberta Institutional Animal Care and Use Committees. Male and female
(150 g) Harlan Sprague-Dawley rats were housed on a 12-h/12-h
light/dark cycle with free access to water. Control animals had free
access to Purina rat chow. Fasted animals were sacrificed after being
without food for 48 h. Refed rats were sacrificed after being fed
Purina rat chow or a high sucrose diet (69.5% sucrose, Dyets, Inc.)
for 24 h after a 48-h fast.
Subcellular Fractionation--
For subcellular localization
experiments, liver cytosol, microsomes, rough and smooth endoplasmic
reticulum, mitochondria-associated membrane (MAM), and mitochondria
were isolated from male rats by a method (19) modified by Vance (20).
Liver total membrane fraction, microsomes, and mitochondria were
isolated from female rats by differential centrifugation (21) in the
presence of protease inhibitors (1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) for subcellular
localization and nutritional regulation experiments. Fractions were
stored in aliquots at Immunoblotting--
Peptides corresponding to regions of rat
ACS1, ACS4, and ACS5 that show poor amino acid conservation (ACS1,
MEVHELFRYFRMPELIDIR; ACS4, EIHSMQSVEELGSKPENSSI; ACS5, KCGIEMLSLHDAENL)
were synthesized, purified, and coupled to keyhole limpet hemocyanin in
the UNC/PMBB Micro Protein Chemistry Facility. Rabbit antibodies to
these peptides and to purified mitochondrial GPAT were raised
commercially in New Zealand White rabbits (ImmunoDynamics, La Jolla,
CA). Antibodies to PEMT2 were made as described previously (22).
Proteins were separated on an 8% (or 12% for PEMT2) polyacrylamide
gel containing 1% SDS and transferred to a polyvinylidene difluoride
membrane (Bio-Rad). For chemiluminescent detection, the immunoreactive bands were visualized by incubating the membrane with horseradish peroxidase-conjugated goat anti-rabbit IgG and PicoWest reagents (Pierce). For quantitation, the polyvinylidene difluoride membrane was
incubated with 0.5 µ Ci of 125I-Protein A (ICN), exposed
to a phosphor screen, and quantified with the Molecular Dynamics Storm
840 and ImageQuant software.
Enzyme Assays--
Diacylglycerol acyltransferase was assayed at
23 °C with 200 µM sn-1,2-diolein and 25 µM [3H]palmitoyl-CoA (23), acyl-CoA
synthetase was assayed at 37 °C with 5 mM ATP, 250 µM CoA, and 50 µM
[3H]palmitate (24), and glycerol 3-phosphate
acyltransferase (GPAT) was assayed at 23 °C with 300 µM [3H]glycerol-3-P and 112.5 µM palmitoyl-CoA in the presence or absence of 2 mM N-ethylmaleimide to inhibit the microsomal
isoform (25). Microsomal GPAT was estimated by subtracting the
N-ethylmaleimide-resistant activity (mitochondrial GPAT)
from the total. All assays measured initial rates.
[3H]Palmitoyl-CoA (26) and [3H]glycerol
3-phosphate (27) were synthesized enzymatically.
ACS1, ACS4, and ACS5 Are Located in Different Subcellular
Fractions--
The relative distribution of ACS activity in rat liver
is 7% in peroxisomes, 20% in mitochondria, and 73% in microsomes
(28). Since measurements of ACS activity do not distinguish among the different ACS isoforms, we localized each isoform in order to determine
whether it is evenly distributed or, instead, located in a specific
subcellular membrane.
We raised isoform-specific rabbit antibodies against unique peptides
present in ACS1, ACS4, or ACS5 because an antibody raised against
purified ACS1 not only recognized purified recombinant ACS1, but also
recognized purified recombinant ACS4 and ACS5 (Fig. 1A). The cross-reactivity is
most likely due to the high degree of sequence similarity found among
ACS1, ACS4, and ACS5. The specificity of each of our peptide antibodies
was verified by immunoblot analysis of each isoform specific antibody
against purified recombinant rat ACS1, ACS4, and ACS5, each with a
C-terminal Flag epitope (29). Peptide antibodies for ACS4 and ACS5 each
recognized only the correct ACS isoform (Fig. 1, C and
D). The ACS1 peptide antibody failed to recognize
recombinant ACS1-Flag (Fig. 1B) because the initial two
amino acids of the N-terminal sequence had been altered to clone the
recombinant protein. However, the recombinant ACS1-Flag protein
detected by the Flag antibody and the major band in rat liver
microsomes detected by the ACS1 peptide antibody migrate to the same
position, showing that the ACS1 peptide antibody recognizes native ACS1
(Fig. 1E).
Rat liver was fractionated by two different methods. One method
used a sucrose gradient to further purify microsomes into ER1 and ER2
fractions that are enriched in rough and smooth ER, respectively (19,
20). A Percoll gradient separated crude mitochondria into MAM and
purified mitochondria (19, 20). The second method used only
differential centrifugation (21) and produced mitochondria that had
very little microsomal contamination and microsomes that primarily
contained ER membranes, as well as the MAM fraction. The purity of each
fraction was ascertained by enzymatic assays for the ER enzymes DGAT
and NEM-sensitive GPAT, and for the mitochondrial enzyme NEM-resistant
GPAT and an by immunoblots for mitochondrial GPAT and for PEMT, the MAM marker. These markers showed that the microsome fraction was free of
mitochondrial contamination because little NEM-resistant GPAT activity
was present (Table I) and no
mitochondrial GPAT protein was detected by immunoblotting (Fig.
2B). In the ER and MAM
fractions purified using gradients, 11-28% of the GPAT activity was
resistant to NEM (Table I), indicating that these fractions were
somewhat contaminated with mitochondria. No PEMT was detected in the ER and purified mitochondria by immunoblot analysis (Fig. 2B),
indicating that these fractions were not contaminated with the MAM
fraction. Very low DGAT and NEM-sensitive GPAT activities were measured in the purified mitochondria (Table I), indicating little contamination with ER. DGAT activity was enriched in the MAM fraction compared with
ER, consistent with previous results (40). The microsome fraction
(100,000 × g pellet) also has high DGAT activity, and it contains the MAM fraction, as evidenced by the presence of the
MAM-specific marker, PEMT (Fig. 2B).
The location of ACS1, ACS4, and ACS5 in rat liver was determined by
Western blot analysis of various subcellular fractions. ACS1 (68 kDa)
was strongly detected in the microsomal fraction, which comprises rough
ER, smooth ER, and MAM (Fig. 2A). The molecular mass is
smaller than that predicted by the cDNA sequence (78 kDa), but in
agreement with the size of the recombinant ACS1 run in the same manner
(Fig. 1). A prominent ACS1 band was also detected in the cytosol, but
cytosolic ACS specific activity was only 2.6% that of microsomal ACS
specific activity (3.5 versus 132 nmol/min/mg of protein).
Although ACS1 was present in crude mitochondria, which contain the MAM
fraction, no ACS1 band was observed in purified mitochondria (Fig.
2A), as we reported previously (9). ACS4 (74 kDa) was
enriched in the MAM fractions from rat liver (Fig. 2A) and
Chinese hamster ovary cells (data not shown), and was only very weakly
detected in microsomal and mitochondrial fractions. In some
preparations, ACS4 was detected as a doublet with a larger 75-kDa
protein (data not shown), consistent with alternative start sites as
described for the human ACS4 homologue (30). The ACS5-specific antiserum detected a protein of 76 kDa in the mitochondrial fraction (Fig. 2A), 73- and 74.5-kDa proteins in ER, and a 74.5-kDa
protein in cytosol and MAM fractions (Fig. 2A). The 76-kDa
protein agrees with the size predicted by the cDNA sequence and
migrates to the same position as recombinant ACS5. Taken together,
these data indicate that ACS1, ACS4, and ACS5 each have unique
distributions in liver subcellular membranes.
Inhibition of ACS Activity in Liver Subcellular
Fractions--
Since ACS1, ACS4, and ACS5 were present in different
subcellular membranes and the recombinant proteins expressed in
Escherichia coli were inhibited to different extents by
triacsin C, thiazolidinediones, and NEM (29), we hypothesized that
these inhibitors would affect ACS activity differently in each
fraction. Triacsin C (10 µM), which inhibits purified
recombinant ACS1 and ACS4 by 60%, and does not inhibit ACS5 (29),
decreased ACS activity in microsomes, mitochondria, and MAM by 60%
(Fig. 3). This result is surprising because the only ACS we identified in mitochondria was ACS5, which is
resistant to inhibition by triacsin C (29). The mitochondria we assayed
were free of contamination by the MAM fraction as determined by
the absence of PEMT on an immunoblot (data not shown). Therefore, the
observed inhibition suggests that mitochondria contain a hitherto undescribed triacsin-sensitive ACS.
Troglitazone and NEM, two specific ACS4 inhibitors, were more selective
inhibitors of ACS activity in liver subcellular fractions. Troglitazone
at 10 µM decreased ACS activity 25% in MAM, but had no
effect on activity in the microsomal or mitochondrial fractions (Fig.
4). With 50 µM
troglitazone, ACS activity was inhibited 45% in MAM, again with little
effect on microsomal or mitochondrial ACS activity. NEM, a second ACS4
inhibitor, decreased ACS activity 50% in MAM and only 27% in the
smooth ER fraction (Fig. 5). These results are consistent with the immunolocalization of ACS4
preferentially to the MAM fraction.
Nutritional Regulation of ACS1, ACS4, and ACS5 in Liver--
If
ACS1, ACS4, and ACS5 are linked to different metabolic pathways, one
might expect that each isoform would be regulated differently under
conditions of fasting and re-feeding. ACS activity and protein
expression were measured in liver microsomes and mitochondria isolated
by differential centrifugation from control rats, from rats fasted for
48 h, and from rats fasted for 48 h and then refed either
Purina rat chow or a high sucrose diet for 24 h. The microsomes contained MAM, and the mitochondria were free of contamination by MAM
as determined by immunoblot with antibody against PEMT, the
MAM-specific marker (data not shown). ACS activity, assayed with
palmitate as the fatty acid substrate, was similar in microsomes and in
mitochondria isolated from control rats (18.5 ±3.5 and 14.3 ± 2.9, respectively), 48-h fasted rats (21.8 ± 0.9 and 11.9 ± 2.9, respectively), and rats fed Purina rat chow for 24 h after a
48-h fast (15.1 ± 4.5 and 14.2 ± 2.4, respectively).
Although total ACS activity was unchanged by nutritional status, ACS1
protein expression increased 1.8- and 2-fold with Purina chow and high
sucrose re-feeding, respectively (Fig.
6). ACS4 protein expression also
increased with re-feeding (50% with Purina chow diet and 64% with
sucrose diet). ACS5 protein expression in mitochondria did not appear
to be altered by re-feeding. After a 48-h fast, ACS1 protein expression
decreased 14%, ACS4 protein decreased 47%, whereas ACS5 protein
expression increased 82% in mitochondria (Fig. 6).
Long chain acyl-CoA synthetase catalyzes the initial step required
for oxidation, elongation, and desaturation of fatty acids; for the
synthesis of complex lipids and acylated proteins; and for a variety of
signals that regulate cellular metabolism (1, 2). It had been thought
that the ACSs synthesize a common pool of acyl-CoAs, which move freely
within cell membrane monolayers and have equal access to the numerous
metabolic pathways in which they participate. However, genetic studies
in yeast link specific ACS isoforms to different pathways that use
acyl-CoAs (12-17). In addition, in cultured cells, inhibitors of ACS
(triacsin and troglitazone) selectively alter the synthesis and
oxidation of cellular lipids, suggesting that the ACS isoforms might be
differentially inhibited, and that triacsin-sensitive isoforms might be
functionally linked to de novo glycerolipid synthesis
whereas triacsin-resistant ACS isoforms might be functionally linked to
phospholipid reacylation pathways and to In this paper, we show that ACS1, ACS4, and ACS5 differ in their
subcellular distribution, suggesting that, as in yeast, acyl-CoAs may
be channeled toward specific metabolic pathways. Functional channeling
could occur if a particular ACS were located in a membrane that
contained only a handful of specific pathways that use acyl-CoAs or if
each ACS were physically associated with the downstream enzymes in a
specific pathway. To examine this question, we investigated the
subcellular locations of the three ACSs expressed in rat liver. ACS1,
the first cloned and best studied ACS isoform, has been reported to be
present in virtually every subcellular fraction. An early study with a
polyclonal antibody raised against ACS1 purified from microsomes
reported that ACS1 protein was present in rat liver microsomes,
peroxisomes, and mitochondria (34). Others have identified ACS1 in
GLUT4 vesicles (35) and in plasma membrane (36) from fat cells. ACS
activity has been reported in nuclei from rat liver (37), and in
cytosol of PC12 neurons (38), but the ACS isoform was not determined in
either case. Our studies showed ACS1 protein in the microsomal fraction
of rat liver. ACS1 does not appear to contribute to the ACS activity present in mitochondria, since our peptide antibody did not detect ACS1
in purified mitochondria under any nutritional condition, consistent
with our previous study (9). Although ACS1 had been identified in rat
mitochondria with an antibody raised against an ACS purified from
microsomes (34), the antibody may have recognized epitopes on non-ACS1
isoforms present in mitochondrial membranes as we showed in Fig. 1.
We also detected a significant amount of ACS1 protein in rat liver
cytosol. Although a cytosolic location is consistent with measurements
of oleic acid ACS activity in the cytosol of PC12 cells, ACS activity
and ACS1 protein expression are absent in the cytosol from 3T3-L1
adipocytes (36). Since, in liver cytosol, ACS specific activity was
barely detectable, our antibody may cross-react with a non-ACS
cytosolic protein of the same molecular mass. Alternatively, cytosolic
ACS1 may be largely inactive, but become active after it translocates
to intracellular membranes as occurs with oleate-activated FadD, the
bacterial ACS (39).
The subcellular locations of ACS4 and ACS5 have not been reported
previously. Some of the mitochondrial ACS activity is accounted for by
ACS5, since we detect a 76-kDa protein (in agreement with the predicted
size of ACS5) specifically in mitochondria. The ACS5 peptide antibody
also detects 73- and 74.5-kDa proteins in ER and MAM, and a 74.5-kDa
protein in cytosol and MAM fractions. Both ACS3 and ACS4 can use
alternative start sites (6, 30), but no alternative start site is
present in the ACS5 sequence that would yield proteins smaller by 1.5 or 3 kDa. The smaller immunoreactive proteins might represent ACS5
after proteolytic cleavage, or it may be that the ACS5 peptide antibody
recognizes a novel ACS isoform with a high degree of sequence similarity.
ACS4 is highly enriched in the MAM fraction, an ER-like membrane that
can be found in mitochondria or ER preparations, depending on the cell
fractionation method employed. The MAM fraction contains PEMT-2,
microsomal triglyceride transfer protein, apoB, and high specific
activities of acyl-CoA:cholesterol acyltransferase, DGAT (40), and
phosphatidylserine synthase-1 and -2 (41). Vance's group has
hypothesized that MAM may be involved in importing lipids into
mitochondria (42) or in very low density lipoprotein assembly (40).
Inhibition studies with purified ACS1, ACS4, and ACS5 showed that
triacsin C inhibited only ACS1 and ACS4, whereas the thiazolidinediones and NEM were more selective and only inhibited ACS4 (29). Consistent with these inhibition studies and the location of the three isoforms in
liver, troglitazone and NEM had their maximum effects in MAM where ACS4
is located. Similarly, triacsin C was a potent inhibitor of ACS
activity in ER and MAM, membranes that contain both ACS1 and ACS4.
Surprisingly, however, triacsin C inhibited ACS activity in
mitochondria to the same extent that it inhibited activity in ER (Fig.
2). Since the triacsin-resistant ACS5 is the only ACS isoform detected
in mitochondria, a hitherto unknown triacsin-sensitive ACS may be
present in mitochondria. Another possibility is that purified
ACS5-Flag, which is not inhibited by triacsin, does not have the same
properties as the in situ enzyme. We do not believe that
this is the case, since the Flag epitope does not interfere with ACS
activity (29) and triacsin C is unable to inhibit ACS activity in an
E. coli membrane fraction containing overexpressed ACS5-Flag
(data not shown).
Because MAM may be a specialized site for very low density lipoprotein
biosynthesis, the presence of ACS4 in this fraction and its inhibition
by triacsin C suggests that ACS4, like ACS1, is linked to
triacylglycerol synthesis. Consistent with this hypothesis, we found
that ACS4 protein expression decreased 47% after a 48-h fast, and was
up-regulated by re-feeding either normal chow (50% increase) or a high
sucrose diet (64% increase). The most remarkable finding was the
specific inhibition of recombinant ACS4 by thiazolidinediones (29) and
the ability of troglitazone to specifically inhibit ACS activity in MAM
(Fig. 3). It has been hypothesized that the mechanism by which
thiazolidinediones produce their anti-diabetic effects is through their
ability to lower plasma fatty acids (43). Further study is needed to
determine whether thiazolidinediones are insulin sensitizers, in part,
because of their inhibitory effect on ACS4.
In humans, the gene for ACS4 lies on the X chromosome and is part of a
large deletion that results in a human disorder that combines Alport
syndrome with elliptocytosis, dysmorphic facies, and mental retardation
(44). It is not known whether the deleted ACS4 contributes to these
problems or whether the disorder is associated with abnormalities in
serum lipids. However, since expression of ACS4 is highest in human
brain, and human and rat ACS4 show a preference for arachidonate, it
has been suggested that, in brain, ACS4 might be critical for recycling
arachidonate into phospholipids that are sources for signaling
molecules related to intellect and coordination (30).
Consistent with its microsomal location and the powerful
triacsin-mediated inhibition of triacylglycerol synthesis in
fibroblasts, hepatocytes, and HepG2 cells (11, 31-33), ACS1 has been
linked to triacylglycerol synthesis. ACS1 mRNA is prominent in
liver and adipose tissue (4), and in 3T3-L1 cells only after they differentiate into adipocytes (45). ACS1 mRNA expression also increases in adipose tissue and muscle after PPAR ACS5 may be linked to the Many unique eukaryotic enzymes catalyze the same lipid-biosynthetic
reaction (50). These enzymes are encoded by different genes and are
often located in distinct cellular locations. Our findings show that
ACS1, ACS4, and ACS5 are located in different liver subcellular
membranes and that nutritional changes regulate each isoform
independently, consistent with the hypothesis that eukaryotic systems
have redundant lipid-biosynthetic enzymes in order to provide
independent regulation of activity and compartmentalization of lipid
pools (50).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation and for the synthesis of triacylglycerol, phospholipids,
cholesterol esters, and sphingomyelin, and are the source of signaling
molecules like ceramide, diacylglycerol, and arachidonic acid (1, 2).
Acyl-CoAs up-regulate uncoupling protein in brown fat and key enzymes
of glycolysis, gluconeogenesis, and
-oxidation; are essential for
vesicle trafficking; and play a critical role in the transport of fatty
acids into cells by making transport unidirectional. Protein
esterification with myristate and palmitate anchors proteins to
specific membranes and enables them to function correctly (3). Thus,
acyl-CoAs participate in a large number of cellular reactions that
involve lipid synthesis, energy metabolism, and regulation, but how
acyl-CoAs are partitioned or directed toward these diverse synthetic,
degradative, and signaling pathways is not understood.
-oxidation (12). In Saccharomyces cerevisiae, the ACS proteins Faa1p and Faa4p
account for 99% of yeast C14-CoA and C16-CoA activity (13) and
activate exogenously derived fatty acids destined for phospholipid
synthesis (14). Faa4p is specifically needed for myristoylation of
protein substrates (15), and Faa2p is required for peroxisomal
-oxidation (16, 17). Yeast ACS isoforms are also differentially
inhibited by triacsin C (18). From these data, Gordon's group (17)
concluded that there are differences in location or accessibility of
those acyl-CoAs that are derived from endogenous synthesis and those acyl-CoAs formed from exogenously provided fatty acids. Thus, genetic
studies in yeast link specific ACS isoforms to different pathways that
use acyl-CoAs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. Protein concentrations were
determined by the BCA method (Pierce) using bovine serum albumin as the standard.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Specificity of ACS1, ACS4, and ACS5
antibodies. Purified recombinant ACS1, ACS4, or ACS5, each with a
C-terminal Flag epitope (5 µg of total protein), were analyzed by
Western blot with antibodies raised against full-length ACS1
(A) or unique peptides from ACS1 (B), ACS4
(C), ACS5 (D), or the Flag epitope
(E). The MC lane represents liver
microsomes blotted with ACS1 peptide antibody. Protein antibody
complexes were visualized by chemiluminescent detection of horseradish
peroxidase linked to goat anti-rabbit or anti-mouse IgG. The molecular
mass values of recombinant ACS1, ACS4, and ACS5 were 68, 74, and 76 kDa, respectively, on this gel system.
DGAT and GPAT activity in liver subcellular fractions
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Fig. 2.
Location of ACS1, ACS4, and ACS5 in rat liver
subcellular fractions. Cytosol (C), microsomes
(MC), fractions enriched in smooth endoplasmic reticulum
(SER), fractions enriched in rough endoplasmic reticulum
(RER), MAM, crude mitochondria (cMT), and pure
mitochondria (pMT) fractions (100 µg of protein) were
analyzed by Western blot with antibodies against ACS1, ACS4, or ACS5
(A) and GPAT or PEMT (B) . Molecular mass is indicated on
the right.
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Fig. 3.
Triacsin C inhibits ACS activity in
microsomes, smooth ER, mitochondria, and mitochondria-associated
membrane fractions. Microsomes, ER1 (enriched in rough ER),
mitochondria, or MAM proteins (0.5 µg) were assayed for ACS activity
in the presence of increasing concentrations of triacsin C (0-10
µM). Data are representative of two independent
experiments and are presented as percentage of ACS activity remaining
after treatment with inhibitor. Control specific activities (100%):
microsomes = 109 nmol/min/mg; ER1 = 211 nmol/min/mg;
mitochondria = 105 nmol/min/mg; MAM = 345 nmol/min/mg.
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Fig. 4.
Troglitazone specifically inhibits ACS
activity in mitochondria-associated membrane. Microsomes,
mitochondria, or MAM proteins (0.5 µg) were assayed for ACS activity
in the presence of increasing concentrations of troglitazone (0-50
µM). Data are representative of two independent
experiments and are presented as percentage of ACS activity remaining
after treatment with inhibitor. Control specific activities (100%):
microsomes = 89 nmol/min/mg; mitochondria = 67 nmol/min/mg;
MAM = 255 nmol/min/mg.
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Fig. 5.
N-Ethylmaleimide inhibits ACS
activity in mitochondria-associated membrane. ER1 (enriched in
rough ER) and MAM fractions (0.5-1.5 µg of protein) were
pre-incubated in the absence or presence of 5 mM NEM for 10 min on ice prior to ACS assay. Control specific activities (100%):
ER1 = 181 nmol/min/mg; MAM = 397 nmol/min/mg.
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Fig. 6.
ACS1, ACS4, and ACS5 protein expression is
regulated differently by fasting and re-feeding. Liver total
microsomes and mitochondria (100 µg of protein) from rats fed
normally (N) (n = 5), fasted for 48 h
(F) (n = 6), fasted for 48 h and refed
normal chow for 24 h (R) (n = 6), and
fasted for 48 h and refed a 69% sucrose diet for 24 h
(S) (n = 4) were analyzed by quantitative
Western blot analysis with peptide antibodies against ACS1, ACS4, or
ACS5 and 125I-Protein A. The blots were exposed to a
phosphor screen for quantitation with a Molecular Dynamics Storm 840 system. The blots shown are representative, and the numbers
below the bands indicate the -fold change compared with
normally fed rats. The mean and S.D. for each are as follows: ACS1,
N (1 ± 0.05), F (0.8 ± 0.05),
R (1.8 ± 0.1), S (1.9 ± 0.02); ACS4,
N (1 ± 0.08), F (0.5 ± 0.07),
R (1.6 ± 0.15), S (2 ± 0.12); ACS5,
N (1 ± 0.04), F (1.8 ± 0.05),
R (1.2 ± 0.1), S (1 ± 0.06).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation (9-11, 31-33).
These data, together with our results showing that triacsin C inhibits
recombinant ACS1 and ACS4, but not ACS5, and that thiazolidinediones
specifically inhibit ACS4 (29), suggested that acyl-CoA entry into
specific metabolic pathways could be mediated by the action of
individual ACS isoforms.
activation (46),
and in liver after high dietary fat re-feeding (4). The presence of
ACS1 in GLUT1 vesicles is hypothesized to be linked to vesicle
trafficking, which requires palmitoyl-CoA (35), but ACS1 could, in
fact, function to increase fatty entry into cells in response to
insulin stimulation. On the other hand, liver ACS1 is regulated by
PPAR
(46-48), whose activation is usually associated with the
up-regulation of enzymes of fatty acid oxidation. Our finding that ACS1
is present in ER, MAM, and possibly
peroxisomes2 provides a
potential explanation, if the various physiological processes result in
changes in the amount of ACS1 protein present in different subcellular
organelles (e.g. peroxisomes versus ER). In fact,
reciprocal differences in ACS activity were observed in mitochondria
and microsomes after stimulation by cytokines, but, unfortunately, the
ACS isoforms involved were not identified (49).
-oxidation pathway because its protein
expression increased 80% after a 48-h fast. This increase in ACS5
protein contrasts with a previous study that reported a 50% decrease
in ACS5 mRNA after a fast (8). It is possible that the rate of ACS5
protein turnover is decreased in response to fasting. Although the
presence of a triacsin-sensitive ACS linked to
-oxidation is
consistent with previous observations that treatment of hepatocytes
with triacsin C (10 µM) results in a 30% decrease in
acid soluble metabolites (9), because recombinant ACS5 is not sensitive
to triacsin (29), there must be a novel triacsin-sensitive ACS in mitochondria.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Paul Watkins for the ACS1 antibody and Dr. David G. Klapper for assistance in synthesizing the peptides for the ACS1, ACS4, and ACS5 antibodies.
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FOOTNOTES |
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* This work was supported by GlaxoSmithKline, by Grants HD 56598 (to R. A. C.) and HD 08431 (to T. M. L.) from the National Institutes of Health, and by a grant from the North Carolina Institute of Nutrition.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: CB 7400, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-7213; Fax: 919-966-7216; E-mail: rcoleman@unc.edu.
These authors contributed equally to this work.
Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.M102036200
2 T. M. Lewin, S. K. Krisans, and R. A. Coleman, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ACS, acyl-CoA synthetase; DGAT, diacylglycerol acyltransferase; ER, endoplasmic reticulum; GPAT, glycerol-3-phosphate acyltransferase; NEM, N-ethylmaleimide; MAM, mitochondria-associated membrane; PEMT, phosphatidylethanolamine methyltransferase.
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