From the Departments of Nutrition and Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599-7400
Received for publication, November 29, 2000, and in revised form, March 6, 2001
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ABSTRACT |
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Inhibition by triacsins and
troglitazone of long chain fatty acid incorporation into cellular
lipids suggests the existence of inhibitor-sensitive and
-resistant acyl-CoA synthetases (ACS, EC 6.2.1.3) that are linked to
specific metabolic pathways. In order to test this hypothesis, we
cloned and purified rat ACS1, ACS4, and ACS5, the isoforms present in
liver and fat cells, expressed the isoforms as ACS-Flag fusion proteins
in Escherichia coli, and purified them by Flag affinity
chromatography. The Flag epitope at the C terminus did not alter the
kinetic properties of the enzyme. Purified ACS1-, 4-, and 5-Flag
isoforms differed in their apparent Km values for
ATP, thermolability, pH optima, requirement for Triton X-100, and
sensitivity to N-ethylmaleimide and phenylglyoxal. The ACS
inhibitor triacsin C strongly inhibited ACS1 and ACS4, but not ACS5.
The thiazolidinedione (TZD) insulin-sensitizing drugs and peroxisome
proliferator-activated receptor Acyl-CoA synthetase
(ACS,1 EC 6.2.1.3) catalyzes
the ligation of long chain fatty acids with coenzyme A (CoA) to produce long chain acyl-CoAs (1). The resulting acyl-CoAs can be further metabolized in pathways of All ACS isoenzymes are members of the luciferase superfamily and have a
common structure that consists of an N terminus, two luciferase-like
regions, a linker connecting the two luciferase-like regions, and a C
terminus. A highly conserved AMP-binding site and a predicted fatty
acid binding site are located in the first and the second
luciferase-like regions, respectively (10-14). ACS1, ACS2, and ACS5
are structurally similar, with more than 60% amino acid identity (11).
ACS3 and ACS4 form a second subgroup with ~30% homology to ACS1 and
68% identity to each other (12). Despite their high degree of
homology, the expression of ACS1 and ACS5 mRNA is regulated
independently. In liver, both ACS1 and ACS5 mRNAs are increased by
high sucrose refeeding whereas high fat refeeding increases only ACS1
mRNA. ACS5 is the sole member of the ACS family whose mRNA
decreases with fasting (11). In 3T3-L1 cells, ACS1 mRNA is detected
only after adipocyte differentiation, whereas ACS5 mRNA is
consistently expressed independent of differentiation status (10, 11).
In the adrenal, ACS4 expression increases with exposure to
adrenocorticotropic hormone and to arachidonate (15). These differences
in regulation, tissue distribution, and substrate preference suggest
that each ACS isoform might function independently in various tissues
for different metabolic purposes.
Use of two inhibitors of ACS has suggested the presence of different
acyl-CoA pools within cells, because these inhibitors are effective in
blocking only some of the metabolic pathways that require long chain
acyl-CoAs. For example, in human fibroblasts, the fungal metabolite
triacsin C almost completely inhibits de novo synthesis of
triacylglycerol and phospholipid from glycerol, but does not prevent
phospholipid synthesis via reacylation of lysophospholipids with oleate
or arachidonate (16). Similar results are observed in rat hepatocytes
in which triacsin C blocks de novo synthesis of TAG 73%,
but inhibits [14C]oleate incorporation into phospholipid
and CE, or metabolism via These differing effects of ACS inhibitors on metabolic end products of
acyl-CoA-dependent pathways suggest the presence of inhibitor-sensitive and -resistant ACSs that are linked to specific pathways. If so, ACS isoforms might play a critical role in
partitioning acyl-CoAs toward lipid synthesis versus
oxidation. In order to determine whether this hypothesis was correct,
we needed to examine the effects of the ACS inhibitors on each of the
three ACS isoforms expressed in liver and fat cells. To do this, we
expressed ACS1, ACS4, and ACS5 as recombinant ACS-Flag fusion proteins,
and purified each ACS by Flag antibody column chromatography.
Materials--
RT-for-PCR kit and Advantage 2 PCR kit were
purchased from CLONTECH. Bacterial Flag expression
kit including pFlag-CTC vector, Flag M2 affinity column, and Flag
peptides, aminophenylboronate, phenylglyoxal,
N-ethylmaleimide, coenzyme A, ATP, palmitate, xenobiotic carboxylic acids, and Triton X-100 were from Sigma. HMS174(DE3) and
pET21a(+) expression vector were from Novagen.
[9,10-3H]Palmitic acid was from PerkinElmer Life
Sciences. Triacsin C was from Biomol. The thiazolidinediones, GW1929
and GW4647 were a gift from Dr. Steven Jacobs, GlaxoSmithKline.
Construction of Recombinant pACS-Flag Plasmids--
cDNA was
synthesized from rat liver total RNA and used as a template for
amplification of the ACS open reading frames
(CLONTECH, RT-for-PCR kit). The primers for
amplification of ACS1, ACS4, and ACS5 were designed to include the
entire open reading frames based on nucleotide sequences obtained from
the GenBankTM data base (accession nos. D90109, D85189, and AB012933
for ACS1, ACS4, and ACS5, respectively) and specific restriction sites. The upper primer for ACS1 was
5'-ACAGACTCGAGGAGGTCCATGAATTGTTCCGTA-3' (recognition site
indicated in boldface type) and the lower primer was
5'-CCTCGGTACCAATCTTGATGGTGGAGTACAG-3'. The upper and lower
primers for ACS4 were 5'-AAAAGCTTGCAAAGAGAATAAAGGCTAAGC-3' and 5'-TTGCGTCGACTTTGCCCCCATACATC-3'. The upper and lower primers for ACS5 were
5'-TATTCCTCGAGCTTTTTATTTTTAACTTCTTGTTTTC-3' and
5'-CTTGGTACCCTCTTCGATGCTCATAGAG-3'. ACS amplification was
performed by PCR with the designed primers and Advantage®2
PCR kit (CLONTECH). The amplified ACS1 PCR product
was digested with XhoI and Acc65I and ligated
into pFlag-CTC vector (Sigma) digested with the same restriction
enzymes. The ACS4 PCR product and the pFlag-CTC vector were digested
with HindIII and SalI and then ligated. To obtain
ACS4 without the Flag sequence, a primer was designed by adding a stop
codon in the position just before the Flag sequence. The upper primer
used was the same as for ACS4-Flag fusion protein, and the newly
designed lower primer was
5'-ACAGTGTCGACTTATTTGCCCCCATACAT-3' (stop codon is
underlined). The pFlag-ACS plasmid constructs contain a tac
promoter, a ribosome binding site, the entire coding region of each ACS
isoforms, and C-terminal Flag sequences with pBR322 origin. For ACS5,
the pET-Flag vector was used. The plasmid pET-Flag was constructed by
cloning the SphI (Klenow-filled)-NheI fragment from pET-21a(+) (Novagen) into pFlag-CTC (Sigma) digested with BamHI, treated with Klenow, and then digested with
NheI. In the pET-Flag vector, the tac promoter
region in pFlag-CTC was replaced with the T7 promoter region from
pET21a(+) (Novagen). ACS5 PCR products and pET-Flag vector were
digested with XhoI and Acc65I and then ligated.
The sequences of ACS-Flag fusion constructs were verified by the
University of North Carolina Automated Sequencing Facility.
Expression of Recombinant ACS1- and ACS4-Flag Proteins in
Escherichia coli--
Recombinant ACS1-Flag, ACS4-Flag, and ACS4 were
expressed in E. coli JM109 after induction with 1 mM IPTG at an A600 of 1.0. JM109 was
grown in Terrific Broth (Life Technologies, Inc.) supplemented with
ampicillin (60 µg/ml) at 30 °C and shaken at 250 rpm. After a 12-h
induction, cells were harvested by centrifugation at 5,000 rpm for 10 min in a Sorvall HS-4 rotor. The cell pellet was resuspended in 10 ml
of 10 mM Tris (pH 7.4), 0.5 mM EDTA (TE)
buffer. The resuspended cells were incubated with 100 µg/ml lysozyme
for 30 min on ice and then sonicated with six 10-s bursts, each
followed by a 10-s rest on ice. Cellular debris was removed from the
cell lysates by centrifugation at 3,000 × g for 10 min. Part of the supernatant was saved (cell extract), and the
remainder was layered over a 2-ml cushion of 55% (w/w) sucrose topped
with 0.5 ml of 5% (w/w) sucrose in TE buffer. After centrifugation in
a Beckman SW41 rotor at 35,000 rpm for 3 h, the supernatant was
removed (soluble fraction). The membrane band at the interface was
collected with a 19-gauge needle and syringe. Protein concentrations
were determined by the BCA method (Pierce) with bovine serum albumin as
the standard.
Purification of the Recombinant ACS1- and ACS4-Flag
Proteins--
ACS1-Flag and ACS4-Flag were purified by Flag M2 column
chromatography. The Flag M2 antibody affinity matrix (1 ml) (Sigma) was
activated with 0.1 M glycine (pH 3.5), 50 mM
Tris (pH 7.4), and 150 mM NaCl (TBS) buffer. JM109 membrane
fractions containing overexpressed ACS1-Flag or ACS4-Flag were
solubilized in TBS containing 1% Triton X-100 and passed over the
column four times. The column was washed three times with 12 ml of TBS
(pH 7.4), and then eluted with five 1-ml aliquots of 100 µg/ml Flag
peptide (Sigma) dissolved in TBS buffer (pH 7.4). Eluates were run on a
10% polyacrylamide gel containing 1% SDS and stained with Gel
Code® blue stain reagent (Pierce). ACS4 and ACS1 were
purified to near homogeneity and migrated as single bands. Compared
with the cell extracts the activities of purified proteins increased
19-fold for ACS1 and 10-fold for ACS4.
Expression and Purification of Recombinant ACS5-Flag
Protein--
Unlike ACS1- or ACS4-Flag, ACS5-Flag protein could not be
expressed under the tac promoter in JM109 or DH5 Assay for ACS Activity--
The ACS assay contained 50 µM [3H]palmitate in Triton X-100, 1 µM EDTA, 10 mM ATP, 250 µM CoA,
175 mM Tris (pH 7.5), 8 mM MgCl2, and 5 mM dithiothreitol in a total of 200 µl (20, 21).
The maximum concentration of Triton X-100 in the assay was 0.03% (0.5 mM). All comparisons of the recombinant ACS isoenzymes
employed identical concentrations of substrates and Triton X-100. The
reaction was initiated with 0.1 µg of protein for ACS1 and ACS4, and
0.5-1.0 µg of protein for ACS5. ACS activity was measured after a
5-min incubation at 37 °C, except for the inhibition kinetic
studies, which were only incubated for 1 min (20). Substrate
concentrations and time allowed measurement of initial rates. The
specific activities of recombinant ACS1-, ACS4-, and ACS5-Flag proteins
from different preparations were in the ranges of 939-1472,
1670-2200, and 98-166 nmol/min/mg, respectively. All the experiments
were repeated three times, and virtually identical results were
obtained from each experiment.
Expression and Purification of Recombinant ACS-Flag
Proteins--
Recombinant rat ACS1, ACS4, and ACS5 had previously been
purified by column chromatography (10-12). To obtain purified ACS isoenzymes more efficiently, we designed plasmid constructs to produce
ACS proteins with Flag epitopes at their C termini. After induction
with IPTG, 10% of the ACS activity was found in the soluble fraction.
This soluble activity is surprising because ACS4 is an integral
membrane protein, which remains associated with rat liver microsomes
after washing with 0.5 M KCl (data not shown).
Nevertheless, soluble ACS4 (21%) was also obtained when ACS4 was
overexpressed in DH5 Characterization of ACS1, ACS4, and ACS5--
To compare the
purified ACS isoenzymes, we studied the kinetics of the three
substrates required for ACS catalysis. The three ACS isoenzymes had
similar apparent Km values for CoA between 2.4 and
6.4 µM and for palmitic acid between 5.0 and 8.6 µM (Table I). ACS4, on the
other hand, had an apparent Km value for ATP that
was almost 20-fold lower than that of ACS1 or ACS5 (34 µM
versus 649 and 666 µM). To test sensitivity to heating, ACS isoenzyme activities were measured after heating at
43 °C for various times (Fig. 1). ACS1
was most stable to heat with a half-life of about 10 min whereas ACS4
had a t1/2 shorter than 2 min, and ACS5
had a t1/2 of ~4 min. To find the
optimum pH for each ACS isoenzyme, MES was used as a buffer for ACS
reaction in the range of pH 6-7, Tris for pH 7-9, and glycine for pH
9 and 9.5. ACS1 and ACS4 had broad pH optima between pH 7.4 and 9.0, but the optima for ACS5 was more narrow (pH 7.4-8.0) (data not shown).
The three purified ACS isoenzymes were differently affected by Triton
X-100 (Fig. 2). ACS4 gradually lost
activity at Triton X-100 concentrations above 0.5 mM, the
concentration normally used for assay, whereas ACS1 maintained maximal
activity up to 1.5 mM Triton X-100. In contrast, ACS5
activity dramatically increased at Triton X-100 concentrations above
0.5 mM, and maintained maximal activity at 3 mM. ACS5 remained highly active even at 10 mM
Triton X-100 (data not shown).
Triacsin C Strongly Inhibited ACS1 and ACS4, but Did Not Affect
ACS5--
Triacsin C, an alkenyl-N-hydroxytriazene fungal
metabolite, was reported to be a potent competitive inhibitor of ACS
(22, 23). Studies in fibroblasts, HepG2 cells, and rat hepatocytes, however, suggest that triacsin C does not equally inhibit all the
pathways in which acyl-CoAs are used. To test the effects of triacsin C
on the purified ACS isoenzymes, triacsin C was added directly to the
reaction mixture (Fig. 3). Triacsin C
selectively and strongly inhibited ACS1 and ACS4 in a
dose-dependent manner, but had little effect on ACS5. The
IC50 for ACS1 and ACS4 was 4-6 µM.
Thiazolidinediones Are Specific Inhibitors of
ACS4--
Thiazolidinediones are oral antidiabetic drugs, generally
believed to act as insulin sensitizers through activation of PPAR
Troglitazone exhibited a mixed type inhibition of ACS4. With respect to
palmitate, the reciprocal plots intersected above the 1/palmitate axis
(Fig. 5A) and the
Ki was 0.1 µM as calculated from the
Km/vmax versus
inhibitor replot (Fig. 5B). Since the 1/v replot
intersected with the Km/vmax replot near the y axis, we can characterize the system as
exhibiting linear non-competitive inhibition. Linear mixed inhibition
also arises when an inhibitor binds at mutually exclusive sites.
Therefore, we examined troglitazone inhibition kinetics of CoA and ATP,
the other two substrates of ACS4. Troglitazone exhibited partial
uncompetitive inhibition with respect to CoA (Fig. 5C). For
ATP, the reciprocal plots intersected below the 1/palmitate axis (Fig.
5D), another linear mixed-type inhibition system.
Non-thiazolidinedione PPAR N-Ethylmaleimide Strongly Inhibited ACS4 and Weakly Inhibited ACS1,
but Had No Effect on ACS5--
To further characterize the ACS
isoenzymes, the effects of amino acid-reactive compounds were
investigated. Before assay, each purified ACS isoenzyme was
pre-incubated with various concentrations of NEM, a sulfhydryl-reactive
compound, for 10 min on ice (Fig. 6). NEM at 3 mM inhibited
ACS4 and ACS1 activities 83% and 25%, respectively, but had no effect
on ACS5, indicating that amino acids containing a sulfhydryl group were
more critical for ACS4 catalysis compared with ACS1 or ACS5 (Fig. 5).
After a 10-min preincubation, the arginine-reactive compound,
phenylglyoxal, at 25 mM inhibited ACS1 38%, ACS4 78%, and
ACS5 65%, suggesting the importance to each isoform of an arginine
residue for catalysis (data not shown).
m-Aminophenylboronate (0-20 mM), a
serine-reactive compound, however, had little effect on ACS1, ACS4, and
ACS5 (data not shown).
We focused on ACS1, ACS4, and ACS5 because these three isoforms
are present in liver, a tissue that metabolizes long chain fatty acids
via a variety of synthetic and degradative pathways. To characterize
ACS1, ACS4, and ACS5 and test the effects of inhibitors, we purified
each ACS isoform as a ACS-Flag fusion protein after confirming that the
Flag sequence at the C terminus does not alter enzymatic kinetic
properties. The ACS5-Flag fusion protein was unique with regard to the
conditions required for its expression, purification, and full recovery
of activity. Although others have expressed ACS5 without reported
difficulty in DH5 The three purified ACS isoenzymes had similar apparent
Km values for CoA and palmitic acid. The apparent
Km for palmitic acid of ACS4 in this study is more
than 10-fold lower than the one reported previously (12), probably
because the concentrations of palmitic acid we used to study kinetics
ranged from 0.33 to 2.0 of the Km, as recommended
(30). ACS4 had approximately a 20-fold higher affinity for ATP than did
ACS1 or ACS5. If these isoenzymes compete for the same pool of ATP, ACS4 might remain active during fasting or when cellular energy levels
are low. On the other hand, however, the subcellular location of ACS4
and its increase after refeeding (31) suggest that ACS activities are
not simply dependent on substrate availability.
Despite their striking homology, the three ACS isoforms exhibited
marked differences in their responses to heating and amino acid-modifying reagents. ACS4 was the most sensitive isoform when heated at 43 °C. ACS1 and ACS4 had broad pH optima from pH 7.4 to pH
9.0, whereas the optimum for ACS5 was from pH 7.4 to pH 8.0. ACS4 was
more severely inhibited by phenylglyoxal than were ACS1 or ACS5,
suggesting that arginine residues may be more critical for ACS4
activity. The marked inhibition of only ACS4 by NEM suggests that
sulfhydryl group-containing amino acids may also be important. On the
other hand, none of the isoforms was affected by
m-aminophenylboronate, indicating that serine is not an
essential residue for catalysis. Taken as a whole, the responses to
site-specific inhibitors probably reflect the relative similarity of
ACS1 and ACS5 in amino acid sequence and the relative dissimilarity of ACS4.
ACS inhibitors such as triacsin C or troglitazone differentially affect
metabolic end products of ACS-dependent pathways (16-19). Thus, we hypothesized that pathways using acyl-CoA substrates might be
initiated by distinct ACS isoforms and that the different degree of
inhibition of TAG, phospholipid, and CE synthesis, or fatty acid
Troglitazone at 10 µM did not alter ACS1 activity, mildly
inhibited ACS5 activity, and almost completely blocked ACS4 activity. Thiazolidinediones appear to be highly specific inhibitors
of ACS4. Troglitazone exhibited mixed-type inhibition of ACS4 with respect to all three substrates. Troglitazone is believed to activate PPAR (PPAR
) ligands, troglitazone,
rosiglitazone, and pioglitazone, strongly and specifically inhibited
only ACS4, with an IC50 of less than 1.5 µM.
Troglitazone exhibited a mixed type inhibition of ACS4.
-Tocopherol,
whose ring structure forms the non-TZD portion of troglitazone, did not
inhibit ACS4, indicating that the thiazolidine-2,4-dione moiety is the
critical component for inhibition. A non-TZD PPAR
ligand, GW1929,
which is 7-fold more potent than rosiglitazone, inhibited ACS1 and ACS4
poorly with an IC50 of greater than 50 µM,
more than 100-fold higher than was required for rosiglitazone, thereby
demonstrating the specificity of TZD inhibition. Further, the PPAR
ligands, clofibrate and GW4647, and various xenobiotic carboxylic acids
known to be incorporated into complex lipids had no effect on ACS1, -4, or -5. These results, together with previous data showing that triacsin
C and troglitazone strongly inhibit triacylglycerol synthesis compared
with other metabolic pathways, suggest that ACS1 and ACS4 catalyze the
synthesis of acyl-CoAs used for triacylglycerol synthesis and that lack
of inhibition of a metabolic pathway by triacsin C does not prove lack
of acyl-CoA involvement. The results further suggest the possibility
that the insulin-sensitizing effects of the thiazolidinedione drugs
might be achieved, in part, through direct interaction with ACS4 in a
PPAR
-independent manner.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-oxidation, glycerolipid synthesis, cholesteryl ester (CE) synthesis, desaturation, elongation, and protein
acylation and can serve as signaling molecules (2-4). Although ACS was
first believed to be a constitutive enzyme because the activity in
liver was not altered by changes in nutritional status or hormonal
stimuli (5-9), the cloning of ACS1 from rat liver in 1990 disclosed
that hepatic ACS1 mRNA expression is sensitively regulated by
fasting and refeeding as well as by specific nutrients provided as the
energy source (10). Later, four additional rat ACS isoforms from
different genes were cloned that differed in tissue distribution of
their mRNA expression and in substrate preference (11-14). Of
these, ACS2 and ACS3 mRNAs are abundantly expressed in brain, but
are not detected in liver (13, 14). The mRNAs of ACS4 and ACS5 are
highly expressed in steroidogenic tissues and in intestine,
respectively, and are also present in liver (11, 12). ACS1 and ACS5
have a broad substrate specificity for saturated fatty acids of 12-18
carbon atoms and unsaturated fatty acids of 16-20 carbon atoms. In
contrast, ACS4 has a marked preference for arachidonic acid and
eicosapentaenoic acid. The presence of three ACS isoforms in liver
suggests that liver ACS activity does not change with physiological
alterations (5-9) because the different ACS isoenzymes compensate for
each other.
-oxidation only 30-40% (17). In HepG2
cells, triacsin D inhibits oleate-induced TAG synthesis 80% without
affecting CE synthesis (18), and in rat primary hepatocytes,
troglitazone at 100 µM inhibits [14C]oleate
incorporation into TAG and oxidation products 50% and 20%,
respectively, whereas incorporation into phospholipid remains unaffected (19). The sulfo-conjugate of troglitazone appeared to be the
likely inhibitory metabolite (19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. To
express ACS5-Flag, we used the pET-Flag vector because the T7 promoter
regulates the expression of foreign proteins more tightly than does the tac promoter. Overexpressed ACS5-Flag was present in the
membrane fraction of HMS174(DE3). ACS5-Flag was maximally expressed at 30 °C after a 12-h induction with 1 mM IPTG beginning at
an A600 of 0.4 in HMS174(DE3), which provides
the T7 polymerase. Purification of active ACS5-Flag was similar to that
described for ACS1 and ACS4, except that 0.1% Triton X-100 was
required in the elution buffer that contained 100 µg/ml Flag peptide.
When Triton X-100 was omitted, only inactive ACS was eluted. ACS5-Flag
migrated as a single band on a 10% polyacrylamide gel containing 1%
SDS and stained with Gel Code® blue stain reagent.
Compared with the cell extracts, the activities of purified proteins
increased 29-fold for ACS5. The purification yield was relatively low
because excess protein applied to the column was eluted in the
flow-through fraction and during the washing procedure.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(12). ACS1- and ACS5-Flag were also
overexpressed and identified in the membrane fraction. Recombinant ACSs
were purified to near homogeneity by one-step Flag-antibody affinity
chromatography. To ensure that the Flag epitope did not alter ACS
function, we compared ACS activity in membrane fractions from JM109
that overexpressed ACS4 or ACS4-Flag. The kinetic constants for
palmitic acid, CoA, and ATP were virtually identical, and the
sensitivity to heating at 43 °C was similar, indicating that the
Flag epitope at the C terminus does not alter the catalytic properties
of ACS and supports the use of ACS-Flag recombinant proteins for
comparative study (data not shown).
Kinetic constants for ACS1, ACS4, and ACS5
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Fig. 1.
ACS1-, ACS4-, and ACS5-Flag proteins differ
in thermolability. Purified ACS1-Flag ( ) and ACS4-Flag (
)
(each 4 µg/ml) and ACS5-Flag (
) (40 µg/ml) were heated at
43 °C in TE buffer. Samples containing 0.1 µg of protein were
removed at the times indicated for immediate assay at 37 °C. Initial
activities were 873 for ACS1, 1491 for ACS4, and 97 nmol/min/mg for
ACS5.
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Fig. 2.
Triton X-100 activates ACS5. The
activities of purified ACS1-Flag ( ) and ACS4-Flag (
) (each 0.1 µg) and ACS5-Flag (
) (1 µg) isoenzymes were measured in the
presence of different concentrations of Triton X-100. ACS activity at
0.5 mM Triton X-100, the concentration regularly used in
the assay, represents 100%. Activities with 0.5 mM Triton
X-100 were 1340 for ACS1, 2220 for ACS4, and 100 nmol/min/mg for
ACS5.
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Fig. 3.
Triacsin C inhibits ACS1 and ACS4.
Purified ACS1-Flag ( ) and ACS4-Flag (
) (each 0.1 µg) and
ACS5-Flag (
) (0.5 µg) were assayed for ACS activity in the
presence of varying concentrations of triacsin C dissolved in
Me2SO (2.5% of final assay reaction). The values are from
an experiment that is representative of three independent
determinations. Activities in the absence of triacsin C were 763 for
ACS1, 1378 for ACS4, and 63 nmol/min/mg for ACS5.
(24-26). Because the sulfo-conjugate of troglitazone was reported to
directly inhibit ACS activity in both microsomes and mitochondria from
rat liver (19), we tested purified recombinant ACS1, ACS4, and ACS5 to
determine which isoform was inhibited by thiazolidinediones. Troglitazone at 1 µM decreased ACS4 activity 50%; with
10 µM troglitazone, the activity was almost completely
blocked (Fig. 4A). In
contrast, ACS1 and ACS5 activities were unaffected by troglitazone at
10 µM. Because troglitazone contains the ring structure
of
-tocopherol linked to the thiazolidinedione moiety, we determined
which part of the molecule was critical for inhibition by testing the
effect of
-tocopherol itself and of other thiazolidinediones on each of the purified ACS isoenzymes.
-Tocopherol had little effect on
three ACS isoforms (Fig. 4B). On the other hand, both
pioglitazone and rosiglitazone, which contain the common
thiazolidinedione group but lack the
-tocopherol moiety, showed
virtually the same effect as troglitazone on the three ACS isoenzymes.
At 10 µM both drugs inhibited ACS4 85-95%, inhibited
ACS5 less than 20%, and did not alter ACS1 activity (Fig. 4,
C and D). At concentrations up to 10 µM, troglitazone had little effect on ACS5 activity, even
after full activation by 3 mM Triton X-100 (data not
shown). Of the thiazolidinediones, rosiglitazone was the strongest
inhibitor of ACS4. The IC50 was 0.5 µM for
rosiglitazone and 1.5 µM for troglitazone and
pioglitazone (Table II). These data
indicate that the thiazolidine-2,4-dione moiety is the critical moiety for ACS4 inhibition.
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Fig. 4.
Thiazolidinediones inhibit ACS4.
Purified ACS1-Flag ( ) and ACS4-Flag (
) (each 0.1 µg) and
ACS5-Flag (
) (1 µg) were assayed for ACS activity. Troglitazone,
pioglitazone, rosiglitazone, and
-tocopherol were dissolved in
Me2SO (2.5% of final assay reaction). The values shown are
from an experiment that is representative of two independent
determinations. A, troglitazone; B,
-tocopherol; C, pioglitazone; D,
rosiglitazone.
Effect of PPAR and PPAR
activators and of selected xenobiotic
carboxylic acids on purified ACS1-, ACS4-, and ACS5-Flag activities
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Fig. 5.
Troglitazone is a mixed-type inhibitor of
ACS4. Purified ACS4-Flag (0.1 µg) was assayed for ACS activity
for 1 min at 37 °C. Troglitazone was dissolved in Me2SO
(DMSO; 2.5% of final assay reaction). A,
Lineweaver-Burk plot with palmitate; B, replots of
Km/vmax and
1/vmax versus troglitazone;
C, Lineweaver-Burk plot with CoA; D,
Lineweaver-Burk plot with ATP.
and PPAR
Activators Do Not Inhibit
ACS4--
N-(2-Benzoylphenyl)L-tyrosine
(GW1929) is a newly identified non-thiazolidinedione PPAR
activator
with a high affinity for human PPAR
(27). The glucose-lowering
effect of GW1929 in rats is 100-fold more potent than that of
troglitazone (27). In contrast, however, GW1929 was a poor inhibitor of
ACS4 with an IC50 of 50 µM versus
1.5 µM for the TZDs) (Table II). PPAR
activators such as clofibric acids or GW4647 up to 50 µM had no effect on
ACS1, ACS4, or ACS5 (Table II) despite the ability of PPAR
ligands to improve insulin sensitivity and reduce adiposity (28). We also
tested a variety of xenobiotic carboxylic acids, which contain hydrophobic and carboxylic acid moieties, are known to be incorporated into complex lipids, and either resemble natural fatty acids or are
aromatic derivatives of short chain fatty acids (29). The long chain
cyclopropane (bridged) analogue of oleic acid,
(±)-cis-9,10-methylene octadecanoic acid, inhibited ACS1
and ACS4 activities 50% at 25 and 30-40 µM,
respectively, but had no effect on ACS5. Other xenobiotic carboxylic
acids, 3-phenoxybenzoic acid, p-coumaric acid, ibuprofen, ferulic acid, and firefly luciferin, at concentrations up to 50 µM had no effect on three ACS isoenzymes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
with a vector containing the lac
promoter (11), our successful ACS5 expression required a vector
containing the T7 promoter, suggesting that the ACS5 protein may be
toxic to E. coli since the T7 promoter-driven vector
suppresses the expression of foreign proteins in uninduced states more
completely. In addition, elution of active ACS5-Flag from the Flag
antibody column required the presence of 0.1% Triton X-100 in the
elution buffer, whereas active ACS1 and ACS4 could be eluted without
adding detergent. Furthermore, the loss of 50% of ACS5 activity,
merely in the process of obtaining bacterial membranes, suggests that
detergent might be helpful at every stage of preparation. Although 0.5 mM Triton X-100 is regularly used in the ACS assay, we
found that maximal activation of ACS5 demanded an ~6-fold higher
concentration of Triton X-100. Thus, optimal assay conditions required
the presence of ~500 Triton X-100 micelles/molecule of ACS1 or ACS4,
and about 3000 micelles/molecule of ACS5. The critical micellar
concentration of Triton X-100 is 0.3 mM, and, although it
remains unclear why ACS5 requires such a large number of Triton X-100
micelles, the detergent may be essential to reconstitute or stabilize
purified ACS5 or to relieve product inhibition by palmitoyl-CoA. In
order to be consistent in our comparisons, we used identical
concentration of Triton X-100 (0.5 mM) and substrates to
assay all recombinant ACS isoenzymes.
-oxidation by ACS inhibitors might result if each ACS isoform had a
different sensitivity to the two inhibitors. If this were true, ACS
isoforms could play a critical role in partitioning their products
toward specific pathways through independent regulation of each ACS
isoform. This hypothesis was supported by our data. The current study
using purified ACS isoenzymes demonstrates that ACS isoforms can be
distinguished by their sensitivity to ACS inhibitors. Triacsin C had
little effect on ACS5, but strongly inhibited ACS1 and ACS4 activities
in a dose-dependent manner. Because studies with cultured
cells show that triacsin C strongly blocks de novo synthesis
of TAG and has little affect on phospholipid reacylation or CE
synthesis, or on
-oxidation (16-18), ACS1 and ACS4 may be closely
linked to TAG synthesis.
-Tocopherol was not an inhibitor, whereas rosiglitazone and
pioglitazone were potent ACS4-specific inhibitors and had little affect
on ACS1 or ACS5. Thus, the critical component for ACS4 inhibition appears to be the thiazolidine-2,4-dione moiety rather than the
-tocopherol ring. It is most likely that ACS4 inhibition by
thiazolidinediones is more closely related to TAG synthesis rather than
to
-oxidation for several reasons. 1) ACS4 protein was most
prominently located in the mitochondrial-associated membrane (31),
which exhibits the highest specific activities for enzymes of TAG
synthesis (32), and was not detected in mitochondria (31). 2) After a
48-h fast followed by 24-h refeeding with chow or sucrose (69%),
hepatic ACS4 protein increased 150% (31). 3) The preference of ACS4 for arachidonic acids is consistent with only a weak link to
-oxidation because arachidonic acid is among the least preferred
fatty acids for
-oxidation during fasting states (33).
because it mimics a fatty acid; therefore, we postulated that
troglitazone might compete with palmitate in the acyl-CoA ligation
reaction. We found that troglitazone displayed linear mixed-type
inhibition (Fig. 5A). This
could arise because the ACS4-troglitazone complex has a lower affinity
than ACS4 for palmitate and the enzyme-substrate(s)-inhibitor complex
is nonproductive. Another possibility is that troglitazone interacts
at more than one site, so we examined troglitazone inhibition
kinetics of ACS4 with CoA and ATP. Troglitazone is a partial
uncompetitive inhibitor of ACS4 with respect to CoA (Fig.
5C), indicating that the affinity for CoA of the
ACS4-troglitazone complex increases by the same degree as the inhibitor
decreases vmax. For ATP, troglitazone displayed
linear mixed-type inhibition with the reciprocal plots intersecting
below the 1/palmitate axis (Fig. 5D), suggesting that the
ACS4-troglitazone complex has an increased affinity for ATP, but that
the enzyme-substrate(s)-inhibitor complex forms product more slowly
than the enzyme-substrate(s) complex. It should be noted that these
types of inhibition kinetic systems were initially characterized using
soluble single substrate enzymes (30), whereas we are extrapolating
them to apply to a membrane-bound enzyme, ACS4, that has three
substrates, only two of which are soluble.
View larger version (22K):
[in a new window]
Fig. 6.
N-Ethylmaleimide inhibits
ACS4. The reaction was initiated by adding ACS1-Flag ( ),
ACS4-Flag (
) (each 0.1 µg), and ACS5-Flag (
) (1 µg) that had
been incubated with different concentrations of NEM for 10 min on ice.
The proteins were preincubated with NEM dissolved in 10 mM
Tris, and 0.5 mM EDTA (pH 7.4) buffer. The values are from
an experiment that is representative of two independent determinations.
Uninhibited activities were 1159 for ACS1, 2477 for ACS4, and 113 nmol/min/mg for ACS5.
GW1929, a potent non-thiazolidinedione PPAR activator, was a weak
inhibitor of both ACS1 and ACS4, with an IC50 value for ACS4 more than 30-fold higher than that observed with
thiazolidinediones. ACS4 was not affected by the PPAR
activators
clofibric acid and GW4647 at concentrations up to 50 µM,
even though these compounds, like the thiazolidinediones, can markedly
lower hyperinsulinemia and hyperglycemia in diabetic animal models
(28). Furthermore, ACS4 was unaffected by a variety of xenobiotic
carboxylic acids that contain hydrophobic rings and are known to be
incorporated into complex lipids.
Although we did not test the effects of troglitazone O-sulfate (TOS), a major troglitazone metabolite, 100 µM TOS was reported to inhibit ACS activity 80% in both microsomes and mitochondria from rat hepatocytes, whereas troglitazone itself had no effect (19). The purity of the membrane fractions used in the reported study was not evaluated. We found that troglitazone at 10 and 50 µM inhibited ACS activity 25% and 50%, respectively, in purified mitochondria-associated membrane fractions (31). The reported poor ability of troglitazone to inhibit ACS activity (19) probably occurred because the membrane fractions used in that study contained relatively little ACS4, but may have contained other ACS isoforms that were sensitive to high concentrations of TOS or to an inhibitory factor elicited by TOS.
Thiazolidinediones are powerful oral antidiabetic drugs that act as
insulin sensitizers. Although it is believed that their efficacy occurs
via activation of PPAR (24-26), some evidence suggests that some
thiazolidinedione actions can occur in a PPAR
-independent manner. In
PPAR
-deficient heterozygous mice, for example, glucose disposal rate
and suppression of hepatic glucose production are greater than in
normal mice, the reverse of what one would expect if thiazolidinediones
ameliorate insulin resistance by activating PPAR
(34). Further, some
compounds exhibit potent antidiabetic effects despite their weak
activation of PPAR
(35). Troglitazone and rosiglitazone decrease
serum triacylglycerol even in aP2/DTA mice, which lack adipose
deposits, a main site for PPAR
action (36-38); troglitazone has
acute effects in rats both on glucose disposal and on hepatic glucose
production (39); finally, TZDs can decrease the expression of tumor
necrosis factor-
and interleukin-6 in macrophages that contain no
PPAR
(40). Our results suggest that ACS4 could contribute to the
insulin-sensitizing and anti-inflammatory effects of the
thiazolidinediones through direct interaction with ACS4 in a
PPAR
-independent manner.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. David G. Klapper for assistance in synthesizing the peptide for the ACS4 antibody and Ping Wang and AnneMarie Earnhardt for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants DK56598 (to R. A. C.) and HD08431 (to T. M. L.) from the National Institutes of Health and by a grant from GlaxoWellcome.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: Rosalind A. Coleman,
CB 7400, Depts. of Nutrition and Pediatrics, University of North
Carolina, Chapel Hill, NC 27599-7400. Tel.: 919-966-7213; Fax:
919-966-7216; E-mail: rcoleman@unc.edu.
Published, JBC Papers in Press, April 23, 2001, DOI 10.1074/jbc.M010793200
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ABBREVIATIONS |
---|
The abbreviations used are:
ACS, acyl-CoA
synthetase;
CE, cholesterol ester;
IPTG, isopropyl-1-thio--D-galactopyranoside;
NEM, N-ethylmaleimide;
PCR, polymerase chain reaction;
PPAR, peroxisome proliferator-activated receptor;
TAG, triacylglycerol;
TOS, troglitazone O-sulfate;
TZD, thiazolidinedione;
TBS, Tris-buffered saline;
MES, 4-morpholineethanesulfonic acid.
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