Departments of Nutrition and Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7400
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ABSTRACT |
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Acyl-CoA synthetase (ACS) catalyzes the
activation of long-chain fatty acids to acyl-CoAs, which can be
metabolized to form CO2, triacylglycerol (TAG),
phospholipids (PL), and cholesteryl esters (CE). To determine whether
inhibiting ACS affects these pathways differently, we incubated rat
hepatocytes with [14C]oleate and the ACS inhibitor
triacsin C. Triacsin inhibited TAG synthesis 70% in hepatocytes from
fed rats and 40% in starved rats, but it had little effect on oleate
incorporation into CE, PL, or -oxidation end products. Triacsin
blocked [3H]glycerol incorporation into TAG and PL 33 and
25% more than it blocked [14C]oleate incorporation,
suggesting greater inhibition of de novo TAG synthesis than
reacylation. Triacsin did not affect oxidation of prelabeled
intracellular lipid. ACS1 protein was abundant in liver microsomes but
virtually undetectable in mitochondria. Refeeding increased microsomal
ACS1 protein 89% but did not affect specific activity. Triacsin
inhibited ACS specific activity in microsomes more from fed than from
starved rats. These data suggest that ACS isozymes may be functionally
linked to specific metabolic pathways and that ACS1 is not associated
with
-oxidation in liver.
triacylglycerol; -oxidation; triacsin
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INTRODUCTION |
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ACTIVATION OF FATTY
ACIDS by acyl-CoA synthetase (ACS; EC 6.2.1.3) provides acyl-CoA
that can be partitioned toward -oxidation or toward the synthesis of
esterified lipids such as phospholipids (PL), cholesterol esters (CE),
and triacylglycerol (TAG). Dysregulation of fatty acyl-CoA partitioning
between oxidative and biosynthetic pathways is linked to metabolic
disorders such as obesity and diabetes. In these diseases acyl-CoAs are
preferentially esterified, thereby leading to TAG accumulation in a
variety of tissues, including adipose, muscle, liver, and pancreas
(19, 31). Increased TAG content in these tissues is
strongly associated with insulin resistance and hyperlipidemia
(12, 28, 32, 35). Thus an understanding of how acyl-CoAs
are selectively used by different metabolic pathways may provide
insight into the pathobiology of disordered energy homeostasis.
Rather than being equally accessible to all enzymes that use acyl-CoA, acyl-CoAs may be channeled into distinct acyl-CoA pools that are functionally linked to specific pathways. This hypothesis is mainly supported by studies that have evaluated the metabolism of long-chain fatty acids in cells exposed to triacsins, a family of competitive inhibitors of ACS (34). In fibroblasts, triacsin C completely blocks de novo synthesis of TAG and PL without inhibiting the reacylation of lysophospholipid (14), and in rat insulinoma cells, triacsin C inhibits de novo synthesis of cellular lipids by 83% (29). Similarly, in HepG2 human hepatoma cells, triacsin D inhibits TAG synthesis 60% without affecting the synthesis of CE (36). These studies strongly suggest that ACS inhibitors have distinct inhibitory effects on different pathways that metabolize acyl-CoAs.
In these studies, the majority of acyl-CoAs were esterified into TAG.
In primary hepatocytes, however, acyl-CoA use is more equally
distributed among diverse pathways, including -oxidation and the
synthesis of lipids secreted in very low-density lipoproteins (VLDL)
(37). Furthermore, in liver, acyl-CoA partitioning among these pathways is regulated nutritionally. Starvation increases the
rates of
-oxidation and ketogenesis (26), whereas
refeeding a high-sucrose diet stimulates TAG synthesis and VLDL
secretion (11). Thus primary hepatocytes provide an ideal
model in which to study regulated trafficking of acyl-CoAs into
oxidative and storage pathways. In the present study, we investigated
the effects of triacsin C on long-chain fatty acyl-CoA metabolism in
hepatocytes that were isolated from either fed or starved rats. These
studies provide strong evidence that, in liver, acyl-CoAs are
functionally channeled toward specific pathways, and they suggest that
one mechanism responsible for selective trafficking of acyl-CoAs might involve regulated expression of ACS1.
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METHODS |
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Materials. Silica gel G plates were from Whatman. [2-3H]glycerol, [9,10-3H]palmitate, and [1-14C]oleate were from Amersham Life Sciences. Glycerol, BSA (essentially fatty acid-free), and sodium oleate were from Sigma. Lipid standards and sn-1,2-dioleoylglycerol were from Serdary. Triacsin C (>95% pure) was from Biomol Research Lab. Tissue culture supplies and fetal bovine serum (FBS) were from Life Technologies. Rat-tail collagen was from Sigma. Western blots were performed using polyvinylidene fluoride (PVDF) membranes from Bio-Rad, horseradish-peroxidase-conjugated goat anti-rabbit immunoglobulin G and a Supersignal chemiluminescence detection kit from Pierce, and 125I-labeled protein A from ICN Pharmaceuticals.
Hepatocyte isolation and incubations. Animal protocols were approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (200-250 g) were maintained on a 12:12-h light-dark cycle with free access to Purina rat chow. Before experiments, animals were given free access to food overnight, and hepatocytes were isolated at 0900; or food was removed at 0900 or 1700 so that rats were deprived of food for 8 or 24 h, respectively, before hepatocytes were isolated at 1500. Hepatocytes were isolated by collagenase perfusion (3). Cell viability, determined by trypan blue exclusion, exceeded 95%. Hepatocytes were seeded at a density of 200,000 cells per well into 24-well dishes pretreated with 0.01% rat-tail collagen and were cultured at 37°C in a humidified atmosphere of 5% CO2, in 1.0 ml of minimal essential medium (MEM), 10% heat-inactivated FBS, 50 U/ml penicillin, and 50 mg/ml streptomycin. After cells had been attached for 3-4 h, medium was replaced with serum-free MEM plus 10 nM dexamethasone and 0.1 mM nonessential amino acids, and hepatocytes were cultured overnight. Experiments were performed on the following morning in serum-free MEM containing 10 mM HEPES, 1.0 mM carnitine, 0.5% fatty acid-free BSA, and 0.2 mM sodium oleate with either [14C]oleate (0.8 mCi/mM) or 0.5 mM [3H]glycerol (1.0 Ci/mM) in the presence or absence of 5.0 or 10.0 µM triacsin C. Control cells were incubated with the dimethylsulfoxide vehicle (0.1% vol/vol). Sodium oleate was dissolved in H2O at 65°C and added to dry [14C]oleate. Then, 0.5% BSA (final medium concentration) in H2O was added. After 1 or 24 h, the medium was transferred to new dishes and assayed for labeled oxidation products [CO2 and acid-soluble metabolites (ASM)] (27). Hepatocytes were washed twice with 1% BSA in PBS at 37°C and scraped in two additions of 0.5 ml ice-cold methanol and 0.5 ml H2O. Then total cell lipids were extracted (4). For pulse-chase experiments, cells were attached to plates and incubated overnight in serum-free MEM (as above) and then in labeled medium containing 0.2 mM [14C]oleate (as above) for 24 h. Finally, cells were washed twice and incubated for an additional 24 h in medium that was identical except for containing 0 or 0.2 mM unlabeled sodium oleate in the presence or absence of 10 µM triacsin C. Media and cells were collected and analyzed as described above.
To determine whether hepatocyte sensitivity to triacsin C is affected by physiological conditions (nutritional status or media fatty acid concentration), hepatocytes were isolated from fed and 24-h-starved rats and incubated in serum-free medium overnight, as described in the previous paragraph. On the following day, cells were incubated for 24 h in medium containing 0.125-1.0 mM [14C]oleate (0.8 mCi/mM) in the presence or absence of 10 µM triacsin C.Lipid analysis. Aliquots of the lipid extracts were spotted on 0.25-mm silica gel G plates and chromatographed with hexane-diethyl ether-acetic acid (80:20:1; vol/vol/vol) (6) in parallel with authentic standards. Lipid products labeled with 3H or 14C were visualized using a BioScan Image 200 System (Washington, DC). The 3H-labeled spots were scraped into vials and counted in a liquid scintillation counter. The 14C-labeled spots were quantified by the BioScan 200 system.
Enzyme assays. Diacylglycerol acyltransferase (DGAT) was assayed with 200 µM sn-1,2-diolein and 25 µM [3H]palmitoyl-CoA; ACS was assayed with 5 mM ATP, 250 µM CoA, and 50 µM [3H]palmitate; and glycerol 3-phosphate acyltransferase (GPAT) was assayed with 300 µM [3H]glycerol 3-phosphate and 112.5 µM palmitoyl-CoA in the presence or absence of 2 mM N-ethylmaleimide to inhibit the microsomal isoform (5). Microsomal GPAT was estimated by subtracting the N-ethylmaleimide-resistant activity from the total. All assays measured initial rates.
ACS1 protein analyses. A 17-residue peptide corresponding to the NH2-terminal region of ACS1, which shows poor amino acid conservation among distinct ACS isozymes (15), was synthesized, purified, and coupled to keyhole limpet hemocyanin in the University of North Carolina/Program in Molecular Biology and Biotechnology Micro Protein Chemistry Facility. Rabbit antibodies to the peptide were raised commercially in New Zealand White rabbits (ImmunoDynamics, La Jolla, CA). Tissue samples were collected at 0900 from rats that were starved for 48 h or starved 48 h and then refed overnight. For Western blots, 100-200 µg of protein prepared from rat liver microsomes or mitochondria were separated by 8% SDS-PAGE, transferred to PVDF membranes, and then incubated overnight with serum from immunized rabbits. Proteins were visualized by horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G with a chemiluminescence Western blotting detection kit. Blots were subsequently incubated with 0.5 µCi 125I-labeled protein A, and protein quantification was performed using ImageQuant Phosphoimager software.
Other methods.
Microsomes and mitochondria were obtained by differential
centrifugation (9) and stored in aliquots at 80°C.
[3H]palmitoyl-CoA was synthesized enzymatically
(25). Protein was measured using BSA as the standard
(21). For each experiment, total cellular DNA content was
measured fluorometrically (20).
Statistics. Data are presented as means ± SD. Differences between control and triacsin-treated groups were analyzed by unpaired Student's t-test or two-way ANOVA at the P < 0.05 level.
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RESULTS |
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Effects of triacsin C on hepatocyte fatty acid partitioning.
When hepatocytes from rats starved for 8 h were incubated with 5 or 10 µM triacsin C for 24 h, the incorporation of
[14C]oleate into total lipid decreased 55% (Fig.
1A). Triacsin's effect differed markedly
for individual lipid species. The presence of 10 µM triacsin
inhibited oleate incorporation into TAG by 73% (Fig. 1A)
and diacylglycerol (DAG) by 78% (Fig. 1B), whereas
incorporation into PL and CE was inhibited only 34 and 44%,
respectively (Fig. 1B). Similarly, triacsin inhibited the
formation of labeled ASM, a measure of -oxidation, by only 33%
(>95% of labeled oxidation products were ASM) (Fig. 1A).
The presence of 5 µM triacsin had only a negligible effect on CE
formation and inhibited DAG only 50%, but it was similar to the effect
of 10 µM triacsin C on all other lipid metabolites.
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Effect of triacsin C on de novo glycerolipid synthesis and
recycling of endogenous lipids.
The incorporation of [14C]oleate into lipids measures de
novo glycerolipid synthesis from glycerol, reacylation of
lysophospholipids, esterification of cholesterol, and the DAG and
monoacylglycerol (MAG) products of TAG lipase, as well as reactivation
and use of hydrolyzed fatty acid. To examine de novo glycerolipid
synthesis alone, hepatocytes were incubated with
[3H]glycerol for 1 h. Hepatocytes isolated from the
same liver were simultaneously incubated with [14C]oleate
for direct comparison. Triacsin blocked glycerol incorporation into TAG
and PL 33 and 25% more than it blocked oleate incorporation (Fig.
2), suggesting less inhibition of
reacylation and/or that the activation of fatty acids hydrolyzed from
labeled TAG or PL stores might be less sensitive to triacsin C. Although we cannot exclude the possibility that triacsin-induced
differences in incorporation of [14C]oleate and
[3H]glycerol resulted from different effects on label
recycling and tracer dilution, recycling of 14C should have
been minimized by the short (1-h) incubation period.
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Effect of feeding status on triacsin's inhibition of fatty acid
metabolism.
To determine whether feeding status affects the ability of triacsin C
to inhibit fatty acid incorporation into different lipid species, we
examined [14C]oleate incorporation in hepatocytes from
fed and 24-h-starved rats (Fig. 4). In
the presence of 0.125 mM oleate, hepatocytes from fed rats incorporated
57% more oleate into TAG (Fig. 4A) and ~47% less oleate
into ASM (Fig. 4B), CE (Fig. 4C), and PL (Fig.
4D) than did cells from starved rats. Increasing oleate to
1.0 mM diminished nutrition-induced differences in hepatocyte synthesis
of complex lipids and markedly enhanced oxidation in hepatocytes from
fed rats. At all oleate concentrations, triacsin inhibited TAG
synthesis in hepatocytes from fed and starved rats by 70 and 40%,
respectively. In comparison, regardless of feeding status, triacsin C
had little effect on incorporation of 0.125 mM oleate into CE or PL.
Only when hepatocytes from starved rats were incubated with 1 mM fatty
acid did triacsin inhibit incorporation into CE (by 44%) (Fig.
4C). In contrast to the oxidation of endogenous fatty acid,
which was unaffected by medium fatty acid concentration (Fig. 3), the
amount of exogenous fatty acid incorporated into ASM varied strongly
with the oleate concentration in the media (Fig. 4B). In the
presence of 0.25 and 0.5 mM oleate, triacsin actually increased
incorporation into ASM in hepatocytes from starved rats, whereas in fed
rats, triacsin decreased incorporation into ASM only at high oleate
concentrations. Differences in partitioning between synthetic and
oxidative pathways are shown as a ratio of label incorporation into TAG
and ASM (Fig. 4E). When the fatty acid supply was low,
acyl-CoAs were preferentially esterified to TAG in fed hepatocytes but
were preferentially oxidized in starved hepatocytes. High
concentrations of oleate stimulated a shift toward TAG synthesis in
starved hepatocytes but stimulated production of ASM by fed
hepatocytes. Thus, in the presence of high (1.0 mM) oleate, hepatocytes
partitioned fatty acid equally between TAG and ASM. In the presence of
triacsin C, changes in nutritional status and medium oleate
concentration no longer altered partitioning between TAG synthesis and
oxidation.
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Analyses of ACS activity and protein abundance in
mitochondria and microsomal fractions.
Rat liver contains at least three ACS isozymes, two of which are known
to be nutritionally regulated (30, 33). To determine whether some of the changes observed in partitioning of fatty acids
between synthesis and oxidation might be related to differences in ACS
activity and/or expression in different subcellular organelles, we
measured total ACS activities and ACS1 protein abundance in mitochondrial and microsomal fractions (Table
1). In microsomes, the mitochondrial
isoform of GPAT contributed only 9% to the total specific activity,
thus showing little contamination of microsomes with mitochondrial
outer membrane (2). Microsomal GPAT contributed 13% to
the total GPAT specific activity in mitochondria, and the specific
activity of DGAT, another microsomal marker (2), was 19-fold higher in microsomes than in mitochondria, thus indicating only
a small degree of contamination of the mitochondrial fraction with
endoplasmic reticulum membrane. The specific activity of ACS was 57%
greater in microsomes than in mitochondria. By immunoblot analysis,
ACS1 protein was abundant in microsomes but barely detectable in
mitochondria, regardless of nutritional status (Fig.
5A). When 15% microsomal
contamination was present, a faint ACS1 band could be observed in some
mitochondrial preparations (data not shown). These results indicate
either that ACS1 is not present in mitochondria or that it contributes
only minimally to total mitochondrial ACS activity. In microsomes,
refeeding increased ACS1 protein abundance 89% (Fig. 5, B
and C), but neither fasting nor refeeding changed ACS
activity in either microsomes or mitochondria. When analyzed in
isolated subcellular fractions, triacsin's relative inhibition of
total ACS activity was greater in microsomes from fed compared with
starved rats (Fig. 6), consistent with
triacsin's potent inhibition of hepatocyte TAG synthesis in
hepatocytes from fed rats. Surprisingly, although triacsin did not
inhibit -oxidation in cultured hepatocytes (Fig. 4B),
triacsin inhibited ACS activity similarly in isolated mitochondria and
microsomes. These results suggest that liver mitochondria contain at
least one ACS isoform that is not directly coupled to
-oxidation.
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DISCUSSION |
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Acyl-CoAs are synthesized from long-chain fatty acids by ACSs, a
family of integral membrane proteins that vary in subcellular location,
substrate preference, and tissue distribution (15, 16).
Liver expresses at least three long-chain ACS isoenzymes, ACS1, ACS4,
and ACS5, and may express others that have yet to be identified
(16, 30, 33). It is not known whether different ACSs
synthesize acyl-CoAs that comprise a common pool, which is available to
all acyl-CoA-utilizing pathways, or separate acyl-CoA pools that are
functionally distinct. If acyl-CoAs were equally available to all
metabolic pathways, one would expect the ACS inhibitor triacsin C to
block production of all acyl-CoA-dependent end products to a similar
degree. Instead, triacsin C preferentially inhibited hepatocyte TAG
synthesis, whereas other acyl-CoA pathways, including -oxidation,
were either less inhibited or unaffected. Furthermore, the magnitude of
triacsin's inhibition of acyl-CoA metabolism depended on the
nutritional status of the rat from which hepatocytes were isolated.
These data provide strong evidence that, in liver, acyl-CoAs do not
move freely within cellular compartments but instead are channeled
toward specific pathways. This model of acyl-CoA channeling is
supported by previous studies in other types of cells that also relied
on the use of ACS inhibitors (10, 14, 29, 36). To account
for these observations, we hypothesize that distinct ACS isozymes vary
in their sensitivities to ACS inhibitors and are functionally complexed
with specific metabolic pathways, a hypothesis which is consistent with
in vitro data demonstrating that triacsin C differentially inhibits
arachidonoyl-CoA synthetase compared with long-chain ACS
(13). Additionally, this hypothesis predicts that the ACS
isoform most tightly coupled to TAG biosynthesis is triacsin sensitive,
whereas the ACS isoform closely associated with -oxidation is
triacsin resistant. If this hypothesis is true, it would explain why
triacsin preferentially inhibits TAG synthesis. Additionally, it might
also explain why triacsin's inhibition of TAG synthesis was more
marked in hepatocytes isolated from fed than from starved rats, because
we predict that feeding might be associated with preferential synthesis
of acyl-CoAs by the ACS linked to TAG synthesis. We found that ACS1
protein is abundant in microsomes, the site of TAG synthesis, but not
in mitochondria, the site of
-oxidation. These results, which are the first to report ACS1 protein abundance in different subcellular fractions, provide strong evidence that ASC1 is not the primary isoform
that activates fatty acids in liver mitochondria. Additional indirect
evidence that ACS1 is closely associated with TAG biosynthesis and that
it might be triacsin sensitive is provided by ACS1's microsomal
localization, refeeding-induced stimulation of microsomal ACS1 protein
expression, and the high potency of triacsin C against TAG synthesis,
but not
-oxidation.
The existence of triacsin-sensitive and triacsin-resistant ACS isozymes has been shown in yeast, which express at least four separate ACSs (faa1-faa4) (18). Genetic studies suggest that yeast ACSs have specific functions. Only FAA1 and FAA4 can activate exogenous fatty acids, and a yet unidentified ACS specifically activates endogenously synthesized fatty acids (17). When faa1/faa4 null yeast are grown in the presence of cerulenin, which inhibits de novo fatty acid synthesis and thus kills the cells, rat ACS1 complements and rescues the cells (17). Triacsin C blocks complementation by ACS1 (17), consistent with our prediction that ACS1 is triacsin sensitive.
Contrary to our expectation, triacsin inhibited ACS similarly in
microsomes and mitochondria, suggesting the presence of at least one
mitochondrial ACS that is triacsin sensitive but perhaps not directly
linked to -oxidation. Additionally, despite increased ACS1 protein
abundance in liver microsomes from fed compared with starved rats,
microsomal ACS specific activity was unaffected by nutritional status.
Others have reported similar inconsistencies between ACS1 mRNA
abundance and enzyme activities in subcellular fractions from hamsters
(24). These data demonstrate the difficulty in
interpreting ACS specific activities, which reflect several ACS
isoenzymes. Discrepancies between changes in ACS1 expression compared
with changes in enzyme activity might be explained if distinct ACS
isozymes are regulated in opposite directions. For example, whereas
starvation decreased the abundance of ACS1 protein, expression of other
ACS isoforms might have been increased, thereby negating measurable
changes in ACS specific activity. Alternatively, because of the
inherent limitations of measuring enzyme activities in vitro, the
specific activity in isolated membrane fractions might not reflect
regulation that occurs in vivo or in intact cells.
The alternative to our hypothesis that distinct ACS isoforms are
functionally linked to specific pathways is that different ACSs
synthesize acyl-CoAs that form a common pool. In this case, adding
triacsin C should uniformly decrease the availability of acyl-CoA to
all pathways. Use of a limited pool of acyl-CoAs would then be
regulated by enzyme kinetics, such that the enzyme with the lowest
Michaelis-Menten constant (Km) for acyl-CoA
would esterify more acyl-CoAs. In a previous study (14) we
reported data that oppose this hypothesis. We found that in rat liver
microsomes, DGAT, which catalyzes the final step in TAG synthesis, and
lysophosphatidylcholine acyltransferase, which catalyzes the
reacylation of lysophosphatidylcholine, have similar dependencies on
palmitoyl-CoA. Thus differences in triacsin's effect on oleate
incorporation into TAG and PL (Fig. 4) probably did not result merely
from differences in apparent Km values for the
acyl-CoA substrates. Other enzymes that compete for acyl-CoA, such as
carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in
-oxidation, and mitochondrial GPAT, which catalyzes the initial step
of glycerolipid synthesis, appear to be acutely regulated by allostery
and reversible phosphorylation (22, 27). Thus direct
comparisons of Km values obtained from in vitro
assays would be difficult to interpret.
A second argument against regulation by enzyme kinetics is based on
reports that feeding decreases liver CPT1 activity and increases GPAT
activity, suggesting that, in the fed state, acyl-CoAs would be
preferentially esterified (1, 7, 23). Acyl-CoAs were, in
fact, preferentially esterified in hepatocytes from fed rats; ~2.5
times more oleate was incorporated into TAG than was oxidized to ASM.
Despite preferential esterification, however, adding triacsin inhibited
TAG synthesis more than -oxidation. Furthermore, if limited
availability of acyl-CoA were critical in determining acyl-CoA
partitioning, then increasing acyl-CoA availability should have
produced a shift in acyl-CoA use. On the contrary, in triacsin-treated
cells from both fed and starved rats, the TAG-to-ASM ratio remained
constant at all concentrations of fatty acid (Fig. 4E).
Together, these data argue that enzyme kinetics alone cannot explain
our observation that acyl-CoAs were selectively channeled in
hepatocytes treated with triacsin C. Definitive evidence supporting the
hypothesis that acyl-CoA trafficking is regulated by coordinated
expression of distinct ACS isozymes awaits further investigation of
acyl-CoA metabolism in transgenic animals or in cells that selectively
express specific ACSs.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. LeMasters, Dr. Ting Qian, and Steve Elmore for their assistance with the hepatocyte isolations. We also thank Dr. David G. Klapper for assistance in synthesizing the peptide for the ACS1 antibody, and Ping Wang for assistance with enzyme assays.
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FOOTNOTES |
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This work was supported by National Institute of Child Health and Human Development Grants HD-56598 (R. A. Coleman) and HD-08431 (T. M. Lewin), a grant from the North Carolina Institute of Nutrition, and a predoctoral fellowship to D. M. Muoio from the American Heart Association-North Carolina Affiliate.
Address for reprint requests and other correspondence: R. A. Coleman, Depts. of Nutrition and Pediatrics, CB #7400, Univ. of North Carolina, Chapel Hill, NC 27599-7400 (E-mail: rcoleman{at}sph.unc.edu).
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.
Received 16 February 2000; accepted in final form 19 July 2000.
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