(Received for publication, December 24, 1996, and in revised form, February 27, 1997)
From the Department of Physiology and Diabetes and
Metabolism Unit, Evans Department of Medicine, Boston University
Medical Center, Boston, Massachusetts 02118, the
¶ Endocrine-Metabolism Division, Department of Medicine and
Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756, and
St. Vincent's Institute of Medical Research, 41 Victoria
Parade, Fitzroy, Victoria 3065, Australia
The concentration of malonyl-CoA, a negative
regulator of fatty acid oxidation, diminishes acutely in contracting
skeletal muscle. To determine how this occurs, the activity and
properties of acetyl-CoA carboxylase (ACC-
), the skeletal muscle
isozyme that catalyzes malonyl-CoA formation, were examined in rat
gastrocnemius-soleus muscles at rest and during contractions induced by
electrical stimulation of the sciatic nerve. To avoid the problem of
contamination of the muscle extract by mitochondrial carboxylases, an
assay was developed in which ACC-
was first purified by
immunoprecipitation with a monoclonal antibody. ACC-
was
quantitatively recovered in the immunopellet and exhibited a high
sensitivity to citrate (12-fold activation) and a
Km for acetyl-CoA (120 µM) similar to
that reported for ACC-
purified by other means. After 5 min of
contraction, ACC-
activity was decreased by 90% despite an apparent
increase in the cytosolic concentration of citrate, a positive
regulator of ACC. SDS-polyacrylamide gel electrophoresis of both
homogenates and immunopellets from these muscles showed a decrease in
the electrophoretic mobility of ACC, suggesting that phosphorylation
could account for the decrease in ACC activity. In keeping with this
notion, citrate activation of ACC purified from contracting muscle was
markedly depressed. In addition, homogenization of the muscles in a
buffer free of phosphatase inhibitors and containing the phosphatase
activators glutamate and MgCl2 or treatment of
immunoprecipitated ACC-
with purified protein phosphatase 2A
abolished the decreases in both ACC-
activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-
activity after the onset of contractions (50% by 20 s) and its slow
restoration to initial values during recovery (60-90 min) were
paralleled temporally by reciprocal changes in the activity of the
2
but not the
1 isoform of 5
-AMP-activated protein kinase (AMPK). In
conclusion, the results suggest that the decrease in ACC activity during muscle contraction is caused by an increase in its
phosphorylation, most probably due, at least in part, to activation of
the
2 isoform of AMPK. They also suggest a dual mechanism for ACC
regulation in muscle in which inhibition by phosphorylation takes
precedence over activation by citrate. These alterations in ACC and
AMPK activity, by diminishing the concentration of malonyl-CoA, could be responsible for the increase in fatty acid oxidation observed in
skeletal muscle during exercise.
In tissues such as liver (1) and heart (2, 3), malonyl-CoA
regulates fatty acid oxidation by inhibiting carnitine palmitoyl
transferase I, the enzyme that catalyzes the transfer of cytosolic long
chain fatty acyl-CoA into mitochondria. Evidence has been presented
that it plays a similar role in skeletal muscle (4-7), although
definitive evidence is still lacking (see "Discussion"). Malonyl-CoA is synthesized from cytosolic acetyl-CoA by a reaction catalyzed by acetyl-CoA carboxylase (ACC).1
In liver, the principal ACC is the isoform with a mass of 265 kDa
(ACC-
); in skeletal muscle, it is a distinct 280-kDa
-isoform (ACC-
) (8-14). Numerous studies have shown that the activity of ACC
in liver is altered by starvation, refeeding, and incubation with
insulin and glucose due to changes in its cellular content and
phosphorylation state (15-22). In contrast, neither nutritional changes in vivo (23, 8) nor incubation with insulin and
glucose (24) have been shown to affect the activity of ACC-
in
skeletal muscle extracts, despite the fact that they produce
substantial changes in malonyl-CoA content. Recent studies suggest that
the rapid (min) increase in malonyl-CoA caused by insulin and glucose in incubated rat soleus muscle is caused by an increase in the cytosolic concentration of citrate, an allosteric activator of ACC and
a substrate for its precursor, cytosolic acetyl-CoA (24).
Alterations in ACC activity due to phosphorylation may occur in
skeletal muscle during voluntary exercise. Thus, Winder and Hardie (25)
have demonstrated that after 5 min of swimming, malonyl-CoA levels and
ACC activity in rat skeletal muscle are diminished and the activity of
5-AMP-activated protein kinase is increased. Direct evidence that the
decrease in ACC activity is due to phosphorylation is still lacking,
however. In addition, it is unclear whether the observed decrease in
ACC activity is a consequence of muscle contraction per se
or a systemic effect of exercise. Finally, whether changes in the
cytosolic concentration of citrate, such as those produced by insulin
and glucose, continue to modulate ACC activity in muscle during
exercise is not known.
To examine these questions, ACC activity and tissue levels of citrate
and malate, an antiporter for citrate efflux from the mitochondria,
were assessed in control rat muscle and in muscle made to contract
intensely by electrical stimulation. Because of difficulties in
assaying ACC in frozen muscle due to the contamination of the extracts
by other carboxylases (12), a novel method for studying ACC- was
developed, in which its properties and activity were assessed after
immunopurification. The relationship between ACC activity and
phosphorylation state was also addressed, as were the potential roles
of the
1 and
2 5
-AMP-activated protein kinase isoforms in
regulating ACC activity.
All radiochemicals were obtained from DuPont NEN. Protein phosphatase 2A was purchased from Upstate Biochemical Inc. (UBI), and okadaic acid was purchased from Calbiochem. Agarose-Protein A/G plus conjugate was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), streptavidin-horseradish peroxidase from Amersham Corp., and PVDF membranes (0.45 µm) from Millipore. For enhanced chemiluminescence (ECL), luminol and p-coumaric acid were obtained from Sigma, and 30% H2O2 was obtained from Fisher. X-ray films were Fuji RX.
Animal CareMale Sprague-Dawley rats weighing 55-67 g were obtained from Charles River Breeding Laboratories (Wilmington, MA). They were kept in the departmental animal house with a light-dark cycle of 6 p.m.-6 a.m. and were fed Purina rat chow ad libitum. In all studies, the rats were fasted for 18-20 h prior to an experiment.
Muscle StimulationRats were anesthetized with sodium
pentobarbital (55 mg/kg body weight intraperitoneally), and 45 min
later the skin from both hindlimbs was removed, and the sciatic nerves
were exposed. The right sciatic nerve was stimulated for 0-5 min with
a bipolar electrode connected to a Grass stimulator (model S48) (5 pps, 100-ms trains of 2.5 V, 50 Hz, and 10-ms duration). The exposed muscle
was kept moist with Ringer's solution maintained at room temperature.
At various times following stimulation, the gastrocnemius-soleus muscles of the stimulated and unstimulated contralateral limbs were
rapidly excised and frozen in liquid nitrogen. All tissues were stored
at 80 °C until analyzed.
Buffer A contained 30 mM
NaHEPES, pH 7.4; 2.5 mM EGTA; 3 mM EDTA; 32%
glycerol; 20 mM KCl; 40 mM
-glycerophosphate; 40 mM NaF; 4 mM
NaPPi, 1 mM Na3VO4,
0.1% Nonidet P-40, 2 mM diisopropyl fluorophosphate, 2 mM phenylmethylsulfonyl fluoride; 5 µM
aprotinin, leupeptin, and pepstatin A; and 1 mM
dithiothreitol.
Buffer B contained 30 mM NaHEPES, pH 7.4; 2.5 mM EGTA; 3 mM EDTA; 70 mM KCl; 20 mM -glycerophosphate; 20 mM NaF; 2 mM NaPPi; 1 mM
Na3VO4; 0.1% Nonidet P-40; 2 mM
diisopropyl fluorophosphate; 2 mM phenylmethylsulfonyl
fluoride; 5 µM aprotinin, leupeptin, and pepstatin A; and
1 mM dithiothreitol.
Buffer C contained 30 mM NaHEPES, pH 7.4; 2.5 mM EGTA; 3 mM EDTA; 32% glycerol; 30 mM KCl; 85 mM NaCl; 0.1% Nonidet P-40; 2 mM diisopropyl fluorophosphate; 2 mM phenylmethylsulfonyl fluoride; 5 µM aprotinin, leupeptin, and pepstatin A; and 1 mM dithiothreitol.
Buffer D contained 100 mM NaCl; 20 mM NaHEPES,
pH 7.4; 60 mM -mercaptoethanol; and 1 mg/ml bovine serum
albumin.
Solution E contained 20 ml of 100 mM Tris-Cl, pH 8.4; 10 µl of 30% H2O2; 200 µl of 250 mM Luminol; and 100 µl of 100 mM p-coumarate. (Note: luminol and coumarate were dissolved in 100% Me2SO and stored at room temperature protected from light.)
Metabolite and Enzyme AssaysMalonyl-CoA (26), citrate, and
malate, were measured in perchloric acid extracts of frozen muscles as
described elsewhere (24). For ACC and 5-AMP-activated protein kinase
(AMPK) activity, frozen muscles were powdered under liquid
N2, homogenized in buffer A, and centrifuged at
100,000 × g for 40 min. The supernatants were then
diluted 1:1 with buffer B and centrifuged at 13,500 × g for 12 min. The resultant supernatants were used for
immunoprecipitation of ACC and AMPK and also for measuring ACC activity
by the 14CO2 fixation assay.
To assay ACC- in immunoprecipitates, equal amounts of supernatant
protein (500-700 µg) were incubated for 3 h with 2.5 µg of
the monoclonal antibody 7AD3 (10) and 24 µl of agarose-Protein A/G
plus beads. The beads were washed once in buffer B, twice in buffer B
containing 250 mM NaCl, and once in buffer B. They were
then assayed for ACC activity by the 14CO2
fixation assay (15). In selected experiments, the concentration of
citrate or acetyl-CoA was varied. Results are expressed as pmol of
14CO2 incorporated into acid-stable products
per min per mg of extract protein subjected to immunoprecipitation.
The AMPK was assayed against the SAMS peptide substrate, as in Ref. 27.
Tissue extracts were immunoprecipitated with nonimmune sera or with
specific antisera directed against either the 1 or
2 catalytic
subunits of the AMPK heterotrimer (28). Following collection of the
immunoprecipitates on Protein A/G beads and extensive washing, the
immobilized enzyme was assayed under standard conditions in a 10-min
assay. Peptide kinase activity in each sample was corrected both for
apparent 32P incorporation in the absence of added
substrate peptide and for apparent incorporation in nonimmune
precipitates. Results are expressed as cpm of 32P
incorporated per min per mg of extract protein subjected to immunoprecipitation.
To assess the effect of dephosphorylation by endogenous phosphatases on ACC activity, muscles were homogenized in Buffer A or Buffer C (no phosphatase inhibitors) or Buffer C plus 50 nM okadaic acid. Supernatants, obtained after centrifuging the homogenates at 13,500 × g for 30 min, were incubated at 37 °C for 50 min with or without the addition of 100 mM sodium glutamate, 10 mM MgCl2. They were then diluted 1:1 with buffer B and centrifuged at 13,500 × g for 15 min. ACC was immunoprecipitated from the resultant supernatant, and its activity was assayed as described above.
To examine the effect of dephosphorylation by protein phosphatase 2A
(PP2A) on ACC activity in vitro, muscles were homogenized in
buffer A and centrifuged at 100,000 × g for 40 min.
The supernatants were then diluted 1:1 with buffer B and centrifuged at
13,500 × g for 12 min. The resultant supernatants were
used to immunoprecipitate ACC- as described under "Metabolic and
Enzyme Assays," but with three additional washes with buffer D. Following the washes, the beads were incubated with 500 milliunits of
PP2A (5 units/ml) at 37 °C for 2.5 h in the presence or absence
of 100 mM sodium glutamate/10 mM
MgCl2. As controls, the same incubations were carried out
with the addition of 10 nM okadaic acid or with buffer D in
the absence of PP2A. At the end of the incubations, the immunopellets were washed twice with buffer B and then assayed for ACC activity as
described above.
Samples were run overnight at 65 V on 6.4 or 7% SDS-polyacrylamide gel electrophoresis. Following the electrophoresis, the protein bands were transferred onto PVDF membrane at 70 V for 1 h and then at 100 V for 2.5 h using Towbin buffer containing 10% methanol and 0.01% SDS. The membranes were blocked with 0.5 mg/ml poly(vinyl alcohol) for 2 s and then probed with streptavidin-horseradish peroxidase for 20 min (1:2000 in phosphate-buffered saline with 0.4% Tween 20). The membranes were washed three times for 5 min with phosphate-buffered saline containing 0.4% Tween 20 and once with phosphate-buffered saline alone for 2 min. They were then developed with enhanced chemiluminescence (ECL) by incubation for 90 s in freshly prepared solution E and exposure onto Fuji RX film for approximately 1 min.
In agreement with previous results (4, 29), malonyl-CoA levels were diminished by 50% after 5 min of intense muscle contraction (1.9 ± 0.2 versus 0.9 ± 0.2 nmol/g of muscle, n = 4, p < 0.05). This was not attributable to a decrease in the cytosolic concentration of citrate, since the whole muscle cell concentration of citrate was increased (190 ± 16 versus 300 ± 34 nmol/g muscle, n = 4, p < 0.05). Furthermore, the concentration of malate, an antiporter for citrate efflux from the mitochondria, was also increased (280 ± 10 versus 390 ± 28 nmol/g of muscle, n = 4, p < 0.05), suggesting the possibility of citrate redistributioon from mitochondria to cytosol (24).
ACC-Attempts to assay ACC activity directly in muscle
supernatants were hindered by the presence of contaminating
carboxylases, presumably pyruvate carboxylase (PC) and propionyl-CoA
carboxylase (PCC) arising as a consequence of mitochondria breakage
(Fig. 1A). Thus, when acetyl-CoA carboxylase
activity was assayed by the 14CO2 fixation
method in 100,000 × g supernatants obtained from frozen muscle extracts, a high but variable activity was noted in the
absence of citrate (50-80% of total in different experiments), and
activation by 10 mM citrate, an index of ACC activity, was modest (382 ± 63 versus 957 ± 78 pmol/min/mg,
n = 4, p < 0.05). Supernatants from
freshly homogenized muscles contained the same amount of ACC as did
frozen muscles, as judged by streptavidin blots (data not shown), but
less of the other carboxylases. They also exhibited less
citrate-independent 14CO2 fixation activity and
a relatively greater activation by 10 mM citrate (86 ± 29 versus 610 ± 48 pmol/min/mg, n = 4, p < 0.05), suggesting that the contaminating
carboxylases account principally for the citrate-independent
activity.
To assay ACC in the absence of PC and PCC, a specific monoclonal
antibody (7AD3) was used to immunoprecipitate ACC- (see "Experimental Procedures"). As shown in Fig. 1A, the
antibody immunoprecipitated the majority of the ACC and left behind PC and PCC. Equally important, carboxylase activity toward acetyl-CoA in
the immunodepleted supernatants was not increased by 10 mM citrate, indicating that it was free of ACC activity (Fig.
1B). Nearly all (80-90%) of the
citrate-dependent ACC activity present in the muscle
supernatant was recovered in the immunoprecipitate, indicating that the
antibody did not inhibit the enzyme. The immunopurified enzyme
exhibited a Michaelis constant for acetyl-CoA (Km = 120 ± 4.2 µM) similar to that reported by others
for ACC-
purified by conventional methods (10). Thus, it appears
that immunoprecipitation with the 7AD3 antibody can be a simple
alternative to more conventional techniques for purifying and assaying
ACC-
.
ACC activity in 7AD3 immunopellets, measured at close to physiological
citrate levels (0.2 mM), was decreased by 90% after 5 min
of intense muscle contractions (78 ± 9 versus 9.1 ± 4 pmol/min/mg). Similar decreases in activity were observed at all
assay citrate concentrations (Fig. 2A). In
addition, the K0.5 for citrate, which was
1.7 ± 0.12 mM (mean ± S.E.) for the control
muscles, was increased to 2.6 ± 0.3 mM. Such
decreases in the sensitivity and responsiveness of ACC to citrate are
suggestive of increased enzyme phosphorylation (12, 15).
Decreased ACC Activity Induced by Contractions Is Attributable to Phosphorylation
Consistent with an increase in enzyme
phosphorylation, the immunoprecipitated enzyme from stimulated muscle
showed a decrease in its electrophoretic mobility when subjected to
SDS-polyacrylamide gel electrophoresis (Fig. 2, B and
C). To obtain more direct evidence that phosphorylation
decreased ACC activity, muscles were homogenized in lysis buffer with
or without the phosphatase inhibitors NaF, NaPPi,
-glycerophosphate, and Na3VO4, or without
these phosphatase inhibitors but with okadaic acid present (another
phosphatase inhibitor, see "Experimental Procedures"). As shown in
Fig. 3A, homogenization in the absence of
phosphatase inhibitors partially reversed the decrease in ACC activity
caused by contractions, and it completely abolished it when the
phosphatase activators glutamate and MgCl2 were added to
the muscle extract (see "Experimental Procedures"). Incubation of
extracts from control muscles with glutamate and MgCl2 in
the absence of phosphatase inhibitors also enhanced ACC activity,
although to a lesser extent. Under all conditions, these increases in
ACC activity were prevented by 50 nM okadaic acid (Fig.
3A), confirming that they were due to the
action of phosphatases.
Similar results were obtained when ACC isolated from stimulated muscles
was treated with PP2A. In these studies, immunopellets from lysates
containing phosphatase inhibitors were washed with phosphatase buffer
(see "Experimental Procedures") and then treated for 2.5 h
with 500 milliunits of PP2A in the presence or absence of glutamate and
MgCl2. As shown in Fig. 3B, when PP2A was added to the immunopellets, it partially abolished the inhibition of ACC
caused by electrical stimulation. This effect, like that of endogenous
phosphatases, was greatly enhanced by the addition of
MgCl2/glutamate and was inhibited by okadaic acid. The
addition of MgCl2/glutamate alone to the immunopellets did
not increase the activity of ACC, further suggesting that these agents
work by activating a phosphatase. Also in keeping with this conclusion, treatment of immunoprecipitated ACC- with PP2A eliminated the observed shift in its electrophoretic mobility caused by contraction (Fig. 2C) in an okadaic acid-inhibitable manner.
A logical candidate for regulating ACC
phosphorylation during contraction is AMPK (25). To assess its role, we
compared the time course of changes in the activities of ACC and the
1 and
2 isoforms of AMPK during and after contraction.
Immunoprecipitable ACC and
1 and
2 AMPK activities were measured
after periods of contraction ranging from 5 s to 5 min. As shown
in Fig. 4A, the decrease in ACC activity was very rapid,
with 50% inactivation evident by 20 s and near maximum (90%)
inhibition in less than a minute. These changes were closely mirrored
by reciprocal increases in
2 AMPK activity. The return of ACC and
2 AMPK activity to base-line values after the cessation of the
contractions was slow (Fig. 4B). Thus, ACC activity was
still diminished by 50% 40 min poststimulation, and only after
1.5 h of recovery did it return to precontraction values. The
return of
2 AMPK activity to precontraction values followed a
similar pattern. In contrast, no detectable activation of
1 AMPK was
observed during the course of stimulation. A transient activation of
its activity poststimulation did not achieve statistical significance
(p > 0.1).
The results indicate that the activity of ACC and the
concentration of malonyl-CoA in skeletal muscle decrease within seconds during intense contractions. They also strongly suggest that the decrease in ACC activity is secondary to phosphorylation, most probably
due, at least in part, to activation of the 2 isoform of AMPK. In
contrast, alterations in the cytosolic concentration of citrate, which
acutely modulate ACC in muscle during changes in its fuel milieu (24),
did not appear to play any role in the modulation of ACC activity
during contraction.
The potential relevance of these findings relates to the regulation of fatty acid oxidation in muscle and the mechanism by which it is increased during and after exercise. Regulation of fatty acid oxidation by malonyl-CoA has been demonstrated in liver (1) and heart (2, 3), and it probably occurs in skeletal muscle (4-7). In the latter, malonyl-CoA levels are decreased by starvation (23) and exercise (25, 30, 31) and increased by refeeding after a fast, in keeping with the directional changes in fatty acid oxidation in these conditions. In addition, the decrease in fatty acid oxidation, observed in human muscle during a 5-h euglycemic-hyperinsulinemic clamp (7) is associated with a decrease in long chain acylcarnitine, suggesting inhibition of long chain fatty acyl-CoA transport into mitochondria. Although not measured, an increase in the concentration of malonyl-CoA is a likely cause for this finding. Unexplained is the observation that the inhibitory constants of malonyl-CoA for the carnitine palmitoyl transferase I isoforms that predominate in skeletal and cardiac muscle are 1-2 orders of magnitude lower than that for the carnitine palmitoyl transferase I isoform of liver (32). Thus, at the measured concentration of malonyl-CoA in these muscles, carnitine palmitoyl transferase I should be totally inhibited. Presumably, much of the malonyl-CoA in heart and skeletal muscle is bound (33), and/or other factors, such as CoA or acetyl-CoA (34), alter its ability to inhibit carnitine palmitoyl transferase I in vivo.
Winder and Hardie (25), investigating the mechanism for the decline of
malonyl-CoA levels in rat muscle during voluntary exercise, found that
ACC activity, assayed in (NH4)2SO4
pellets, was diminished. The findings of the present study strongly
suggest that this decrease in ACC activity is due to local changes
induced by contraction rather than systemic factors altered by
exercise. They also suggest that the decrease in ACC activity caused by contraction is due to phosphorylation of the enzyme. Evidence in
support of this conclusion includes 1) the decreased electrophoretic mobility of ACC after contractions (Fig. 2B), 2) the
concurrent decrease in responsiveness and sensitivity of ACC to citrate
activation (Fig. 2A; Refs. 12 and 15), and 3) the
restoration the electrophoretic mobility and activity of ACC following
dephosphorylation by endogenous or exogenous phosphatases, (Fig.
2C; Ref. 3). In addition, the reactivation by phosphatases
was most complete when glutamate and MgCl2 were added to
the medium. Gaussin et al. (35) have described an ACC
phosphatase in liver that is strongly activated (29-fold) by
Mg2+/glutamate and inhibited by okadaic acid and that
exhibits other properties of a type 2A phosphatase. In our studies,
when both PP2A and MgCl2/glutamate were added to ACC-
immunopellets, ACC-
activity was increased to values equal to that
of the untreated control (Fig. 3B). However, it did not
completely reach the activity of ACC-
from control muscles treated
with these agents, suggesting either that other phosphatases, in
addition to PP2A, might be involved or that longer incubations are
required to achieve maximum activation.
Several protein kinases have been shown to phosphorylate ACC obtained
from liver and other tissues, with the greatest inhibition occurring
when ACC is phosphorylated by the AMPK (36-45). Most studies indicate
that AMPK is the major regulatory kinase acting on hepatic ACC and that
ACC regulation by several effectors (glucagon, substrate depletion,
inhibitors of oxidative phosphorylation, ischemia/hypoxia, arsenite,
and heat shock) is due to to the phosphorylation of specific serine
residues (Ser-79, -1200, and -1215) on ACC (36-45). The regulation of
AMPK activity occurs through alterations in cellular free 5-AMP
(36-45), such as occurs in skeletal muscle during during contraction
(46, 47). The mechanism by which 5
-AMP activates AMPK is complex,
involving allosteric regulation of AMPK subunits and modulation of AMPK
phosphorylation by AMPK kinase(s) and phosphatase(s) (48, 49).
Despite the fact that 5-AMP is closely regulated in skeletal muscle,
the effects of AMPK on muscle ACC have not been well studied. Winder
and Hardie (25) have reported a modest activation of AMPK, accompanied
by a decrease in ACC activity, in rat skeletal muscle following several
minutes of swimming. However, the time courses of these changes in AMPK
and ACC activity were not necessarily coincident (25). In the perfused
rat heart, reciprocal changes in AMPK and ACC activity have been
described during stop-flow ischemia/reperfusion, although again the
time course of these changes leaves open the question of their
cause-effect relationship (45). In both of these studies, AMPK activity
was measured in crude extracts (concentrated by polyethylene glycol or
(NH4)2SO4 precipitation) by SAMS
peptide phosphorylation and without taking into account AMPK
heterogeneity. AMPK is a heterotrimeric protein consisting of an
catalytic subunit associated with noncatalytic
and
subunits,
which are essential for optimal enzyme activity (27, 50-53). For each
enzyme subunit, there is a recognized isoprotein family; in the case of
the catalytic subunits,
1 and
2 polypeptides are known, each with
wide tissue distribution (50). Rat skeletal muscle expresses both
1
and
2 subunits, as well as the
1 and
1 noncatalytic subunits
(50, 52). For this reason, we analyzed the effect of electrical
stimulation on the two AMPK isoforms using specific antibodies to the
1 and
2 catalytic subunits. As shown in Fig. 4, the decrease in
ACC activity during contractions and its restoration during the
recovery period were closely paralleled by reciprocal changes in
2-AMPK activity. In contrast, no significant change of
1 AMPK
activity was observed. Why these AMPK isoforms are differentially
regulated by contraction remains to be determined. Nevertheless, the
data suggest that the
2-AMPK could be responsible, at least in part,
for ACC phosphorylation under the conditions of these experiments. They
do not rule out the possibility that activation of the
1 isozyme by
5
-AMP also occurred but was not reflected in our assays, which were
conducted at saturating concentrations of 5
-AMP (27, 28). For this
reason, studies of the independent effects of the
1 and
2
isozymes on ACC-
activity in vitro need to be performed.
The role of other factors, such as ACC phosphatase and/or other
kinases, regulating the contraction-induced changes in ACC-
activity
also requires examination.
In contrast to the changes observed during contraction, the acute
(minutes) increases in malonyl-CoA caused by incubating muscles with
insulin and glucose are not due to increases in stable ACC activity
(24). Rather, they correlate with increases in the cytosolic
concentration of citrate, an allosteric activator of ACC, and a
precursor of its substrate, cytosolic acetyl-CoA (24). In contracting
muscle, however, decreases in malonyl-CoA were associated with
increases in whole cell citrate and its mitochondrial antiporter,
malate, suggesting if anything an increase in cytosolic citrate. Thus,
when the energy expenditure of muscle is substantially increased,
phosphorylation appears to overcome the effects of citrate and becomes
the dominant mechanism of ACC regulation (Fig. 5). Since
free 5-AMP levels in muscle increase markedly during contraction (46,
47), a logical conclusion is that these increases initiate ACC
phosphorylation by activating AMPK. In that it is regulated by both
citrate and by 5
-AMP via AMPK, ACC-
resembles another crucial
metabolic enzyme, namely phosphofructokinase-I (PFK-I). PFK-I is
inhibited by ATP, and this inhibition is potentiated by citrate and
diminished by 5
-AMP, although without intermediation by AMPK (54, 55).
An attractive hypothesis is that, by virtue of their dual effects on
ACC-
and PFK-I, citrate and 5
-AMP complement each other in
coordinating the regulation of these enzymes and secondarily the use of
glucose and fatty acids as fuels for muscle (Fig. 5).
The standard assay for ACC activity measures the difference in
acid-stable products generated by a CO2 fixation reaction
in the presence and absence of exogenous acetyl-CoA. Such assays can be
hindered by the presence of contaminating carboxylases (12). The most
important of these appears to be propionyl-CoA carboxylase, which can
catalyze the carboxylation of acetyl-CoA (to give malonyl-CoA) at
the rate it carboxylates propionyl-CoA (56). In contrast
to ACC, propionyl-CoA carboxylase is not activated by citrate
(56).2 Thus, when present in large amounts,
due to leakage from mitochondria, it could give an erroneously high
value for ACC activity when assayed either in the absence of citrate or
when citrate is present at low concentrations (0-0.3 mM).
In keeping with this notion, extracts immunodepleted of ACC showed a
large residual 14CO2 fixation activity that was
not activated by citrate (Fig. 1). To avoid the problem of
contaminating carboxylases, ACC was assayed after immunopurification
with the monoclonal antibody 7AD3. The antibody recovered nearly all of
the citrate-dependent ACC activity and protein present in
the muscle supernatant, indicating that it did not inhibit ACC (Fig.
1). Finally, activation of ACC by citrate is classically portrayed as
due to citrate-induced polymerization of protomeric ACC subunits (57,
58). If so, when ACC is immobilized on beads during
immunoprecipitation, one would not expect citrate to activate it
12-fold, as in the present study (Fig. 1). Thus, citrate activation of
ACC may be dissociated from polymerization, as has been suggested by
studies of chicken ACC (59-61).
In conclusion, we report here a simple assay that makes use of the
monoclonal antibody 7AD3 to assay ACC- activity in skeletal muscle,
free of contaminating mitochondrial carboxylases. Using this assay,
we were able to show that muscle contractions markedly diminish the
activity of ACC-
within seconds. The data strongly suggest that such
decreases in ACC activity are due to phosphorylation and that they are
reversible by phosphatases, probably of type 2A. They also indicate
that the
2 but not the
1 isoform of AMPK follows a pattern of
activation/inactivation during and following contraction that closely
mirrors that of ACC. Finally, the data suggest the existence of a dual
mechanism for ACC regulation in muscle, in which inhibition by
phosphorylation (e.g. during contraction) can take
precedence over activation by citrate.
We thank Fotini Vavva for technical assistance and Prof. Joseph Avruch (Harvard University) and Prof. E. Shafrir (Hebrew University, Israel) for useful discussions about the manuscript.
After this manuscript was accepted for publication Hutber et al. (Hutber, C. A., Hardie, D. G., and Winder, W. W. (1997) Am. J. Physiol. 35, E262-E267) published a report describing inactivation of acetyl-CoA carboxylase and increases in AMP-activated protein kinase in electrically stimulated muscle.