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All Theses and Dissertations
2004-12-01
Phosphorylation of Skeletal Muscle Acetyl-CoA
Carboxylase by AMPK Enhances Palmitoyl-CoA
Inhibition
Dustin S. Rubink
Brigham Young University - Provo
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Rubink, Dustin S., "Phosphorylation of Skeletal Muscle Acetyl-CoA Carboxylase by AMPK Enhances Palmitoyl-CoA Inhibition"
(2004). All Theses and Dissertations. Paper 210.
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PHOSPHORYLATION OF SKELETAL MUSCLE ACETYL-COA CARBOXYLASE
BY AMPK ENHANCES PALMITOYL-COA INHIBITION
by
Dustin S. Rubink
A thesis submitted to the faculty of
Brigham Young University
in partial fulfillment of the requirements for the degree of
Master of Science
Department of Physiology and Developmental Biology
Brigham Young University
December 2004
BRIGHAM YOUNG UNIVERSITY
GRADUATE COMMITTEE APPROVAL
of a thesis submitted by
Dustin S. Rubink
This thesis has been read by each member of the following graduate committee
and by majority vote has been found to be satisfactory.
Date
William W. Winder, Chair
Date
James P. Porter
Date
Gerald D. Watt
BRIGHAM YOUNG UNIVERSITY
As chair of the candidate’s graduate committee, I have read the thesis of Dustin S. Rubink
in its final form and have found that (1) its format, citations, and bibliographical style are
consistent and acceptable and fulfill university and department style requirements; (2) its
illustrative materials including figures, tables, and charts are in place; and (3) the final
manuscript is satisfactory to the graduate committee and is ready for submission to the
university library.
Date
William W. Winder
Chair, Graduate Committee
Accepted for the Department
___________________________________
James P. Porter
Department Chair
Dixon Woodbury
Graduate Coordinator
Accepted for the College
R. Kent Crookston
Dean, College of Agriculture and Biology
ABSTRACT
PHOSPHORYLATION OF SKELETAL MUSCLE ACETYL-COA CARBOXYLASE
BY AMPK ENHANCES PALMITOYL-COA INHIBITION
Dustin S. Rubink
Department of Physiology and Developmental Biology
Master of Science
Acetyl-CoA carboxylase (ACC) catalyzes the formation of malnoyl-CoA, which in
turn controls the rate of fatty acid metabolism. ACC beta or 2 has been shown to be
localized on the mitochondria in close proximity to carnintine palmitoyl transferase 1
(CPT-1), the enzyme responsible for the influx of acyl-CoA into the matrix where beta
oxidation takes place. CPT-1 is inhibited by malonyl-CoA produced by ACC. It has been
well documented that AMP activated kinase (AMPK) when activated phosphorylates and
inactivates ACC. ACC is controlled allosterically by citrate, which activates, and by
palmitoyl-COA, which inhibits. In this study, we asked the question, “Does
phosphorylation by AMPK effect the inhibition of ACC by palmitoyl-CoA?” ACC was
isolated and then subjected to phosphorylation and activity was measured in varying
concentrations of acetyl-CoA and citrate. Phosphoryation reduced the substrate (acetylCoA) saturation activity curves for ACC at all levels of palmitoyl-CoA. The Ki for
palmitoyl-CoA inhibition of ACC was reduced from 1.7 ± 0.25 µM to 0.85 ± 0.13 uM
(p<0.05) as a consequence of phosphorylation. In addition the citrate activation curves for
ACC were greatly reduced in the presence of palmitoyl-CoA. The data show that skeletal
muscle ACC or ACC-beta is more potently inhibited by palmitoyl-CoA after
phosphorylation by AMPK. During long-term exercise when AMPK is activated and
muscle palmitoyl-CoA is elevated this may contribute to the low malonyl-CoA and
increased fatty acid oxidation.
Key Words: acetyl-CoA, exercise, fatty acid oxidation, malonyl-CoA
ACKNOWLEDGEMENTS
These studies were supported by NIH Grant AM41438. The authors thank Dr. D.
Grahame Hardie for the AMPK used in the phosphorylation studies. Thanks are also
given to Dr. William W. Winder for mentoring Dustin S. Rubink during the completion
of this project.
TABLE OF CONTENTS
TITLE PAGE…………………………………………...………….…...…………..……....i
GRADUATE COMMITTEE APPROVAL……………………...……………..……...…..ii
FINAL READING APPROVAL AND ACCEPTANCE…………….………..……...…..iii
ABSTRACT……………………………………………………………..……..…...……..iv
ACKNOWLEDGEMENTS….………….………………………………………..……….vi
TABLE OF CONTENTS………………………………………………..….....…...….….vii
LIST OF FIGURES………………………………………………………….…....….….viii
BODY OF THE WORK……………………………………………………….....….…1-28
INTRODUCTION………………………….…………………………....….…......1
MATERIALS AND METHODS……………………………………..…………....3
RESULTS…………………………………………………………..…….…..……7
DISCUSSION………………………………….…………………………….…….9
REFERENCES………………………………………………………...…...……..15
vii
LIST OF FIGURES
FIGURE 1: Western Blot Time Analysis………………………………………………..23
FIGURE 2: ACC Activity Curves…………………………………………….….…...…24
FIGURE 3: Apparent Vmax………………………………………………….….............25
FIGURE 4: Citrate Activation Curves………………………………………...………....26
FIGURE 5: Activity at Two Citrate Concentrations…………...…………….….……….27
FIGURE 6: Inhibition Curves…………………………………………………………….28
viii
1
INTRODUCTION
Acetyl-CoA carboxylase (ACC) controls an important regulatory step in fatty acid
metabolism (25). ACC catalyzes carboxylation of acetyl-CoA to form malonyl-CoA
(5,6,20). There are two major isoforms of ACC in mammals, including humans (2).
ACC alpha, found predominately in the liver and adipose tissue, catalyzes a rate limiting
step in fatty acid biosynthesis. The product malonyl-CoA is the substrate for fatty acid
synthase (FAS) in lipogenic tissues. There has been increasing evidence that ACC beta
found in muscle, considered a non-lipogenic tissue, is involved in controlling the rate of
fatty acid oxidation(5-7,34,37,44-50). Here the malonyl-CoA formed inhibits carnitine
palmitoyl transferase-1, the enzyme responsible for the regulation of the transport of fatty
acids into the mitochondria by forming the carnitine derivative (22). In the mitochondrial
matrix, the CoA derivative is regenerated by CPT-2 and can then be oxidized to acetylCoA by the enzymes of beta oxidation (22).
There is increasing evidence that ACC-beta has potential in therapeutically
regulating metabolic diseases such as obesity and diabetes. This was demonstrated
recently by creation of an ACC-beta -/- null mutant mouse phenotype (2). These mice
had less fat mass and higher rates of fatty acid oxidation, while continuing to be fertile
and have a normal life span(2). It has also been demonstrated that the null mutant mice
had reduced obesity and delayed onset of insulin-insensitivity while being fed obesity
inducing diets, specifically high fat or high carbohydrate diets (1).
There has been skepticism regarding the importance of malonyl-CoA in the
regulation of fatty acid oxidation. This is due to the fact that there has been a lack of
evidence of decreases in malonyl-CoA content in human muscle during exercise (19,27-
2
28). Recent data have made regulation of fatty acid oxidation by malonyl-CoA more
feasible due to elucidation of the cellular localization of ACC beta (2). ACC beta is a
larger protein (272 kD vs 265 kD) than ACC alpha due primarily to an N terminal
extension that has a hydrophobic mitochondrial targeting sequence (1,9,18). ACC beta
has been shown to be associated with the mitochondria by immunohistochemical
techniques and confocal microscopy (2). The malonyl-CoA that is produced by the ACC
beta is closely associated with the CPT-1 and can have an inhibitory effect without
drastic changes to the total cellular content of malonyl-CoA (2). In rat the total muscle
content of malonyl-CoA is higher than in human muscle, particularly in the fast-twitch
red muscle fibers (8,13,27-28,32,47). Malonyl-CoA declines markedly in this tissue in
response to exercise or electrically stimulated muscle contraction (17,31,33,46).
Hindlimb perfusion studies demonstrate a good correlation between the rate of fatty acid
oxidation and the muscle malonyl-CoA content (23,24,50).
ACC is allosterically regulated by citrate, which activates ACC, and also by
palmitoyl-CoA, an inhibitor of ACC (6,25,40). There is also the phosphorlyation /
dephosphorlytion by AMP activated protein kinase (AMPK) and acetyl CoA carboxylase
phosphatase on specific serine residues (particularly ser 221) (10,14,51). AMPK has
previously been shown to phosphorylate the muscle isoform of acetyl-CoA carboxylase
(ACC beta) at the site equivalent to serine 79 in ACC-1 (ser221)(16,48,51). It has been
demonstrated that AMPK is activated in response to muscle contraction (17,30,42,48). It
has also been shown that ACC beta is phosphorylated and inactivated in exercising or
electrically stimulated muscle (13,17,30,31,33,38,42). Phosphorylation of ACC beta by
AMPK results in a shift of the citrate activation curve to the right, making the enzyme
3
insensitive to allosteric activation at physiological citrate concentrations (30,48,51).
This coupled with removal of malonyl-CoA by malonyl-CoA decarboxylase results in a
marked decrease in malonyl-CoA concentration in the muscle (35,37,38,48). This is
thought to remove inhibition of CPT1, allowing fatty acid oxidation to proceed as
exercise continues.
In addition to activation by citrate, ACC has been shown to be inhibited by
palmitoyl-CoA (29,41). The content of long chain acyl-CoA in muscle increases during
long-term exercise (43). No data is available regarding the effect of phosphorylation of
ACC by AMPK on palmitoyl-CoA inhibition of ACC. This study was designed to
determine if phosphorylation of muscle ACC by AMPK influences the extent of
palmitoyl-CoA inhibition.
MATERIALS AND METHODS
ACC Isolation. Rats (Sprague-Dawley, Sasco, Charles River) were given free
access to food and water and were kept in a room with a 12:12 light dark cycle. Rats
were anesthetized with sodium pentobarbital (52 mg/100 g body weight). Quadriceps,
hamstrings, and gastrocnemius muscles from 5-6 rats (Body weight 400-600 grams) were
removed and placed between stainless steel blocks at ice temperature for quick cooling.
Visible fat tissue was removed so as to only isolate skeletal muscle ACC. ACC was
isolated as described previously (48). Muscles were placed in buffer (9 ml/gram muscle,
wet weight) containing 225 mM mannitol, 75 mM sucrose, 10 mM tris-HCl, 0.05 mM
EDTA, 5 mM potassium citrate, and 2.5 mM MnCl2 at pH 7.5. Leupeptin (10 mg/liter),
4
antitrypsin (10 mg/liter) and aprotonin (10 ml/liter) were added immediately prior to use.
To optimize collection of non-phosphorylated ACC, phosphatase inhibitors were not
utilized in the homogenizing medium. Muscles were minced with scissors and then
homogenized with a Brinkmann Polytron PT 3000 homogenizer. After centrifugation at
17,000 X g for 30 min at 4 ºC, the supernatant was combined with ammonium sulfate
granules (200 grams/liter) and stirred at 4 ºC for one hour. After centrifugation (17,000
X g for 30 min at 4 ºC) the supernatant was poured off and the precipitate was suspended
in a minimal volume of buffer B (100 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA, 0.1 mM
DTT, and 10% glycerol at pH 7.5). Proteolytic enzyme inhibitors were added at the same
concentration indicated for buffer A. A homogenizer was utilized to resuspend the pellet.
This resuspended solution was dialyzed for 3 hr against buffer C (100 mM Tris-HCl, 0.5
M NaCl, 1 mM EDTA, 0.1 mM DTT, 5% glycerol, pH 7.5). The dialyzed solution was
centrifuged at 40,000 X g for 15 min at 4 ºC to remove any insoluble material. The
supernatant was applied to a freshly regenerated column of Promega Soft-Link, Softrelease avidin resin (Promega, Madison, WI). The column was washed with
approximately 50 ml of buffer C. Elution buffer (100 mM Tris-HCl, 0.5 M NaCl, 1 mM
EDTA, 0.1 mM DTT, 5 mM biotin, and 5% glycerol, pH 7.5) was then added to the
column and allowed to stand at 4 ºC for two hr. ACC was then eluted from the column
with elution buffer at a rate of 2-3 ml/hr. Elutions fractions were analyzed for ACC
activity. Active fractions were pooled and kept at -90 ºC until utilized in assays. ACC
activity from five different isolations was analyzed separately with respect to effect of
phosphorylation on inhibition by palmitoyl-CoA .
5
ACC Phosphorylation. Globulin free albumin was added to aliquots of the ACC
preparation to a concentration of 1 mg/ml. The presence of albumin greatly enhanced the
yield of active enzyme collected in subsequent steps. ACC was then precipitated by
addition of an equal volume of saturated ammonium sulfate solution. Samples were
mixed, placed on ice 15 min and then centrifuged at 48,000 X g 15 min at 4 ºC. After
discarding the supernatant, ACC was resuspended at 0.32 times the original volume in
suspension buffer (68 mM HEPES, 126 mM NaCl, 13.6% glycerol, 1.36 mM EDTA,
1.36 mM EGTA, 1.36 mM DTT, pH 7.4). Final concentrations in the phosphorylation
mix were 34 mM Hepes, 68 mM NaCl, 0.68 mM EDTA, 0.68 mM EGTA, 0.68 mM
DTT, 6.8% glycerol, 0.12 mM ATP, 3 mM MgCl2, 0.2 mM AMP, and 5 units/ml AMPK,
pH 7.0. The AMPK, provided by Dr. D.Grahame Hardie, was purified from rat liver as
described previously (48). In preliminary experiments, tubes were incubated for 0, 15,
30, and 47 min in a 30 ºC water bath to determine the incubation time required for
maximal phosphorylation (determined by western blot, described below). For ACC used
for activity assays, the 30 min incubation time was used routinely in the phosphorylation
step of the procedure.
Electrophoresis and Western Blots. To verify phosphorylation, the incubation
mixes from above were added to Laemmli’s solution and proteins separated on 5% SDSPAGE gels (Tris-HCl Ready Gels, Bio-Rad, Hercules, CA) and then transferred to
nitrocellulose membranes at 100 V for 50 min. Membranes were blocked in 5% dried
milk (Bio-Rad) in PBST (139 mM NaCl, 2.7 mM KH2PO4, 9.9 mM Na2HPO4, and 0.1%
Tween 20) for one hour. The membrane designated for phospho-ACC determination was
left overnight with phospho-ACC polyclonal antibody (Cell Signaling Technology,
6
Beverly, MA) in 5% nonfat dried milk solution (1:5,000 dilution). Both membranes were
washed twice in PBST and twice in PBS. The membrane designated for ACC
determination was then exposed to streptavidin-horseradish peroxidase conjugate
(Amerham Life Sciences, Arlington Heights, IL) in PBST (1:5,000 dilution) for 1 hr at
room temperature. The membrane designated for phospho-ACC determination was
exposed to horseradish peroxidase conjugated donkey anti-rabbit IgG (Amersham Life
Sciences) in 3% nonfat dried milk in PBST (1:1,500 dilution) for 1 hr at room
temperature. After being washed twice with PBST and twice with PBS, the membranes
were incubated in enhanced chemoluminescence-detection reagent and then visualized on
enhanced chemoluminescence hyperfilm (Amersham Life Sciences). Relative amounts
of ACC and phospho-ACC were then quantified using SigmaGel software (SPSS,
Chicago, IL).
ACC Activity Measurements. The ACC activity assay was performed on
phosphorylated ACC and non-phosphorylated ACC. Final concentrations in the assay
mix were 50 mM Hepes, pH 7.5, 1.5 mM MgSO4, 2 mM DTT, 4 mM ATP, 12.5 mM
KHCO3, 2 µCi [14C-bicarbonate], 20 mM magnesium acetate, 0.75 mg/ml fatty acid free
bovine serum albumin with varying amounts of citrate, acetyl-CoA and palimtoyl-CoA in
a final volume of 0.19 ml. In substrate (acetyl-CoA) activation curves citrate
concentration was 20 mM and acetyl-CoA concentration varied with values at 0, 0.0025,
0.005, 0.010, 0.025, 0.050, 0.100, 0.250, and 0.500 mM and the palmitoyl-CoA
concentration varied at 0, 0.001, 0.010 and 0.100 mM. For the citrate activation assay,
citrate concentration was varied at concentrations of 0.00, 0.20, 0.50, 1.00, 2.50, 5.00,
10.00, and 20.00 mM, acetyl-CoA was held at 0.500 mM, and two palmitoyl-CoA
7
concentrations of 0.00 and 0.01 mM were used. The reaction was started by addition of
0.01 ml phosphorylated or non-phosphorylated ACC. After 4 min incubation at 37 ºC the
reaction was stopped by addition of 50 µl 5 M HCl. After mixing and centrifugation 200
µl was added to a 7 ml scintillation vial and evaporated to dryness at 80 ºC. The residue
(containing malonyl-CoA) was dissolved in 0.4 ml water and mixed with 5.5 ml Ecolite
(ICN, Costa Mesa, CA) for determination of radioactivity. Preliminary experiments
indicated linearity with time and enzyme concentration in this range. Sigma Plot 8.0
Software (Jandel Scientific) for Enzyme Kinetics was utilized for analysis of the data and
preparation of curves of best fit.
Statistical Analysis. Statistical data for the family of inhibition or activity curves
was obtained by the Sigma Plot 8.0 Software (Jandel Scientific) including standard error
and R2 correlation coefficient for Enzyme Kinetics or Number Crunching Statistical
Software (NCSS) was used to perform analysis of variance and Fisher least significant
difference test (for comparison of 3 or more means). The paired t-test was used for
comparison of two means.
RESULTS
In order to determine the incubation time required for maximal phosphorylation
of ACC by AMPK, western blot technique was used. Using a phospho-ACC antibody,
membranes were probed to show the extent of phosphorylation as a function of time. As
a control, duplicate blots were probed with streptavidin to show total ACC, which
showed no change in ACC content as a function of time. The data from four
8
determinations are shown in Figure 1, a representative blot is also shown. From this
analysis it was determined that an incubation time of 30 min would be sufficient for
maximal phosphorylation of ACC by AMPK. Therefore this time was used for all
subsequent phosphorylations of ACC by AMPK.
In Figure 2 there are two families of ACC activity curves which were generated
by incubating purified skeletal muscle with varying acetyl-CoA concentrations and four
palmitoyl-CoA concentrations. Each curve represents five runs, each one from a separate
isolation of ACC. Using the Sigma Plot Enzyme Kinetic Software, the mixed partial
form of inhibition gave the best fit analysis with an R2 coefficient of 0.98. This implies
both competitive and non-competitive components of inhibition by palmitoyl-CoA on
ACC. As seen in the plots it is apparent that palmitoyl-CoA is a more effective inhibitor
after ACC has been phosphorylated by AMPK.
Table 1 shows the values generated from the mixed partial inhibition analysis.
Vmax, and Ka for acetyl-CoA were unaffected by AMPK phosphorylation while the Ki
for palmitoyl-CoA was not only statistically different but also markedly reduced. This
implies that when ACC is phosphorylated by AMPK less palmitoyl-CoA is needed for
greater inhibition.
In Figure 3, Vmax as a function of acetyl-CoA concentration for the four different
palmitoyl-CoA concentrations is shown. The reduction in the Vmax is proportionally
lower in the phosphorylated ACC compared to non-phosphorylated. These differences
were statistically significant for all of the palmitoyl-CoA concentrations (p<0.05).
Figure 4 shows ACC activity as a function of citrate concentration. It is shown
for non-phosphorylated and AMPK phosphorylated ACC with or without the presence of
9
0.01 mM palmitoyl-CoA. As is apparent in these curves there is a marked decline in
phosphorylated ACC activity in the presence of palmitoyl-CoA. In fact, statistically,
only the curve with both phosphorylation and an inhibitor present is significantly
different from the other three curves.
Figure 5 shows the effect of phosphorylation and palmitoyl-CoA at two specific
citrate concentrations of 20 and 0.5 mM. The effect of palmitoyl-CoA inhibition is
greater in the AMPK phosphorylated state than non-phosphorylated and this effect is
even more exaggerated at the lower citrate level which was used to demonstrate
physiological conditions. Statistically there was no difference at the high citrate level
between the phosphorylated and non-phosphorylated without palmitoyl-CoA present but,
there was a significant difference for all other phosphorylated and non-phosphorylated
pairs.
DISCUSSION
For the results, CPM/min was used to show activity instead of the more standard
protein concentration per rate. The reason why this was used was because of the inability
to maintain the stability of the ACC enzyme as seen in activity. During storage of ACC
after isolation the absolute activity would decrease in time. It was unfeasible to perform
activity assays directly after isolation because of the enormous time constraints both
processes required. As a result, activity would be lost even though protein concentration
would remain the same. This would make the calculation of protein content inconsistent
with activity. Therefore CPM/min was adopted as the form to be used for demonstrating
activity instead of converting to concentration of protein. An activity assay would be run
10
on a sample right before the actual assay and concentration adjustments would be made
from these results instead of using protein concentration for concentration adjustments.
The mixed partial form of inhibition was shown to have the highest correlation by
having the highest R2 coefficient. Not only does the mixed form of this inhibition infer
competitive and non-competitive components but it is also conceivable the enzyme can
produce product with the inhibitor attached. In fact competitive partial and
uncompetitive partial models also gave R2 coefficients above 0.9. This demonstrates a
strong partial component suggesting as stated in the results that there exists a palmitoylCoA inhibition site away from the active site where palmitoyl-CoA is able to influence
the activity of the enzyme. These data differ from the past data in which it was thought
palmitoyl-CoA influenced ACC in a completely competitive inhibitory manner (41).
In looking at metabolic diseases such as obesity and diabetes the study of
regulation of ACC has become more important. The recent formation of ACC-beta -/null mutant mouse phenotype demonstrated why there needs to be an understanding of
the natural regulatory mechanisms (2). These mice had less fat mass and higher rates of
fatty acid oxidation, while continuing to be fertile and have a normal life span(2). Even
when being fed high fat and high carbohydrate reduced weight gain and insulinsensitivity were attenuated (1).
In looking at the above data, using the natural methods of inactivating ACC beta
are plausible and important. Muscle contraction has been demonstrated to activate
AMPK, which then phosphorylates and inactivates ACC beta (17,29,31,42,48).
Contraction induced by electrical stimulation in rat muscle has been shown to decrease
malonyl-CoA levels (31,33,46-48). This decline in malonyl-CoA has been postulated to
11
be important in allowing fat oxidation to proceed in the working muscle (44-46,49). In
hindlimb perfusion studies, fatty acid oxidation increases when AMPK is chemically
activated using AICAR (23,24,50). A good correlation was observed between the rate of
palmitate oxidation and the malonyl-CoA content of muscle (23,24,50).
Palmitoyl-CoA has been reported to be an allosteric inhibitor of ACC isolated
from skeletal muscle (41). Skeletal muscle is not considered to be a lipogenic tissue and
the ACC beta is thought to be involved primarily in regulation of fatty acid oxidation.
During fasting the mechanism of increased fatty acid oxidation to provide the energy
needs of skeletal muscle is thought to be mediated through a decline in malonyl-CoA.
During prolonged exercise, it is also important that malonyl-CoA content of the muscle
be reduced to relieve inhibition of CPT1 and allow fatty acid oxidation to proceed to
provide ATP for muscle contraction. This contraction-induced decrease in malonyl-CoA
is thought to be mediated by AMPK phosphorylation/inactivation of ACC beta and by
AMPK induced activation of malonyl-CoA decarboxylase (37,38,44,45,49). Results of
the present study suggest that ACC phosphorylation not only produces direct covalent
modulation/inactivation, but also enhances the ability of palmitoyl-CoA to inhibit ACC
which reduces malonyl-CoA synthesis. The dual control insures that ACC will be
inactivated, allowing fatty acid oxidation to proceed.
Clark and colleagues (12) demonstrated that elevated fatty acids (palmitate and
oleate) can cause increased phosphorylation/activation of AMPK in perfused heart in the
absence of changes of the energy status of the cell as seen in content of AMP, ADP, ATP
and creatine phosphate. It is not known if this mechanism exists in skeletal muscle. It
would seem possible that elevated fatty acids would have a mechanism to facilitate their
12
own oxidation by phosphorylation and inactivation of ACC. While Clark used palmitate
and oleate to look at effects of AMPK and ACC activity, we only used palmitoyl-CoA as
a representative of fatty acids in our inhibition of ACC. Similar results of palmitoyl-CoA
activation of AMPK in liver were seen by Carling (11).
Fasting, high-fat diets, and exercise have been shown to increase rat skeletal
muscle long chain fatty acyl-CoA (15,43). Values in the range of 1.6 to 8.3 µmol/Kg
have been reported for rat muscle (15). A six fold increase of long chain acyl-CoA from
18 to 82 µmol/Kg dry mass has been demonstrated in humans during long term exercise
(43). From this study the sensitivity range of muscle acetyl-CoA carboxylase thus
appears to be in the range of fluctuation of long-chain acyl-CoA concentrations observed
in muscle. It is also apparent that a phosphorylation-induced decrease in the Ki for
palmitoyl-CoA inhibition of ACC would be expected to have physiologically relevant
consequences in inhibiting malonyl-CoA synthesis.
High glucose levels are present in skeletal muscle in situations of insulin
resistance and type 2 diabetes. Ruderman has demonstrated that by infusing rats with
high gluscose and insulin there were increases in the concentration of malonyl-CoA and
citrate (21,39). These high glucose levels are part of a fuel-sensing mechanism proposed
by Ruderman which lead to increases in citrate concentration that activates ACC, and to
high malonyl-CoA levels thus reducing fatty acid oxidation (36). It is apparent from our
data that the combination of AMPK phosphorylation with palmitoyl-CoA inhibits ACC
in the presence of high and physiological citrate concentrations, therefore possibly
shifting the fuel use of the cell when fatty acids are available and AMPK is activated.
13
Malonyl-CoA has previously been shown to be a competitive inhibitor for ACC in
our lab by Trumble et al (41). In that study the Ki for malonyl-CoA was found to be 10.6
± 1.0 µM (41). We did not assay the effect of malonyl-CoA on AMPK phosphorylated
ACC at this time. Future research in this area would provide valuable information on the
effect of AMPK phosphorylation of ACC activity when malonyl-CoA levels are elevated.
Elevated malonyl-CoA levels occur in insulin resistance. Ruderman specifically showed
elevated malonyl-CoA levels in hyperglycemic and hyperinsulinemic rodents (39).
Malonyl-CoA concentration should be noted as only one mechanism controlling
the rate of fatty acid oxidation in the muscle. Fatty acid concentration, availability of
carnitine, concentration of acetyl-CoA (feedback inhibitor of fatty acid oxidation via the
thiolase reaction), the availability of CoA in the mitochondrial matrix, and the overall
energy charge all must be considered factors influencing total muscle fatty acid oxidation
rate (19,45).
Another way to look at the data was to make inhibition curves for specific
concentrations of acetyl-CoA. This is very similar to the apparent Vmax data, but it
provides the opportunity to look at the lower concentrations of acetyl-CoA. This is
advantageous for two of reasons; first this information is obscured in the activity curves.
These graphs show the results and effectiveness of inhibition in lower acetyl-CoA
concentrations. The second reason that this information is important is it allows us to
look at the data in the physiological range of acetyl-CoA. This information is presented
in figure 6 which shows the inhibition curves for 0.005mM and 0.01mM. The slope of
the curve from the 0.0mM to 0.001 palmitoyl-CoA concentration is steeper for the nonphosphorylated curve compared to the phosphorylated curve. This demonstrates that at
14
low acetyl-CoA concentrations the AMPK phosphorylated ACC is already maximally
inhibited and increasing amounts of palmitoyl-CoA are required to exhibit the same
inhibitory influence. This not only demonstrates physiological importance but also
shows that the effects of these two separate inhibitory methods are not merely additive
but when combined produce and overall greater inhibition. The curves come together in
the high Palmitoyl-CoA concentration. This is probably due to the low acetyl-CoA
concentration and high palmitoyl-CoA concentrations.
In summary, palmitoyl-CoA was able to inhibit both non-phosphorylated and
AMPK-phosphorylated ACC isolated and purified from rat skeletal muscle. The
importance of palmitoyl-CoA as an inhibitor is seen by the dramatic decrease in Ki. Not
only did AMPK phosphorylation have a direct effect but also increased the sensitivity of
ACC to be inhibited by palmitoyl-CoA. There is also a decreased ability of citrate to
activate AMPK phosphorylated ACC when palmitoyl-CoA is present. This is important
during long-term exercise when AMPK is activated and there is elevated fatty acyl-CoA
in the muscle. Therefore exercise is an effective natural means to cause inactivation of
ACC beta, providing a method to prevent or reverse the effects of obesity and type 2
diabetes.
15
REFERENCES
1.
Abu-Elheiga L, Almarza-Ortega DB, Baldini A, and Wakil SJ. Human
acetyl-CoA carboxylase 2. Molecular cloning, characterization, chromosomal
mapping, and evidence for two isoforms. J Biol Chem 272:10669-10677, 1997.
2.
Abu-Elheiga L, Brinkley WR, Zhong L, Chirala SS, Woldegiorgis G, and
Wakil SJ. The subcellular localization of acetyl-CoA carboxylase 2. PNAS
97:1444-1449, 2000.
3.
Abu-Elheiga L, Matzuk MM, Abo-Hashema KAH, Wakil SJ. Continuous
fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA
carboxylase 2. Science 291:2613-2616, 2001.
4.
Abu-Elheiga L, Oh W, Kordari P, and Wakil SJ. Acetyl-CoA carboxylase 2
mutant mice are protected against obesity and diabetes induced by high-fat/highcarbohydrate diets. PNAS 100:10207-10212, 2003.
5.
Alam N, and Saggerson ED. Malonyl-CoA and the regulation of fatty acid
oxidation in soleus muscle. Biochem J 334:233-241, 1998.
6.
Allred JB and Reilly KE. Short-term regulation of acetyl-CoA carboxylase in
tissues of higher animals. Prog Lipid Res 35:371-385, 1996.
7.
Awan MM and Saggerson ED. Malonyl-CoA metabolism in cardiac myocytes
and its relevance to the control of fatty acid oxidation. Biochem J 295:61-66,
1993.
8.
Bavenholm PN, Pigon J, Saha AK, Ruderman NB, Efendic S. Fatty acid
oxidation and the regulation of malonyl-CoA in human muscle. Diabetes
49:1078-1083, 2000.
16
9.
Bianchi A, Evans JL, Everson AJ, Nordlund AC, Watts TD, and Witters LA.
Identification of an isozymic form of acetyl-CoA carboxylase. J Biol Chem
265:1502-1509, 1990.
10.
Boone AN, Rodriques B, and Brownsey RW. Multiple-site phosphorylation of
the 280 kDa isoform of acetyl-CoA carboxylase in rat cardiac myocytes:
evidence that cAMP-dependent protein kinase mediates effects of β-adrenergic
stimulation. Biochem J 341:347-354, 1999.
11.
Carling D, Zammit VA, Hardie DG. A common bicyclic protein kinase cascade
inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis.
FEBS Lett. 223(2):217-22, 1987
12.
Clark H, Carling D, and Saggerson D. Covalent activation of heart AMPactivated protein kinase in response to physiological concentrations of long-chain
fatty acid. Eur J Biochem 271:2215-2224, 2004.
13.
Dean D, Daugaard JR, Young ME, Saha A, Vavvas D, Asp S, Kiens B, Kim
K-H, Witters L, Richter EA, and Ruderman N. Exercise diminishes the
activity of acetyl-CoA carboxylase in human muscle. Diabetes 49:1295-1300,
2000.
14.
Dyck JR, Kudo N, Barr AJ, Davies SP, Hardie DG, and Lopaschuk GD.
Phosphorylation control of cardiac acetyl-CoA carboxylase by cAMP-dependent
protein kinase and 5’-AMP activated protein kinase. Eur J Biochem 262:184-190,
1999.
15.
Ellis BA, Poynten A, Lowy AJ, Furler SM, Chisholm DJ, Kraegen EW, and
Cooney GJ. Long-chain acyl-CoA esters as indicators of lipid metabolism and
insulin sensitivity in rat and human muscle. Am J Physiol 279:E554-E560, 2000.
17
16.
Hardie DG, and Carling D. The AMP-activated protein kinase. Fuel gauge of
the mammalian cell? Eur J Biochem 246:259-273, 1997.
17.
Hutber CA, Hardie DG, and Winder WW. Electrical stimulation inactivates
muscle acetyl-CoA carboxylase and increases AMP-activated protein kinase. Am
J Physiol 272:E262-E266, 1997.
18.
Iverson AJ, Bianchi A, Nordlund AC, and Witters LA. Immunological
analysis of acetyl-CoA carboxylase mass, tissue distribution and subunit
association. Biochem J 269:365-371, 1990.
19.
Jeukendrup AE. Regulation of fat metabolism in skeletal muscle. Ann NY Acad
Sci 967:217-235, 2002.
20.
Kim KH. Regulation of mammalian acetyl-CoA carboxylase. Annu Rev Nutr
17:77-99, 1997.
21.
Laybutt, D R, Schmitz-Peiffer S, Ruderman NB, Chisholm D, Biden T, and
Kraegen EW. Activation od protein kinase CЄ may contributeto muscle insulin
resistance induced by lipid accumulation during chronic glucose infusion in rats
(Abstract). Diabetes 46:214A, 1997
22.
McGarry JD, and Brown NF. The mitochondrial carnitine palmitoyltransferase
system from concept to molecular analysis. Eur J Biochem 244:1-14, 1997.
23.
Merrill GF, Kurth EJ, Hardie DG, and Winder WW. AICA riboside
increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake
in rat muscle. Am J Physiol 273:E1107-E1112, 1997.
24.
Merrill GF, Kurth EJ, Rasmussen BB, and Winder WW. Influence of
malonyl-CoA and palmitate concentration on rate of palmitate oxidation in rat
muscle. J Appl Physiol 85:1909-1914, 1998.
18
25.
Munday, MR. Regulation of mammalian acetyl-CoA carboxylase. Biochem Soc
Trans. 30: 1059-1064. 2002
26.
Odland LM, Heigenhauser GJ, and Spriet LL. Effects of high fat provision on
muscle PDH activation and malonyl-CoA content in moderate exercise. J Appl
Physiol, 89:2352-2358, 2000.
27.
Odland LM, Heigenhauser GJ, Lopaschuk GD, and Spriet LL. Human
skeletal muscle malonyl-CoA at rest and during prolonged submaximal exercise.
Am J Physiol 270:E541-E544, 1996.
28.
Odland LM, Howlett RA, Heigenhauser GJ, Hultman E, and Spriet LL.
Skeletal muscle malonyl-CoA content at the onset of exercise at varying power
outputs in humans. Am J Physiol 274:E1080-E1085, 1998.
29.
Ogiwara H, Tanbe T, Nikawa J, and Numa S. Inhibition of rat-liver acetylcoenzyme A carboxylase by palmitoyl-Coenzyme A. Eur J Biochem 89:33-41,
1978.
30.
Park SH, Gammon SR, Knippers JD, Paulsen SR, Rubink DS, and Winder
WW. Phosphorylation-activity relationships between AMPK and acetyl-CoA
carboxylase in muscle. J Appl Physiol 92:2475-2482, 2002.
31.
Rasmussen BB, Hancock CR, and Winder WW. Post-exercise recovery of
skeletal muscle malonyl-CoA, acetyl-CoA carboxylase, and AMP-activated
protein kinase. J Appl Physiol 85:1629-1634, 1998.
32.
Rasmussen BB, Holmback UC, Volpi E, Morio-Liondore B, Paddon-Jones D,
and Wolfe RR. Malonyl-Coenzyme A and the regulation of functional carnitine
palmitoyltransferase-1 activity and fat oxidation in human skeletal muscle. J Clin
Invest 110:1687-1693, 2002.
19
33.
Rasmussen BB, and Winder WW. Effect of exercise intensity on skeletal
muscle malonyl-CoA and acetyl-CoA carboxylase. J Appl Physiol 83:1104-1109,
1997.
34.
Rasmussen BB, and Wolfe RR. Regulation of fatty acid oxidation in skeletal
muscle. Annu Rev Nutr 19:463-484, 1999.
35.
Ruderman NB, Saha AK, Vavvas D, Kurowski T, Laybutt DR, SchmitzPeiffer C, Biden T, and Kraegen EW. Malonyl-CoA as a metabolic switch and
regulator of insulin sensitivity. Adv Exp Med Biol 441:263-270, 1998.
36.
Ruderman NB, Saha AK, Vavvas D, and Witters LA. Malonyl CoA, fuel
sensing and insulin resistance. Am J Physiol. 276:E1-E18. 1999
37.
Saha AK, and Ruderman NB. Malonyl-CoA and AMP-activated protein
kinase: an expanding partnership. Mol Cell Biochem 253:65-70, 2003.
38.
Saha AK, Schwarsin AJ, Roduit R, Μassé F, Kaushik V, Tornheim K,
Prentki M, and Ruderman NB. Activation of malonyl-CoA decarboxylase in
rat skeletal muscle by contraction and the AMP-activated protein kinase activator
aminoimidazole-4-carboxamide-1-β-D-ribofuranoside. J Biol Chem 275:2427924283, 2000.
39.
Saha AK, Vavvas D, Laybutt D, Chisholm D, Kraegen E, and Ruderman NB.
Increases in cytossolic citrate regulate malonyl CoA levels in insulin-resistant
muscles in vivo (Abstract). Diabetes 46: 235A, 1997
40.
Thampy, KG and SJ Wakil. Regulation of acetyl-coenzyme A carboxylase. I.
Purification and properties of two forms of acetyl-coenzyme A carboxylase from
rat liver. J Biol Chem. 263: 6447-6453. 1988
20
41.
Trumble GE, Smith MA, and Winder WW. Purification and characterization
of rat skeletal muscle acetyl-CoA carboxylase. Eur J Biochem 231:192-198,
1995.
42.
Vavvas D, Apazidis A, Saha AK, Gamble J, Patel A, Kemp BE, Witters LA,
and Ruderman NB. Contraction-induced changes in acetyl-CoA carboxylase
and 5'-AMP-activated kinase in skeletal muscle. J Biol Chem 272:13256-13261,
1997.
43.
Watt MJ, Heigenhauser GJF, O’Neill M, Spriet LL. Hormone-sensitive lipase
activity and fatty acyl-CoA content in human skeletal muscle during prolonged
exercise. J Appl Physiol 95:314-321, 2003.
44.
Winder WW. Energy sensing and signaling by AMP-activated protein kinase in
skeletal muscle. J Appl Physiol 91:1017-1028, 2001.
45.
Winder WW. Intramuscular mechanisms regulating fatty acid oxidation during
exercise. Adv Exp Med Biol 441:239-248, 1998.
46.
Winder WW. Malonyl-CoA - Regulator of fatty acid oxidation in muscle during
exercise. Exer Sports Sci Rev 26:117-132, 1998.
47.
Winder WW, Arogyasami J, Elayan IM and Cartmill D.
Time course of the
exercise-induced decline in malonyl-CoA in different muscle types. Am J Physiol
259: E266-E271, 1990.
48.
Winder WW, and Hardie DG. Inactivation of acetyl-CoA carboxylase and
activation of AMP-activated protein kinase in muscle during exercise. Am J
Physiol 270: E299-E304, 1996.
21
49.
Winder WW, and Hardie DG. AMP-activated protein kinase, a metabolic
master switch: possible roles in Type 2 diabetes. Am J Physiol 277:E1-E10,
1999.
50.
Winder WW, and Holmes BF. Insulin stimulation of glucose uptake fails to
decrease palmitate oxidation in muscle if AMPK is activated. J Appl Physiol
89:2430-2437, 2000.
51.
Winder WW, Wilson HA, Hardie DG, Rasmussen BB, Hutber CA, Call GB,
Clayton RD, Conley LM, Yoon S, and Zhou B. Phosphorylation of rat muscle
acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A. J
Appl Physiol 82:219-225, 1997.
22
Table 1. Effect of phosphorylation of ACC with AMP-Activated Protein Kinase on
calculated Vmax, Km, and Ki
Non-Phosphorylated
Phosphorylated
Vmax (cpm/min)
3158 ± 61
2768 ± 65
Km (acetyl-CoA) (µM)
45 ± 3
54 ± 4
Palmitoyl-CoA Ki (µM)
1.7 ± 0.25
0.85 ± 0.13*
*Significantly different from Non-Phosphorylated ACC, p < 0.01 (n = 5)
23
5
Relative Units
4
Phospho-ACC
3
2
ACC
1
0
0
15
30
45
Time (min)
Figure 1. Western blots of Phospho-ACC (top blot) and ACC (bottom blot) at times
following addition of AMPK buffer or buffer contanig no AMPK to the reaction
mix. The graph shows summary of mean data from 4 determinations (n = 4 at each
point). Values are means +/- sem and are significantly different at 15, 30, and 45
min (p<0.001)
24
3500
Non-Phosphorylated ACC
Rate (cpm/min)
3000
2500
2000
1500
1000
0 mM Palmitoyl-CoA
0.001 mM Palmitoyl-CoA
0.01 mM Palmitoyl-CoA
0.1 mM Palmitoyl-CoA
500
0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.4
0.5
0.6
3500
Phosphorylated ACC
Rate (cpm/min)
3000
2500
2000
1500
1000
500
0
0.0
0.1
0.2
0.3
Acetyl- CoA (mM)
Figure 2. Substrate saturation curves of non-phosphorylated ACC and of AMPKphosphorylated ACC at different concentrations of palmitoyl-CoA (n = 5 ACC
activity assay each from a separate isolations at each point).
25
Vmax (cpm/min)
3000
Non-Phosphorylated
Phosphorylated
2500
*
2000
1500
*
*
1000
500
0
0.0
1.0x10-3
10.0x10-3
100.0x10-3
[Palmitoyl-CoA], mM
Figure 3. Effect of palmitoyl-CoA concentration on Vmax of non-phosphorylated
ACC and of AMPK-phosphorylated ACC (n = 5 isolations of ACC which were used
for non-phosphorylated and phosphorylated ACC at each palmitoyl-CoA
concentration). * Significant difference from non-phosphorylated pair, p<0.05.
26
1200
Non-Phos & 0.00 mM Palm
1000
Rate (cpm)
Phos & 0.00 mM Palm
800
Non-Phos & 0.01 mM Palm
600
400
* Phos & 0.01 mM Palm
200
0
0
5
10
15
20
25
citrate (mM)
Figure 4. Citrate activation saturation curves of non-phosphorylated ACC and of
AMPK- phosphorylated ACC at 0.0 mM and 0.01 mM concentrations of palmitoylCoA (n = 5 ACC activity runs all from the same isolation batch). * Significant
difference from non-phosphorylated pair at same palmitoyl-CoA concentrations,
p<0.05.
27
20 mM Citrate
400
Non-Phosphorylated
Phosphorylated
1500
1000
*
Rate (cpm/min)
Rate (cpm/min)
2000
0.5 mM Citrate
300
200
*
500
100
*
0
0.0 mM Palm
0.01 mM Palm
0
0.0 mM Palm
0.01 mM Palm
Figure 5. Activity of non-phosphorylated and AMPK phosphorylated ACC at 0.0
mM and 0.01 mM palmitoyl-CoA concentrations and at 20 mM and 0.5 mM citrate
concentrations. (n=5 different runs under the same conditions from same ACC
isolation batch for each point). * Significant difference from non-phosphorylated
pair at the same citrate and palmitoyl-CoA concentrations, p<0.05.
28
0.005 mM Acetyl-CoA
Activity in CPM
400
Non-Phosphorylated
Phosphorylated
300
200
100
0
0
1e-3
1e-2
1e-1
Palmitoyl-CoA Concentration
0.01mM Acetyl-CoA
800
Non-Phosphorylated
Phosphorylated
Activity in CPM
600
400
200
0
0
1e-3
1e-2
1e-1
Palmitoyl_CoA Concentration
Figure 6. Inhibition Curves. These are inhibition curves for two concentrations of
Acetyl-CoA one at top 0.05mM and bottom 0.01mM against four palmitoyl-CoA
concentration of 0.00, 0.10, 0.01 and 0.001mM. The curves show activity vs.
palmitoyl-CoA concentration. Open circles are non-phosphorylatyed closed circle
phosphorylated. Analysis of variance showed no significant difference but paired Ttest for phosphorylated vs. non-phosphorylated at the same palmitoyl-CoA showed
significant difference (p<0.05).