Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 82: 219-225, 1997;
8750-7587/97 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Winder, W. W.
Right arrow Articles by Zhou, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Winder, W. W.
Right arrow Articles by Zhou, B.

Journal of Applied Physiology
Vol. 82, No. 1, pp. 219-225, January 1997
EXERCISE AND MUSCLE

Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A

W. W. Winder1, H. A. Wilson1, D. G. Hardie2, B. B. Rasmussen1, C. A. Hutber1, G. B. Call1, R. D. Clayton1, L. M. Conley1, S. Yoon1, and B. Zhou1

1 Department of Zoology, Brigham Young University, Provo, Utah 84602; and 2 Department of Biochemistry, The University of Dundee, Dundee DD1 4HN, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

Winder, W. W., H. A. Wilson, D. G. Hardie, B. B. Rasmussen, C. A. Hutber, G. B. Call, R. D. Clayton, L. M. Conley, S. Yoon, and B. Zhou. Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A. J. Appl. Physiol. 82(1): 219-225, 1997---This study was designed to compare functional effects of phosphorylation of muscle acetyl-CoA carboxylase (ACC) by adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) and by AMP-activated protein kinase (AMPK). Muscle ACC (272 kDa) was phosphorylated and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Functional effects of phosphorylation were determined by measuring ACC activity at different concentrations of each of the substrates and of citrate, an activator of the enzyme. The maximal velocity (Vmax) and the Michaelis constants (Km) for ATP, acetyl-CoA, and bicarbonate were unaffected by phosphorylation by PKA. Phosphorylation by AMPK increased the Km for ATP and acetyl-CoA. Sequential phosphorylation by PKA and AMPK, first without label and second with label, appeared to reduce the extent of label incorporation, regardless of the order. The activation constant (Ka) for citrate activation was increased to the same extent by AMPK phosphorylation, regardless of previous or subsequent phosphorylation by PKA. Thus muscle ACC can be phosphorylated by PKA but with no apparent functional effects on the enzyme. AMPK appears to be the more important regulator of muscle ACC.

carnitine palmitoyl transferase; fatty acid oxidation by muscle; malonyl-CoA

INTRODUCTION

IN LIVER, acetyl-CoA carboxylase (ACC) catalyzes the first committed step in lipogenesis (10). The product, malonyl-CoA, allosterically inhibits carnitine palmitoyl transferase 1 (CPT-1), thereby inhibiting fat oxidation when fatty acid synthesis is occurring at high rates (14, 16). The liver and adipose tissue isoforms of the enzyme can be phosphorylated by several different kinases, including adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), AMP-activated protein kinase (AMPK), and protein kinase C (PKC) (4, 10, 11). Phosphorylation of ACC by AMPK and PKA has been demonstrated to produce changes in functional characteristics of the enzyme, including decreases in maximal velocity (Vmax) and increases in the Michaelis constant (Km) (10, 11). At physiological concentrations of ATP, acetyl-CoA, bicarbonate, and citrate, the changes in these kinetic properties would be expected to produce marked decreases in ACC activity and the rate of malonyl-CoA synthesis.

Previous studies have demonstrated the presence of a 272- to 275-kDa isoform of ACC in rat and human skeletal muscle (2, 21, 22, 34), which is considered to be a nonlipogenic tissue. The muscle ACC appears to be regulated differently than the principal liver isoforms, in that it does not increase from the condition of fasting to a refed state (with high-carbohydrate diet) (33). This system has been postulated to be present in muscle for the purpose of regulation of fatty acid oxidation. In support of this hypothesis, malonyl-CoA has been found to decrease in skeletal muscle during exercise and in response to fasting and electrical stimulation (8, 28). More recently, in vitro studies have demonstrated that avidin affinity column-purified skeletal muscle ACC can be phosphorylated by liver AMPK (31). This phosphorylation induces a decrease in ACC Vmax and an increase in the citrate concentration required to produce half-maximal activation (K0.5) of the enzyme. Similar changes in kinetic constants of ACC occur in hindlimb muscle of the rat during a single bout of exercise. The increase in K0.5 for citrate and decrease in Vmax of muscle ACC were accompanied by an increase in the activity of AMPK partially purified from muscle extracts of the exercised rats. These results imply that ACC is phosphorylated and inactivated in exercising skeletal muscle. The muscle and heart CPT-1 is even more sensitive to malonyl-CoA inhibition than is the liver CPT-1 (13-15, 25). It has been suggested (27, 28, 31) that the decrease in malonyl-CoA is one of the important signals for increased fatty acid oxidation (19) in muscle during exercise.

Previous studies have also demonstrated that cAMP increases in skeletal muscle during prolonged exercise and that this increase is due to an increase in plasma epinephrine (27, 30). Hence, phosphorylation of ACC by PKA (as well as by AMPK) could also be responsible in part for the changes noted in ACC activity in extracts from exercising muscle. The purpose of the present study was to determine whether muscle ACC can be phosphorylated by PKA. We also studied the effects of PKA and AMPK phosphorylation on kinetic constants of ACC for each of the substrates and for the activator citrate. Possible interaction between effects of the two kinases was investigated by studying sequential phosphorylation by PKA and AMPK.

MATERIALS AND METHODS

Isolation of ACC. ACC was isolated from quadriceps and gastrocnemius muscles of the rat hindlimb. Rats (body wt = 350-500 g) were anesthetized (pentobarbital sodium intraperitoneally) for at least 30 min before removing muscles. The muscles were cooled rapidly between aluminum blocks at near 0°C. Muscle groups were separated and fat and connective tissue were removed. Muscle was minced with scissors and suspended in cold buffer A [(in mM) 225 mannitol, 75 sucrose, 10 tris(hydroxymethyl)aminomethane (Tris) · HCl, 0.05 EDTA, 5 potassium citrate, 2.5 MnCl2, pH 7.5, with 10 mg/l of leupeptin and antitrypsin and 10 ml/l of aprotinin (Sigma Chemical, St. Louis, MO)] in a ratio of 6-9 ml buffer per gram muscle. The muscle (from 10 to 12 rats) was then homogenized by using a Brinkmann PT-1000 tissue homogenizer using a PT-DA 3020/2 generator. After centrifugation at 17,000 g for 40 min, the supernatant was collected. The ACC was precipitated by adding 200 g ammonium sulfate/l and stirring at 4°C for 1 h. The precipitate was collected by centrifuging at 17,000 g for 30 min and then resuspended in minimal volume of buffer containing 100 mM Tris · HCl, 0.5 M NaCl, 1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol, pH 7.5, to which was added 10 mg antitrypsin (Sigma Chemical), 10 mg leupeptin (Sigma Chemical), and 10.0 ml aprotinin (9 trypsin inhibitor units/ml) (Sigma Chemical) per liter. After centrifugation to remove insoluble material, the resuspended precipitate was dialyzed (by using Spectra/Por CE membrane; mol wt cut off: 15,000) for at least 5 h at 4°C against column buffer (100 mM Tris · HCl, 0.5 M NaCl, 1 mM EDTA, 0.1 mM dithiothreitol, 5% glycerol, pH 7.5) and then purified by avidin-Sepharose affinity chromatography, using Promega SoftLink Soft Release Avidin Resin (Fisher Scientific, Pittsburgh, PA). After addition of the resuspended precipitate to the column, the column was washed with 30 volumes of column buffer to remove nonbiotin-containing proteins. The ACC was then eluted with column buffer containing 5 mM biotin. Fractions containing ACC activity were pooled, mixed, and then stored frozen at -80°C in 100-µl aliquots. Purity was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on each lot. The principal isoform was the 272-kDa species as previously reported (22).

Phosphorylation of ACC. For in vitro phosphorylation studies, globulin-free albumin (Sigma Chemical) was added (1 µg/µl) to the purified enzyme (1-2 µg protein in 0.1 ml), followed by an equal volume of cold, saturated ammonium sulfate. Poor yields were noted in the absence of albumin. The mixture was allowed to stand for 15 min on ice and was then centrifuged at 48,000 g for 15 min to collect the precipitate. After the supernatant was discarded, the precipitate was resuspended and utilized for phosphorylation studies. Final concentrations in the assay tubes were (in mM) 34 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 68 NaCl, 0.68 EDTA, 0.68 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.68 dithiothreitol, 6.8% glycerol, 0.12 ATP, 3 MgCl2, pH 7.0. To this mixture was added 20 µCi of [gamma -32P]ATP ± 6 units of the catalytic subunit of bovine heart PKA (Sigma Chemical). For the AMPK studies, the reaction mix was the same as for PKA with or without the addition of AMPK (5 U/ml) and 0.2 mM AMP. AMPK was isolated from rat liver as far as the gel filtration step (3). The final volume in the assay mix was 60 µl. After incubating at 30°C for 1 h, the reaction was terminated by addition of 60 µl saturated ammonium sulfate. The mixture was allowed to stand for 15 min and then was centrifuged at 48,000 g for 15 min. The supernatant was discarded, and the precipitate was washed with 1 ml 1:1 saturated ammonium sulfate, HEPES buffer. For electrophoresis, the final precipitate was resuspended in 25 µl of HEPES buffer to which 50 µl of electrophoresis sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 2.5% SDS, 5% beta -mercaptoethanol, 0.025% bromphenol blue) were added. After being heated at 95°C for 4 min, the samples were subjected to PAGE with the Bio-Rad Mini-Protean II Dual Slab Vertical Electrophoresis System, using Mini-Protean II 4-15% gradient precast gels (Bio-Rad, Richmond, CA). Gels were run in the presence of 0.1% SDS, 25 mM Tris, 192 mM glycine, pH 8.3 at 160 V for 75 min. Gels were stained by using a silver stain kit (Sigma Chemical) and were then dried for autoradiography (X-OMAT AR Scientific Imaging Film, Kodak).

An additional experiment was done to make certain the PKA was responsible for the phosphorylation of ACC. PKA inhibitor (rabbit sequence, Sigma Chemical) (2 µg) was added to the phosphorylation medium before addition of PKA. After a 60-min incubation, the mixture was precipitated with ammonium sulfate, resuspended, and subjected to SDS-PAGE followed by autoradiography.

Two other kinases, phosphorylase kinase and PKC (Sigma Chemical), were also tested to determine whether ACC is a substrate. Conditions were the same as for PKA except for addition of calcium (1.5 mM for phosphorylase kinase and 2.0 mM for PKC). PKC tubes also had 50 µg/ml phosphatidyl serine and 10 µM diacylglycerol.

ACC activity measurements. To determine functional effects of phosphorylation, ACC was incubated with and without PKA or with and without AMPK and AMP in the same reaction mix in the absence of radiolabeled ATP. ACC activity was determined at the end of the phosphorylation, with no ammonium sulfate precipitation. ACC activity was determined by measuring rate of incorporation of [14C]bicarbonate into acid-stable compounds (malonyl-CoA) at 37°C for 2 min. Final concentrations of reagents were (in mM) 50 HEPES buffer, pH 7.5, 1.5 MgSO4, 2 dithiothreitol, 0.25 acetyl-CoA, 4 ATP, 12.5 KHCO3, 2 µCi [14C]bicarbonate, and 0.75 mg/ml fatty acid free bovine serum albumin. Citrate and magnesium acetate were added in equimolar concentrations ranging from 0 to 20 mM. Substrate concentrations were varied singly (with other substrates at saturating concentrations) for determination of kinetic constants. ATP/MgSO4 concentrations were varied between 0 and 4 mM. In the case of ATP, an additional precipitation of the enzyme was required after the phosphorylation reaction for removal of ATP from the medium before ACC activity measurement. Alternatively, the ATP added to the incubation tube along with the ACC was accounted for in the final calculation of concentration. Bicarbonate was varied between 0 and 12.5 mM. Acetyl-CoA was varied between 0 and 500 µM. The reaction was started by addition of ACC. The final reaction volume was 200 µl. The reaction was stopped by addition of 50 µl of 5 N HCl. After centrifugation, 200 µl were transferred to a scintillation vial and evaporated to dryness at 80°C. The residue was dissolved in 0.4 ml water and then mixed with 5.5 ml Scintiverse (Fisher Scientific) or Ecolite (ICN) for determination of radioactivity. Preliminary experiments indicated linearity with time and enzyme concentration in this range. The citrate data were fitted to the Hill equation using the Grafit program (Sigma Chemical). This program allows determination of the activation constant (Ka) for citrate, the maximal activity as a function of citrate concentration (Vmax), and the citrate concentration required for K0.5 of ACC. The enzyme activity data for substrates were fitted to the Michaelis-Menton equation; Vmax and Km were determined using the Grafit software. Kinetic constants for both nonphosphorylated and phosphorylated ACC (from the same lot of ACC) were determined in parallel on any 1 day. Because the calculated Vmax varied, depending on the protein content and activity of each lot of enzyme, means of kinetic constants for the two treatment groups were compared, by using Student's t-test for paired observations.

Sequential phosphorylation by PKA and AMPK. When it was found that PKA phosphorylation had no effect on activity of ACC, a series of sequential phosphorylations was done to determine whether prior PKA phosphorylation would influence effects of AMPK phosphorylation on the enzyme. In this series, the same incubation conditions were used as described above except that the labeled ATP was not included for the first 30 min of incubation with or without PKA or AMPK. At the end of 30 min, the other kinase was added along with labeled ATP. This design allows determination whether prior phosphorylation by one kinase alters extent of phosphorylation by the other kinase. If the two kinases share one or more phosphorylation sites, or if phosphorylation by one kinase sterically interferes with phosphorylation by the other kinase, we would expect to see a diminished label incorporation by the second kinase. This can be detected after SDS-PAGE followed by autoradiography. The same protocol was followed for a study on citrate dependence to determine whether sequential phosphorylation has functional effects on the enzyme.

RESULTS

In vitro phosphorylation studies. Figure 1 shows the silver-stained SDS-PAGE gels (A and B, left) and autoradiographs (A and B, right) of corresponding lanes after the phosphorylation reaction. Little phosphorylation was noted in the absence of PKA (B). Addition of PKA to the medium resulted in significant incorporation of the labeled phosphate into the 272-kDa-band region, indicated on the autoradiograph (Fig. 1, A, right). No significant accumulation of labeled phosphate was seen at any other position on the gel, with the exception of some that remained at the origin in both PKA and control lanes. PKA inhibitor (rabbit sequence) completely inhibited phosphorylation of ACC by PKA (autoradiographs not shown).
Fig. 1. Silver-stained gel and autoradiograph of the dried gel from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of acetyl-CoA carboxylase (ACC) isolated from hindlimb skeletal muscle by avidin-Sepharose affinity chromatography and subjected to phosphorylation for 60 min, as described in MATERIALS AND METHODS. Lane A: ACC was exposed to all reagents in the phosphorylation mixture, including protein kinase A (PKA). Lane B: ACC was exposed to all reagents except PKA. Similar results were observed on 4 different preparations of ACC.
[View Larger Version of this Image (94K GIF file)]

Figure 2 shows the autoradiograph of six lanes of a dried SDS-PAGE gel. Lanes A, C, and E represent ACC incubated with the phosphorylation mix without kinase. Lane B shows phosphorylation of ACC with PKA. Lane D indicates that ACC is not a substrate for phosphorylase kinase under these conditions. Lane F shows a slight degree of phosphorylation of skeletal muscle ACC by PKC. The dark bands indicating labeled phosphate incorporation into lower molecular weight proteins show that both phosphorylase kinase and PKC were active under these conditions.
Fig. 2. Autoradiograph of dried gel from SDS-PAGE of ACC isolated from hindlimb muscle by avidin-Sepharose affinity chromatography and subjected to phosphorylation for 60 min, as described in MATERIALS AND METHODS. Lanes A, C, and E: ACC was treated with phosphorylation mixture in absence of kinase. Lane B: ACC was treated with PKA. Lane D: ACC was treated with phosphorylase kinase. Lane F: ACC was treated with PKC.
[View Larger Version of this Image (99K GIF file)]

Figure 3 shows the effect of phosphorylation by PKA on ACC activity in the presence of variable concentrations of each substrate. The curves represent the theoretical relationship between substrate concentration and activity, based on the Michaelis-Menton equation. Each point on each curve represents five determinations. For clarity, SE are not shown on these curves, but means and SE for kinetic constants are shown in Table 1. It seems clear that no significant changes occurred in any of the kinetic constants for any of the substrates in response to phosphorylation by PKA.
Fig. 3. Effect of phosphorylation by PKA (bullet ) on muscle ACC activity in presence of variable concentrations of substrate. open circle , No PKA treatment. Each point represents 5 determinations. The fitted curves are generated from the Michaelis-Menton equation, using Grafit.
[View Larger Version of this Image (18K GIF file)]

Table 1. Effect of phosphorylation of skeletal muscle acetyl-CoA carboxylase by PKA on kinetic constants for substrates

Nonphosphorylated Phosphorylated with PKA
Vmax   7.17 ± 0.81 µmol ·   mg-1 · min-1   7.27 ± 0.76 µmol ·   mg-1 · min-1
Km, acetyl-CoA 37.1 ± 2.5 µM 37.7 ± 2.9 µM
Km, ATP 61.7 ± 2.4 µM 59.4 ± 2.8 µM
Km, bicarbonate 2.87 ± 0.23 mM 2.95 ± 0.24 mM
Values are means ± SE for 5-6 determinations except for maximum velocity (Vmax) of enzyme reactions, where n = 22. PKA, protein kinase A; Km, Michaelis constant. No significant differences were noted using Student's t-test for paired observations.

Figure 4 shows the effect of phosphorylation by AMPK on ACC activity in the presence of variable concentrations of the substrates. In contrast to the effects of PKA, marked changes in the Km for ATP and acetyl-CoA occurred in response to phosphorylation by AMPK (Table 2).
Fig. 4. Effect of phosphorylation by AMP-activated protein kinase (AMPK; bullet ) on muscle ACC activity in presence of variable concentrations of each substrate. open circle , No AMPK treatment. Each point represents 5-6 determinations. Fitted curves are generated from the Michaelis-Menton equation, using Grafit.
[View Larger Version of this Image (18K GIF file)]

Table 2. Effect of phosphorylation of skeletal muscle acetyl-CoA carboxylase by AMPK on kinetic constants for substrates

Nonphosphorylated Phosphorylated with AMPK
Vmax   5.84 ± 0.83 µmol ·   mg-1 · min-1   4.92 ± 0.61 µmol ·   mg-1 · min-1
Km, acetyl-CoA 35.5 ± 1.3 µM 51.5 ± 2.5 µM
Km, ATP 54.4 ± 2.3 µM 147.1 ± 11.0 µM
Km, bicarbonate 2.84 ± 0.07 mM 3.15 ± 0.15 mM
Values are means ± SE for 5-6 determinations. AMPK, AMP-activated protein kinase. All values for phosphorylated acetyl-CoA carboxylase (ACC) are significantly different from those of the nonphosphorylated ACC (P < 0.05) using t-test for paired observations.

Figure 5 shows the effect of sequential phosphorylation by PKA and AMPK. Prior treatment of muscle ACC with PKA in the absence of labeled ATP (nonlabeled ATP in reaction mix) resulted in diminished incorporation of label by AMPK (compare lanes A and B). Radioactivity in the 272-kDa band was reduced by 31%. Similarly, prior treatment of ACC with AMPK in the absence of labeled ATP caused decreased incorporation of label (-36%) into ACC catalyzed by PKA (compare lanes C and D). Incorporation of label into the 272-kDa band by PKA was 57% of that induced by AMPK.
Fig. 5. Sequential phosphorylation of muscle ACC. Muscle ACC was treated with no kinase (lane A) or with PKA (lane B) for 30 min in absence of 32P-labeled ATP and then for an additional 30 min with AMPK in presence of 32P-labeled ATP. ACC was treated with no kinase (lane C) or with AMPK (lane D) for 30 min in absence of 32P-labeled ATP and then with PKA for an additional 30 min in presence of 32P-labeled ATP. Similar results were obtained with 6 additional determinations on 3 different enzyme preparations.
[View Larger Version of this Image (83K GIF file)]

Figure 6 shows functional effects of sequential phosphorylation on citrate dependence of muscle ACC. Note that AMPK phosphorylation produces a marked change in the Ka for citrate activation and that PKA does not. AMPK phosphorylation produces nearly identical changes in Ka for citrate activation, regardless of the order of kinase treatment.
Fig. 6. Effect on citrate dependence curves and activation constants (Ka) for citrate (mM) of phosphorylation of muscle ACC by PKA alone (second 30 min; E), AMPK alone (second 30 min; B), and in sequence with AMPK first (30 min; D) and then with PKA first (30 min) followed by incubation with the other kinase an additional 30 min (F). A: purified enzyme not incubated with phosphorylation medium. B: enzyme incubated with AMPK. C: enzyme incubated for 60 min without kinase. Curves and Ka were generated from activity data, using a modified form of Hill equation. Each point on each curve represents the mean of 4 separate determinations on 2 different enzyme preparations. All Ka for AMPK-treated ACC are significantly different (P < 0.001) from those not treated with AMPK. ACC treated with PKA as well as with AMPK (D and F) showed no significant differences from ACC treated with AMPK alone (B).
[View Larger Version of this Image (27K GIF file)]

DISCUSSION

Previous studies have demonstrated that PKA phosphorylates liver ACC at serines -77 and -1200 (7, 11). Phosphorylation at these sites was accompanied by an increase in K0.5 for citrate activation and a decrease in Vmax for ACC as a function of citrate concentration. The critical site for PKA-induced changes in activity of the 260-kDa isoform of ACC appears from site-directed mutagenesis studies to be serine 1200 (9). A more recent report giving the partial amino acid sequence of human 272-kDa ACC shows serine 1200 to be missing in this isoform (26). In intact liver cells in culture, the inactivation of ACC by glucagon was found to be due to phosphorylation by the AMPK (which phosphorylates at serines 79, 1200, and 1215) instead of by the PKA (17). Hence, the role of the PKA in regulation of liver ACC is also not well defined.

Heart ACC (280 kDa) is also inactivated by phosphorylation, evidenced by an increase in activity after treatment with a phosphatase (20). AMPK increases in isolated perfused working rat hearts concurrently with a decrease in ACC activity (12). In isolated perfused rat hearts, the malonyl-CoA content correlates negatively with the rate of fatty acid oxidation (15). Fatty acid oxidation in isolated myocytes increases in response to inclusion of epinephrine in the medium (containing insulin and glucose), implying a role for PKA in regulation of rat heart ACC (1). The possibility of epinephrine regulation at other sites, such as malonyl-CoA decarboxylase modulation, is also recognized.

It is clear from the present study that the muscle ACC (272 kDa) is a substrate for PKA under in vitro conditions. It was anticipated that with phosphorylation by PKA, conformational changes would occur in the ACC, resulting in alteration of accessibility of substrates to the active site. This would be expected to alter the kinetic properties of the enzyme with respect to the three substrates and to the activator citrate. The fact that the Vmax, Km for substrates, and the K0.5 for citrate activation were not significantly altered implies that neither the active site nor the citrate-binding domain of ACC was affected by PKA phosphorylation.

The current study is consistent with results obtained from adrenodemedullated exercising rats (29). To determine the role of epinephrine in causing the decrease in malonyl-CoA in muscle during exercise, rats were adrenodemedullated or sham operated and then subjected to treadmill running. The adrenodemedullated rats, which showed no increase in plasma epinephrine during exercise, had the same decrease in malonyl-CoA as sham-operated rats, which showed an increase in epinephrine during exercise. Previous studies (30, 32) have demonstrated that the rise in muscle cAMP seen during exercise or during insulin-induced hypoglycemia is completely prevented in adrenodemedullated rats. Thus the increase in epinephrine is not only unessential for decreasing malonyl-CoA, but the present study indicates that PKA has no detectable effect on catalytic properties of ACC. It thus appears that the AMPK is more likely responsible for the regulation of ACC activity in muscle.

The AMPK gene is highly expressed in skeletal muscle (18, 23), although the enzyme activity in skeletal muscle extracts is quite low (5). During exercise, the AMPK is activated in skeletal muscle (31). This activation of AMPK is accompanied by a decreased ACC activity and a decrease in malonyl-CoA. We postulated that in response to a contraction-induced increase in free calcium or free 5'-AMP in the muscle, an AMPK kinase is activated which then phosphorylates and activates the AMPK (31). A similar mechanism has already been described for activation of liver AMPK (24). It has also been determined recently that 5'-AMP decreases the rate of inactivation of AMPK by protein phosphatase-2C and by protein phosphatase-2A (6). AMPK activation by whatever mechanism would ultimately result in the decrease in malonyl-CoA and an increase in fatty acid oxidation.

Two calcium-activated kinases were tested with respect to capacity for phosphorylating muscle ACC. The results indicate that if phosphorylase kinase is involved in control of ACC, it is not likely to be via direct phosphorylation of the enzyme. Possible phosphorylation of the AMPK has not been ruled out by these experiments. The small degree of phosphorylation by the PKC is unlikely to have regulatory effects (see Ref. 7), although this has not been examined carefully.

A previous report from our laboratories showed marked effects of phosphorylation by AMPK on citrate dependence of ACC (31). Particularly marked decreases in ACC activity occurred at physiological concentrations of citrate. The current studies demonstrate marked effects of phosphorylation of ACC by AMPK on Km for acetyl-CoA and ATP. The changes in kinetic properties of ACC for these substrates may also contribute to inactivation of ACC and decrease in malonyl-CoA production in skeletal muscle during exercise.

Sequential phosphorylation of liver ACC by PKA followed by AMPK results in decreased phosphorylation at the serine-79 site, one that is unique for the AMPK (7). Presumably, phosphorylation of serine 77 by the PKA interferes sterically with phosphorylation of serine 79 by AMPK. The decrease in ACC activity seen with AMPK phosphorylation is partially attenuated when ACC is first treated with PKA. Although specific phosphorylation sites have not been identified for the muscle isoform of ACC, the possibility remained that AMPK effects may be modulated by PKA. In searching for a physiological role for muscle ACC phosphorylation by PKA, we considered the possibility of modulation of AMPK effects. The mutual interference of each kinase on phosphorylation by the other implies either the existence of at least one phosphorylation site common to both PKA and AMPK or possibly closely adjacent sites which sterically interfere. The data showing activity changes only with AMPK, regardless of the phosphorylation order, clearly indicate the existence of a unique site for AMPK that is uninfluenced by phosphorylation by PKA at a common site or at a site unique to PKA. The muscle ACC appears to differ in this respect from the liver ACC.

In summary, PKA phosphorylates ACC isolated from rat skeletal muscle without any detectable change in catalytic function. AMPK phosphorylation of muscle ACC is accompanied by increases in Km for acetyl-CoA, ATP, and bicarbonate, and a more than twofold increase in the Ka for citrate activation. Phosphorylation by PKA has no detectable modulatory effects on AMPK regulation of muscle ACC. We conclude that phosphorylation of ACC by AMPK is more important for regulation of malonyl-CoA and hence fatty acid oxidation in skeletal muscle than is phosphorylation by PKA.

ACKNOWLEDGEMENTS

This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-41438 and by a Program Grant from the Wellcome Trust.

FOOTNOTES

Address for reprint requests: W. W. Winder, Dept. of Zoology, 545 WIDB, Brigham Young Univ., Provo, UT 84602 (E-mail: winderw{at}acd1.byu.edu).

Received 5 July 1996; accepted in final form 18 September 1996.

REFERENCES

1. Awan, M. M., and E. D. Saggerson. Malonyl-CoA metabolism in cardiac myocytes and its relevance to the control of fatty acid oxidation. Biochem. J. 295: 61-66, 1993.
2. Bianchi, A., J. L. Evans, A. J. Iverson, A. Nordlund, T. D. Watts, and L. A. Witters. Identification of an isozymic form of acetyl-CoA carboxylase. J. Biol. Chem. 265: 1502-1509, 1990. [Abstract/Free Full Text]
3. Carling, D., P. R. Clarke, V. A. Zammit, and D. G. Hardie. Purification and characterization of the AMP-activated protein kinase. Eur. J. Biochem. 186: 129-136, 1989. [Medline]
4. Corton, J. M., J. G. Gillespie, and D. G. Hardie. Role of the AMP-activated protein kinase in the cellular stress-response. Curr. Biol. 4: 315-324, 1994. [Medline]
5. Davies, S. P., D. Carling, and D. G. Hardie. Tissue distribution of the AMP-activated protein kinase and lack of activation by cyclic-AMP-dependent protein kinase, studied using a specific and sensitive peptide assay. Eur. J. Biochem. 186: 123-128, 1989. [Medline]
6. Davies, S. P., N. R. Helps, P. T. W. Cohen, and D. G. Hardie. 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2Calpha and native bovine protein phosphatase-2AC. FEBS Lett. 377: 421-425, 1995. [Medline]
7. Davies, S. P., A. T. R. Sim, and D. G. Hardie. Location and function of three sites phosphorylated on rat acetyl-CoA carboxylase by the AMP-activated protein kinase. Eur. J. Biochem. 187: 183-190, 1990. [Medline]
8. Duan, C., and W. W. Winder. Nerve stimulation decreases malonyl-CoA in skeletal muscle. J. Appl. Physiol. 72: 901-904, 1992. [Abstract/Free Full Text]
9. Ha, J., S. Daniel, S. S. Broyles, and K.-H. Kim. Critical phosphorylation sites for acetyl-CoA carboxylase activity. J. Biol. Chem. 269: 22162-22168, 1994. [Abstract/Free Full Text]
10. Hardie, D. G. Regulation of fatty acid synthesis via phosphorylation of acetyl-CoA carboxylase. Prog. Lipid Res. 28: 117-146, 1989. [Medline]
11. Kim, K. H., F. Lopez-Casillas, D. H. Bai, X. Luo, and M. E. Pape. Role of reversible phosphorylation of acetyl-CoA carboxylase in long-chain fatty acid synthesis. FASEB J. 3: 2250-2256, 1989. [Abstract]
12. Kudo, N., A. J. Barr, R. L. Barr, S. Desai, and G. D. Lopaschuk. High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5'-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J. Biol. Chem. 270: 17513-17520, 1995. [Abstract/Free Full Text]
13. Lopaschuk, G. D., and J. Gamble. Acetyl-CoA carboxylase: an important regulator of fatty acid oxidation in the heart. Can. J. Physiol. Pharmacol. 72: 1101-1109, 1994. [Medline]
14. McGarry, J. D., S. E. Mills, C. S. Long, and D. W. Foster. Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Biochem. J. 214: 21-28, 1983. [Medline]
15. Saddik, M., J. Gamble, L. A. Witters, and G. D. Lopaschuk. Acetyl-CoA carboxylase regulation of fatty acid oxidation in the heart. J. Biol. Chem. 268: 25836-25845, 1993. [Abstract/Free Full Text]
16. Saggerson, D., I. Ghadiminejad, and M. Awan. Regulation of mitochondrial carnitine palmitoyl transferases from liver and extrahepatic tissues. Adv. Enzyme Regul. 32: 285-306, 1992. [Medline]
17. Sim, A. T. R., and D. G. Hardie. The low activity of acetyl-CoA carboxylase in basal and glucagon-stimulated hepatocytes is due to phosphorylation by the AMP-activated protein kinase and not cyclic AMP-dependent protein kinase. FEBS Lett. 233: 294-298, 1988. [Medline]
18. Stapleton, D., K. I. Mitchelhill, G. Gao, J. Widmer, B. J. Michell, T. The, T. Cox, L. A. Witters, and B. E. Kemp. Mammalian AMP-activated protein kinase subfamily. J. Biol. Chem. 271: 611-614, 1996. [Abstract/Free Full Text]
19. Terjung, R. L., and H. Kaciuba-Uscilko. Lipid metabolism during exercise: influence of training. Diabetes Metab. Rev. 2: 35-51, 1986. [Medline]
20. Thampy, K. G. Formation of malonyl coenzyme A in rat heart. J. Biol. Chem. 264: 17631-17634, 1989. [Abstract/Free Full Text]
21. Trumble, G. E., M. A. Smith, and W. W. Winder. Evidence of a biotin dependent acetyl-coenzyme A carboxylase in rat muscle. Life Sci. 49: 39-43, 1991. [Medline]
22. Trumble, G. E., M. A. Smith, and W. W. Winder. Purification and characterization of rat skeletal muscle acetyl-CoA carboxylase. Eur. J. Biochem. 231: 192-198, 1995. [Medline]
23. Verhoeven, A. J. M., A. Woods, C. H. Brennan, S. A. Hawley, D. G. Hardie, J. Scott, R. K. Beri, and D. Carling. The AMP-activated protein kinase gene is highly expressed in rat skeletal muscle. Eur. J. Biochem. 228: 236-243, 1995. [Medline]
24. Weekes, J., S. A. Hawley, J. Corton, D. Shugar, and D. G. Hardie. Activation of rat liver AMP-activated protein kinase by kinase kinase in a purified, reconstituted system. Effects of AMP and AMP analogues. Eur. J. Biochem. 219: 751-757, 1994. [Medline]
25. Weis, B. C., V. Esser, D. W. Foster, and J. D. McGarry. Rat heart expresses two forms of mitochondrial carnitine palmitoyltransferase I. J. Biol. Chem. 269: 18712-18715, 1994. [Abstract/Free Full Text]
26. Widmer, J., K. S. Fassihi, S. C. Schlichter, K. S. Wheeler, B. E. Crute, N. King, N. Nutile-McMenemy, W. W. Noll, S. Daniel, J. Ha, K.-H. Kim, and L. A. Witters. Identification of a second human acetyl-CoA carboxylase gene. Biochem. J. 316: 915-922, 1996.
27. Winder, W. W., J. Arogyasami, R. J. Barton, I. M. Elayan, and P. R. Vehrs. Muscle malonyl-CoA decreases during exercise. J. Appl. Physiol. 67: 2230-2233, 1989. [Abstract/Free Full Text]
28. Winder, W. W., J. Arogyasami, I. M. Elayan, and D. Cartmill. Time course of the exercise-induced decline in malonyl-CoA in different muscle types. Am. J. Physiol. 259 (Endocrinol. Metab. 22): E266-E271, 1990. [Abstract/Free Full Text]
29. Winder, W. W., R. W. Braiden, D. C. Cartmill, C. A. Hutber, and J. P. Jones. Effect of adrenodemedullation on decline in muscle malonyl-CoA during exercise. J. Appl. Physiol. 74: 2548-2551, 1993. [Abstract/Free Full Text]
30. Winder, W. W., and C. Duan. Control of fructose 2,6-diphosphate in muscle of exercising rats. Am. J. Physiol. 262 (Endocrinol. Metab. 25): E919-E924, 1992. [Abstract/Free Full Text]
31. Winder, W. W., and D. G. Hardie. Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am. J. Physiol. 270 (Endocrinol. Metab. 33): E299-E304, 1996. [Abstract/Free Full Text]
32. Winder, W. W., P. S. MacLean, S. L. Chandler, W. Huang, and R. H. Mills. Role of epinephrine during insulin-induced hypoglycemia. J. Appl. Physiol. 77: 270-276, 1994. [Abstract/Free Full Text]
33. Winder, W. W., P. S. MacLean, J. C. Lucas, J. E. Fernley, and G. E. Trumble. Effect of fasting and refeeding on acetyl-CoA carboxylase in rat hindlimb muscle. J. Appl. Physiol. 78: 578-582, 1995. [Abstract/Free Full Text]
34. Witters, L. A., J. Widmer, A. N. King, K. Fassihi, and F. Kuhajda. Identification of human acetyl-CoA carboxylase isozymes in tissue and in breast cancer cells. Int. J. Biochem. 26: 589-594, 1994. [Medline]
0161-7567/97 $5.00 Copyright © 1997 the American Physiological Society


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
R. W. Schwenk, J. J.F.P. Luiken, A. Bonen, and J. F.C. Glatz
Regulation of sarcolemmal glucose and fatty acid transporters in cardiac disease
Cardiovasc Res, July 15, 2008; 79(2): 249 - 258.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. E. Osler and J. R. Zierath
Minireview: Adenosine 5'-Monophosphate-Activated Protein Kinase Regulation of Fatty Acid Oxidation in Skeletal Muscle
Endocrinology, March 1, 2008; 149(3): 935 - 941.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
D. M. Thomson, J. D. Brown, N. Fillmore, B. M. Condon, H-J. Kim, J. R. Barrow, and W. W. Winder
LKB1 and the regulation of malonyl-CoA and fatty acid oxidation in muscle
Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1572 - E1579.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
M. E. Sandstrom, S.-J. Zhang, H. Westerblad, and A. Katz
Mechanical load plays little role in contraction-mediated glucose transport in mouse skeletal muscle
J. Physiol., March 1, 2007; 579(2): 527 - 534.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
M. C. Towler and D. G. Hardie
AMP-Activated Protein Kinase in Metabolic Control and Insulin Signaling
Circ. Res., February 16, 2007; 100(3): 328 - 341.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. J. An, Y. Rhee, S. H. Kim, D. M. Kim, D.-H. Han, J. H. Hwang, Y.-J. Jin, B. S. Cha, J.-H. Baik, W. T. Lee, et al.
Peripheral Effect of {alpha}-Melanocyte-stimulating Hormone on Fatty Acid Oxidation in Skeletal Muscle
J. Biol. Chem., February 2, 2007; 282(5): 2862 - 2870.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
W. J. Ellingson, D. G. Chesser, and W. W. Winder
Effects of 3-phosphoglycerate and other metabolites on the activation of AMP-activated protein kinase by LKB1-STRAD-MO25
Am J Physiol Endocrinol Metab, February 1, 2007; 292(2): E400 - E407.
[Abstract] [Full Text] [PDF]

Home page
 Exp PhysiolHome page
C. U. Niesler, K. H. Myburgh, and F. Moore
Muscle: The changing AMPK expression profile in differentiating mouse skeletal muscle myoblast cells helps confer increasing resistance to apoptosis
Exp Physiol, January 1, 2007; 92(1): 207 - 217.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
V. Saks, R. Favier, R. Guzun, U. Schlattner, and T. Wallimann
Molecular system bioenergetics: regulation of substrate supply in response to heart energy demands
J. Physiol., December 15, 2006; 577(3): 769 - 777.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. L. Hurley, L. K. Barre, S. D. Wood, K. A. Anderson, B. E. Kemp, A. R. Means, and L. A. Witters
Regulation of AMP-activated Protein Kinase by Multisite Phosphorylation in Response to Agents That Elevate Cellular cAMP
J. Biol. Chem., December 1, 2006; 281(48): 36662 - 36672.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
J.-M. Ye, N. Dzamko, A. J. Hoy, M. A. Iglesias, B. Kemp, and E. Kraegen
Rosiglitazone Treatment Enhances Acute AMP-Activated Protein Kinase-Mediated Muscle and Adipose Tissue Glucose Uptake in High-Fat-Fed Rats.
Diabetes, October 1, 2006; 55(10): 2797 - 2804.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. P. Berasi, C. Huard, D. Li, H. H. Shih, Y. Sun, W. Zhong, J. E. Paulsen, E. L. Brown, R. E. Gimeno, and R. V. Martinez
Inhibition of Gluconeogenesis through Transcriptional Activation of EGR1 and DUSP4 by AMP-activated Kinase
J. Biol. Chem., September 15, 2006; 281(37): 27167 - 27177.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
B. Viollet, M. Foretz, B. Guigas, S. Horman, R. Dentin, L. Bertrand, L. Hue, and F. Andreelli
Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders
J. Physiol., July 1, 2006; 574(1): 41 - 53.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. Sriwijitkamol, J. L. Ivy, C. Christ-Roberts, R. A. DeFronzo, L. J. Mandarino, and N. Musi
LKB1-AMPK signaling in muscle from obese insulin-resistant Zucker rats and effects of training
Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E925 - E932.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
T. Toyoda, S. Tanaka, K. Ebihara, H. Masuzaki, K. Hosoda, K. Sato, T. Fushiki, K. Nakao, and T. Hayashi
Low-intensity contraction activates the {alpha}1-isoform of 5'-AMP-activated protein kinase in rat skeletal muscle
Am J Physiol Endocrinol Metab, March 1, 2006; 290(3): E583 - E590.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
G. P. Holloway, V. Bezaire, G. J. F. Heigenhauser, N. N. Tandon, J. F. C. Glatz, J. J. F. P. Luiken, A. Bonen, and L. L. Spriet
Mitochondrial long chain fatty acid oxidation, fatty acid translocase/CD36 content and carnitine palmitoyltransferase I activity in human skeletal muscle during aerobic exercise
J. Physiol., February 15, 2006; 571(1): 201 - 210.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Moreau, E. Dizin, H. Ray, C. Luquain, E. Lefai, F. Foufelle, M. Billaud, G. M. Lenoir, and N. D. Venezia
BRCA1 Affects Lipid Synthesis through Its Interaction with Acetyl-CoA Carboxylase
J. Biol. Chem., February 10, 2006; 281(6): 3172 - 3181.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
D. G. Hardie and K. Sakamoto
AMPK: A Key Sensor of Fuel and Energy Status in Skeletal Muscle
Physiology, February 1, 2006; 21(1): 48 - 60.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
X Fang, R Palanivel, X Zhou, Y Liu, A Xu, Y Wang, and G Sweeney
Hyperglycemia- and hyperinsulinemia-induced alteration of adiponectin receptor expression and adiponectin effects in L6 myoblasts
J. Mol. Endocrinol., December 1, 2005; 35(3): 465 - 476.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
A. C. Smith, C. R. Bruce, and D. J. Dyck
AMP kinase activation with AICAR simultaneously increases fatty acid and glucose oxidation in resting rat soleus muscle
J. Physiol., June 1, 2005; 565(2): 537 - 546.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
D. S. Rubink and W. W. Winder
Effect of phosphorylation by AMP-activated protein kinase on palmitoyl-CoA inhibition of skeletal muscle acetyl-CoA carboxylase
J Appl Physiol, April 1, 2005; 98(4): 1221 - 1227.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Z. Hu, S. H. Cha, G. van Haasteren, J. Wang, and M. D. Lane
From the Cover: Effect of centrally administered C75, a fatty acid synthase inhibitor, on ghrelin secretion and its downstream effects
PNAS, March 15, 2005; 102(11): 3972 - 3977.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Zang, A. Zuccollo, X. Hou, D. Nagata, K. Walsh, H. Herscovitz, P. Brecher, N. B. Ruderman, and R. A. Cohen
AMP-activated Protein Kinase Is Required for the Lipid-lowering Effect of Metformin in Insulin-resistant Human HepG2 Cells
J. Biol. Chem., November 12, 2004; 279(46): 47898 - 47905.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
D. L. Coven, X. Hu, L. Cong, R. Bergeron, G. I. Shulman, D. G. Hardie, and L. H. Young
Physiological role of AMP-activated protein kinase in the heart: graded activation during exercise
Am J Physiol Endocrinol Metab, September 1, 2003; 285(3): E629 - E636.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
J. F. P. Wojtaszewski, C. MacDonald, J. N. Nielsen, Y. Hellsten, D. G. Hardie, B. E. Kemp, B. Kiens, and E. A. Richter
Regulation of 5'AMP-activated protein kinase activity and substrate utilization in exercising human skeletal muscle
Am J Physiol Endocrinol Metab, April 1, 2003; 284(4): E813 - E822.
[Abstract] [Full Text] [PDF]