HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 32, 24279-24283, August 11, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/32/24279    most recent C000291200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Saha, A. K. Articles by Ruderman, N. B. Search for Related Content PubMed PubMed Citation Articles by Saha, A. K. Articles by Ruderman, N. B. Social Bookmarking                      What's this?

Activation of Malonyl-CoA Decarboxylase in Rat Skeletal Muscle by Contraction and the AMP-activated Protein Kinase Activator 5-Aminoimidazole-4-carboxamide-1--D-ribofuranoside^*

Asish K. Saha^§, Alexandria J. Schwarsin, Raphael Roduit^¶, Frédéric Massé^¶, Virendar Kaushik, Keith Tornheim, Marc Prentki^¶**, and Neil B. Ruderman

From the  Diabetes Unit, Section of Endocrinology, and Departments of Medicine, Physiology, and Biochemistry, Boston Medical Center, Boston, Massachusetts 02118 and the ^¶ Molecular Nutrition Unit, Department of Nutrition and the CR-CHUM, University of Montreal, Montreal, Quebec H2L 4M1, Canada

Received for publication, May 1, 2000, and in revised form, June 2, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Alterations in the concentration of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, have been linked to the regulation of fatty acid oxidation in skeletal muscle. During contraction decreases in muscle malonyl-CoA concentration have been related to activation of AMP-activated protein kinase (AMPK), which phosphorylates and inhibits acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in malonyl-CoA formation. We report here that the activity of malonyl-CoA decarboxylase (MCD) is increased in contracting muscle. Using either immunopurified enzyme or enzyme partially purified by (NH₄)₂SO₄ precipitation, 2-3-fold increases in the V_max of MCD and a 40% decrease in its K_m for malonyl-CoA (190 versus 119 µM) were observed in rat gastrocnemius muscle after 5 min of contraction, induced by electrical stimulation of the sciatic nerve. The increase in MCD activity was markedly diminished when immunopurified enzyme was treated with protein phosphatase 2A or when phosphatase inhibitors were omitted from the homogenizing solution and assay mixture. Incubation of extensor digitorum longus muscle for 1 h with 2 mM 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside, a cell-permeable activator of AMPK, increased MCD activity 2-fold. Here, too, addition of protein phosphatase 2A to the immunopellets reversed the increase of MCD activity. The results strongly suggest that activation of AMPK during muscle contraction leads to phosphorylation of MCD and an increase in its activity. They also suggest a dual control of malonyl-CoA concentration by ACC and MCD, via AMPK, during exercise.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The role of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, in regulating the oxidation of fatty acids in rat skeletal (1, 2) and cardiac (3, 4) muscle has been intensively investigated. Recent studies have demonstrated that its concentration in rat muscle is governed, at least in part, by changes in the activity of the muscle isoform of acetyl-CoA carboxylase (ACC)¹ (5), the enzyme that catalyzes malonyl-CoA synthesis. Thus, in keeping with their observed effects on malonyl-CoA concentration and fatty acid oxidation, insulin and glucose appear to activate ACC in muscle by increasing the cytosolic concentration of citrate, an allosteric activator of ACC and a precursor of its substrate, cytosolic acetyl-CoA. Conversely, decreases in malonyl-CoA concentration and increases in fatty acid oxidation in muscle during exercise (contraction) have been linked to decreases in ACC activity, attributable to its phosphorylation and inhibition by the ₂ isoform of AMP-activated protein kinase (AMPK) (5). AMPK can also be activated and the concentration of malonyl-CoA decreased by exposing resting muscle to 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which is taken into the muscle and phosphorylated to form the 5'-AMP analogue ZMP (6).

Whether a change in malonyl-CoA turnover contributes to the alterations in its concentration in muscle during exercise and other conditions is not known. In a lipogenic tissue such as liver, the de novo synthesis of fatty acids is thought to be the major mechanism by which malonyl-CoA is utilized. In contrast, in skeletal muscle fatty acid synthesis occurs at a very low rate, if at all (7), and attention has been focused on malonyl-CoA decarboxylase (MCD) for removal of malonyl-CoA (1). Evidence has been presented that MCD is present in both cardiac (8, 9) and skeletal (1, 10, 11) muscle. In skeletal muscle, its activity is similar to that of ACC (1). In heart, in which MCD activity is substantially greater than in skeletal muscle, a decrease in the K_m of MCD for malonyl-CoA has been reported following an increase in its work load (9). On the other hand, no change in activity has been observed following ischemia-reperfusion of the heart, a situation in which AMPK is activated (8). The question of whether MCD is acutely regulated in skeletal muscle and, if so, how has not been studied previously.

In this study, we describe the characteristics of purified MCD from rat skeletal muscle and contraction-induced changes in its maximal activity and affinity for malonyl-CoA. In addition, the effects of the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and of treatment with phosphatases on MCD activity were examined.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Animals-- Male Harlan Sprague-Dawley rats weighing approximately 55-70 g obtained from Charles River Laboratories (Wilmington, MA) were used except as indicated. They were housed in individual cages in a temperature-controlled room on a 12-h light cycle and fed standard purina rat chow and water ad libitum for 6 days prior to an experiment unless noted otherwise.

Muscle Stimulation-- Rats were anesthetized with sodium pentobarbital (5.5 mg/100 g of body weight intraperitoneally), and 45 min later the skin from both hindlimbs was removed and the sciatic nerves exposed. The sciatic nerve of one limb was then stimulated for 2 and 5 min with a bipolar electrode connected to a Glass stimulator (model S48) (5 pulses/s, 100-ms trains of 2.5 V, 50 Hz, and 10-ms duration) to induce muscle contractions (5). The gastrocnemius muscles from this limb and the unstimulated contralateral limb were then rapidly excised and frozen in liquid nitrogen. All tissues were stored at 80 °C until assay.

Incubation with AICAR-- Rats were anesthetized with sodium pentobarbital and extensor digitorum longus (EDL) muscles were tied to stainless steel clips. Muscles were preincubated for 20 min at 37 °C in 12 × 75-mm test tubes containing 3.0 ml of Krebs-Henseleit solution containing: 5.5 mM glucose, 50 microunits/ml insulin, and 0.2% fatty acid-free bovine serum albumin as described previously (12). The media were gassed continuously with a 95% O₂, 5% CO₂ mixture. Muscles were then transferred to different test tubes and incubated for 60 min with fresh medium with or without 2 mM AICAR. At the end of the incubation, muscles were removed, blotted on gauze pads, and frozen in liquid N₂. Muscles were then homogenized in 0.1 M Tris-HCl (pH 8.0), 2 mM phenylmethylsulfonyl fluoride (PMSF), 5 µM aprotinin, 5 µM leupeptin, and 5 µM pepstatin A containing 40 mM -glycerophosphate, 40 mM NaF, 4 mM NaPP_i, and 0.1 Na₃VO₄. To examine the effect of protein phosphatase 2A (PP2A), immunopurified MCD was washed twice with 0.1 M Tris-HCl (pH 8.0) containing 2 mM PMSF, 5 µM aprotinin, 5 µM leupeptin, and 5 µM pepstatin A and then incubated with 200 milliunits of PP2A (Upstate Biotechnology Inc., Lake Placid, NY; 1 unit = 1 nmol of phosphate released from 15 µM phosphorylase/min) at 37 °C for 2.5 h.

Purification of MCD by (NH₄)₂SO₄ Precipitation-- Frozen muscles (500 mg) were powdered in liquid nitrogen, weighed, and then homogenized in a glass homogenizer in 30 volumes of a buffer composed of 0.1 M Tris-HCl (pH 8.0), 2 mM PMSF, 5 µM aprotinin, 5 µM leupeptin, and 5 µM pepstatin A (13, 14), with the addition of 40 mM -glycerophosphate, 40 mM NaF, 4 mM NaPP_i, and 1 mM Na₃VO₄ to inhibit phosphatase activity unless otherwise indicated. The homogenized muscles were then centrifuged at 500 × g for 10 min. Partial purification of MCD was initially carried out by a modification of the method of Dyck et al. (8). To the supernatant, powdered (NH₄)₂SO₄ was slowly added with stirring until 40% saturation was achieved. The mixture was stirred for 1 h on ice and centrifuged at 14,000 × g for 10 min. The supernatant from this spin was treated with additional (NH₄)₂SO₄ until 55% saturation was achieved. The mixture was recentrifuged at 14,000 × g. The resultant pellet fraction was dissolved in 0.1 M Tris-HCl (pH 8.0) and stored at 4 °C for use in all further studies. The amount of protein from control and stimulated muscles was essentially the same after (NH₄)₂SO₄ fractionation.

Purification of MCD by Immunoprecipitation-- The supernatants from frozen gastrocnemius muscles (300-350 µg of protein) were incubated for 3 h with 4 µg of an antibody to the N-terminal region of MCD and 20 µl of protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) beads. Affinity-purified antibody from rabbits immunized with the N-terminal region of MCD lacking mitochondrial and peroxisomal targeting sequences (11) was used. The beads were washed twice with 0.1 M Tris-HCl (pH 8.0) containing 2 mM PMSF, 5 µM aprotinin, 5 µM leupeptin, and 5 µM pepstatin A. They were then assayed for MCD activity. Immunopurified enzyme was also used to examine the effect of dephosphorylation by PP2A on MCD activity. For these studies immunopurified enzyme samples from stimulated and unstimulated muscles were incubated with 200, 400, or 600 milliunits of PP2A with or without 10 nM okadaic acid at 37 °C for 2.5 h. For the MgCl₂/glutamate study, the washed beads from muscle extract containing no phosphatase inhibitors were incubated with 100 mM sodium glutamate, 10 mM MgCl₂ at 37 °C for 2.5 h. At the end of the incubation, the immunopellets were washed twice with 0.1 M Tris-HCl (pH 8.0) and then assayed for MCD activity. MCD activity is expressed per milligram of extract protein in the 500 × g supernatant subjected to immunoprecipitation.

MCD Assay-- MCD activity was measured spectrophotometrically (13, 14) using a Hewlett-Packard model 8450A diode array spectrophotometer as described previously (10). In brief, the (NH₄)₂SO₄-purified fraction from 150 µl of the muscle homogenate was added to a 700-µl reaction mixture composed of 0.1 M Tris-HCl (pH 8.0), 0.5 mM dithiothreitol, 10 mM L-malate, 0.5 mM NAD⁺, and 10 µg of malate dehydrogenase (1.0 unit) and preincubated for 10 min at room temperature, in the presence of phosphatase inhibitors (40 mM -glycerophosphate, 40 mM NaF, 4 mM NaPP_i, and 1 mM Na₃VO₄) except as noted. Citrate synthase (10 µg; 1.7 units) was added, and the preincubation was continued for an additional 2 min. Malonyl-CoA (0.3 mM) was then added to start the MCD reaction and the rate of NADH formation was measured over 7 min. Controls were run to correct for the small rate of NADH oxidation obtained when malonyl-CoA was not added. The optimum pH range for MCD was determined using the following buffers: 0.1 M sodium acetate (pH 4.0-6.5), 0.1 M sodium phosphate (pH 6.0-8.0), 0.1 M Tris-HCl (pH 7.5-9.0), and 0.1 M glycine NaOH (pH 8.0-10.0). Protein concentration was determined by the method of Bradford with bovine serum albumin as the standard (15). Immunopurified MCD was assayed in an identical manner except that the immunoprecipitate (~20 µl) was added to 100 µl of the 0.1 M Tris-HCl buffer before addition to the reaction mixture. Activity in all instances is expressed per mg protein in the 500 × g supernatant of the whole tissue extract.

Statistical Analysis-- Results are expressed as means ± S.E. Statistical differences between multiple groups were determined by analysis of variance followed by the Student-Newman-Keuls multiple comparison test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Studies of MCD Activity Using Enzyme Purified by (NH₄)₂SO₄ Precipitation-- After partial purification of skeletal muscle MCD with (NH₄)₂SO₄, the maximal rate of malonyl-CoA decarboxylation was found between pH 7 and 8 (data not shown), a range similar to that previously reported for MCD in rat liver (13, 16), brain (14), and heart (8, 9). The rate of formation of acetyl-CoA increased with increasing concentrations of malonyl-CoA, and a typical Michaelis-Menten type substrate saturation pattern was observed (data not shown). From linear double-reciprocal plots, a K_m for malonyl-CoA of 190 ± 13 µM (n = 5) was obtained for control muscle extracted in the presence of phosphatase inhibitors, a value similar to that found by Prentki and his co-workers for skeletal muscle MCD² but 2-3-fold higher than that reported for MCD in rat heart (8, 9), liver (13), and brain (14). The effect of contraction on MCD activity was studied next. As shown in Fig. 1, MCD activity was increased more than 3-fold in gastrocnemius muscles after 5 min of electrically induced contractions. The K_m of MCD for malonyl-CoA from these muscles was decreased to 119 ± 14 µM (n = 5) after stimulation of contractile activity. When the tissue was processed and the MCD assay performed in the absence of phosphatase inhibitors, the increase in activity was still observed; however, it was diminished by 50% (Fig. 1)

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Effect of contractions induced by sciatic nerve stimulation on MCD activity in rat gastrocnemius muscle. Rats were anesthetized, and the sciatic nerve was either sham-operated (control) or electrically stimulated for 5 min (5 pulses/s, 100-ms trains of 2.5 V, 50 Hz frequency, and 10-ms duration). Gastrocnemius muscles were then excised and frozen in liquid N2. They were homogenized in 0.1 M Tris-HCl (pH 8.0) buffer in the absence (PI) or presence (+PI) of phosphatase inhibitors (40 mM -glycerophosphate, 40 mM NaF, 4 mM NaPPi, and 1 mM Na3VO4). (NH4)2SO4-purified enzyme from these muscles was assayed for MCD activity. Results are means ± S.E. for eight muscles. *, p < 0.05 and **, p < 0.001 (significantly different from control muscles); +, p < 0.001 (significantly different from muscles homogenized and MCD assayed in the absence of phosphatase inhibitors).

Studies of MCD Purified by Immunoprecipitation-- We next examined whether it was possible to assay MCD when it was immunoprecipitated by a N-terminal affinity-purified antibody. As shown in Fig. 2, the antibody immunoprecipitated nearly 75% of the MCD activity (V_max) present in the muscle supernatant. In addition, the antibody immunoprecipitated over 90% of the MCD in the 500 × g supernatant as judged by Western blotting (data not shown). MCD activity following immunoprecepitation was very similar to that observed after (NH₄)₂SO₄ precipitation (Fig. 1). For instance in control unstimulated muscles MCD activity was 3.5 ± 0.35 nmol/min/mg of initial supernatant protein when assayed after (NH₄)₂SO₄ precipitation (Fig. 1) and 5.0 ± 0.9 and 3.8 ± 0.3 nmol/min/mg of protein (Figs. 2 and 3, respectively) when measured after immunoprecipitation.

View larger version (46K): [in this window] [in a new window]   Fig. 2.   An antibody to the N-terminal region of MCD quantitatively immunoprecipitates MCD activity in an extract of rat muscle. Extracts from frozen gastrocnemius (300-350 µg of protein) were incubated for 3 h with 4 µg of antibody and 20 µl of A/G-agarose beads. MCD activity was assayed in the extract prior to (PRE) and after (POST) immunoprecipitation or directly in the immunoprecipitate (I.P.). Results are means ± S.E. of three separate studies. MCD activity in all fractions is expressed per milligram of extract protein in the original 500 × g supernatant subjected to immunoprecipitation. One mg of protein in the supernatant corresponds to 6 mg of tissue wet weight.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effect of PP2A on the activity of MCD immunoprecipitated from contracting muscle. Muscles were stimulated and isolated as described in the legend to Fig. 1. MCD immunopellets were incubated at 37 °C for 2.5 h as described under "Experimental Procedures." A, experiments with 200 milliunits of PP2A ± 10 nM okadaic acid (O.A.). B, dose dependence of PP2A action. Results are means ± S.E. of three separate sets of muscles.

As shown in Fig. 3, MCD activity in the immunoprecipitate was increased 2-fold from 3.8 ± 0.3 to 8.0 ± 0.8 nmol/min/mg of protein in gastrocnemius muscles after 5 min of electrically induced contractions. When immunoprecipitates from these muscles were treated with PP2A (200 milliunits), the observed increase in activity was markedly diminished (Fig. 3A), an effect prevented when the phosphatase inhibitor okadaic acid (10 nM) was added to the medium. As shown in Fig. 3B, treatment with higher concentrations of PP2A caused further decreases in MCD activity in both control and contracting muscle. The addition of phosphatase activators (sodium glutamate and MgCl₂) to muscle extracts containing no phosphatase inhibitors decreased MCD activity both in control (4.0 ± 0.3 versus 3.0 ± 0.2 nmol/min/mg) and stimulated (8.0 ± 0.5 versus 4.5 ± 0.3 nmol/min/mg) muscles, further suggesting that the increase in MCD activity caused by contraction is related to phosphorylation.

Changes in MCD Activity in Relation to Activation of AMPK-- A logical candidate for regulating MCD phosphorylation during contraction is AMPK (5). As shown in Fig. 4, MCD activity is increased even as early as after 2 min of muscle contraction, which correlates well with previously published data from our laboratory (5) and that of Winder (6) showing activation of AMPK and inhibition of ACC at these times. To illustrate the comparison, we have included in Fig. 4 our previously published data (5) for AMPK activation and ACC inhibition after electrically induced contractions. Similar alterations in AMPK and ACC activity have also been observed when muscle is perfused (6) or incubated (17)³ with the cell-permeable AMPK activator AICAR. As shown in Fig. 5, incubation of the EDL with 2 mM AICAR for 1 h increased immunoprecipitable MCD activity 2-fold. Furthermore when PP2A was added to the immunopellets, it reversed the increase in MCD activity caused by AICAR (Fig. 5), much as it did the increase in MCD activity caused by contraction. Parenthetically, the lower activity of MCD in the EDL than in the gastrocnemius (Fig. 1) has been observed previously (1).

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Time course of changes in the activities of MCD, ACC, and the 2 isoform of AMPK during contractions. The sciatic nerve of one hindlimb was stimulated for periods ranging from 2 to 5 min, and the other limb was used as the control. Immunoprecipitates were used to measure the activities of MCD, ACC, and 2 AMPK. Results are means ± S.E. of four to five sets of muscles (data for ACC and 2 AMPK are from Ref. 5).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of AICAR on immunoprecipitable malonyl-CoA decarboxylase activity. Extensor digitorum longus muscles were incubated in the absence or the presence of AICAR (2 mM) for 1 h. MCD immunopellets from AICAR incubated muscles were incubated at 37 °C for 2.5 h with 200 milliunits of PP2A. They were then frozen in liquid nitrogen, homogenized in 0.1 M Tris-HCl (pH 8.0) buffer in the presence of phosphatase inhibitors, and assayed for MCD activity as described in the legend to Fig. 1. Means ± S.E. of 10-12 experiments; *, p < 0.05 versus control; **, p < 0.05 versus AICAR.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The principal findings of this study are: 1) that MCD activity in skeletal muscle is acutely increased after contraction and 2) that this is likely due to its phosphorylation by AMPK.

Acute decreases in the concentration of malonyl-CoA in rat skeletal muscle during exercise (18-20) and during electrically induced contractions in hindlimb muscle (5, 21) very likely contribute to the increase in fatty acid oxidation in these situations. Thus, studies from our laboratory (12, 21-23) and that of Winder (18, 19) indicate that such decreases in malonyl-CoA concentration are associated with reduced ACC activity. As shown by Vavvas et al. (5), ACC is phosphorylated and inhibited by the ₂ isoform of AMP-activated protein kinase within seconds of the onset of muscle contraction, and its activity is diminished by 80% after as little as 2 min. The results of a present study suggest that another factor contributing to a decrease in malonyl-CoA and increase in fatty acid oxidation in muscle tissue could be an increase in MCD activity. Thus, MCD activity was increased 2-3-fold after 2 and 5 min of contraction.

The increase in MCD activity observed during contraction was twice as great when phosphatase inhibitors were added to the homogenizing solution, suggesting it was attributable to phosphorylation (Fig. 1). In keeping with this conclusion, the increase in MCD activity was substantially reduced when immunoprecipitate enzyme was incubated with protein phosphatase 2A (Fig. 3, A and B) or when phosphatase inhibitors were omitted from the homogenizing solution (Fig. 1), an effect magnified when the phosphatase activators MgCl₂ and glutamate were added. The data also strongly suggest that the activation and phosphorylation of MCD during contraction is mediated by AMPK. Several lines of evidence support this conclusion: 1) the time course of MCD activation during contraction parallels that of AMPK activation and ACC inhibition (Fig. 4) reported previously by us (5) and others (6), 2) MCD activity was increased in the EDL when it was incubated with the AMPK activator AICAR (Fig. 5), and 3) the increase in MCD activity was diminished by incubation of the immunopellet with PP2A. Consistent with possible regulation by AMPK, rat MCD possesses 34 serine residues (11), including several that could be in a recognition motif for AMPK (24). Definitive studies showing phosphorylation of specific sites on purified MCD by AMPK have not yet been reported, however. Interestingly, treatment of semi-purified MCD from rat heart with alkaline phosphatase has been shown to increase MCD activity (8). Thus, phosphorylation of MCD on sites other than that phosphorylated by AMPK probably inhibit the enzyme. The observation that immunoprecipitable MCD activity is still somewhat higher in contracting and AICAR-treated muscles than in control muscles following PP2A treatment suggest either that dephosphorylation was incomplete or that some other factors are involved in MCD regulation.

The finding that both contraction and AICAR activate MCD and inhibit ACC (5, 6) suggests that the two enzymes are jointly regulated by AMPK (Fig. 6). It also supports the view that both ACC and MCD participate in regulating the concentration of malonyl-CoA in skeletal muscle. The latter could be difficult to prove, since molecular biological (11) and cell fractionation (25) studies suggest localization of MCD isoforms in mitochondrial, peroxisomal, and possibly cytosolic fractions in various cells. Thus, the activity of MCD in these three fractions, as well as the relative effect of AMPK activation on MCD activity in each of these locations, will need to be evaluated.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Dual regulation of malonyl-CoA decarboxylase and acetyl-CoA carboxylase by AMP-activated protein kinase. As pictured, increases in AMPK activity caused by exercise and AICAR lead to the phosphorylation of both ACC and MCD. According to the proposed schema this results in inhibition of ACC and activation of MCD. Such dual regulation could magnify the effects of changes in AMPK activity on malonyl-CoA concentration and fat oxidation.

The relevance of the changes in MCD activity reported here to other tissues remains to be determined. Dyck et al. (8) did not observe an increase in MCD activity in rat heart during ischemia-reperfusion, a situation in which they had previously observed an increase in AMPK activity. In contrast, Goodwin and Taegtmeyer (9) found a 40% increase in MCD activity at subsaturating concentrations of malonyl-CoA in a perfused rat heart, when its work load was increased by the combination of 1 µM epinephrine and a 40% increase in afterload from 100 to 140 cm H₂O. However, they did not observe an increase in the V_max of the enzyme as we did here. Whether this reflects a difference in the tissue studied, the nature of the increase in muscle work, or the different MCD assays used in the two studies remains to be determined. Also requiring further study is the observation of Goodwin and Taegtmeyer (9) that the increase in fatty acid oxidation that occurs when heart work is increased correlates with an increase in MCD activity, but not with a decrease in assayed ACC activity. ACC activity in that study (9) was measured on the basis of citrate activation of ¹⁴CO₂ fixation, without first purifying the enzyme. As previously noted by Thampy (26), when this is done the ACC assay may be unreliable due to the presence of high but variable activities of propionyl-CoA carboxylase. Thus, the conclusion that MCD can regulate malonyl-CoA concentration and fatty acid oxidation independently of ACC remains open to question.

In conclusion, the results presented here indicate that MCD activity in muscle is increased by contraction and by incubation with AICAR. Previous work has shown that ACC is phosphorylated and inhibited under these conditions (5). Since a common factor in these situations is an increase in AMPK activity, this suggests that MCD and ACC may be jointly regulated by AMPK. The importance of this dual control of MCD and ACC activities to the regulation of the cytosolic concentration of malonyl-CoA and secondarily to fatty acid oxidation remains to be determined.

	ACKNOWLEDGEMENT

We gratefully acknowledge the expert technical assistance of Holly Couture.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grants DK 19514 and DK 49147 and a grant from the Juvenile Diabetes Foundation (to N. B. R. and A. K. S.) and by grants from the Medical Research Council of Canada, the Canadian Diabetes Association, and the Juvenile Diabetes Foundation International (to M. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Diabetes and Metabolism Unit, Boston Medical Center, 650 Albany St., EBRC-827, Boston, MA 02118. Tel.: 617-638-7169; Fax: 617-638-7094; E-mail: aksaha@bu.edu.

Recipient of a postdoctoral fellowship of the Juvenile Diabetes Foundation.

^** Medical Research Council of Canada Scientist.

Published, JBC Papers in Press, June 14, 2000, DOI 10.1074/jbc.C000291200

² M. Prentki, R. Roduit, and F. Massé, unpublished data..

³ V. Kaushik, M. Young, D. Dean, T. Kurowski, A. K. Saha, and N. B. Ruderman, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; AICAR, 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside; ZMP, 5-aminoimidazole-4-carbonamide riboside monophosphate; MCD, malonyl-CoA decarboxylase; EDL, extensor digitorium longus; PP2A, protein phosphatase 2A; PMSF, phenylmethylsulfonyl fluoride.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				G. R. Steinberg and B. E. Kemp AMPK in Health and Disease Physiol Rev, July 1, 2009; 89(3): 1025 - 1078. [Abstract] [Full Text] [PDF]

				C. Frosig, C. Roepstorff, N. Brandt, S. J. Maarbjerg, J. B. Birk, J. F. P. Wojtaszewski, E. A. Richter, and B. Kiens Reduced malonyl-CoA content in recovery from exercise correlates with improved insulin-stimulated glucose uptake in human skeletal muscle Am J Physiol Endocrinol Metab, April 1, 2009; 296(4): E787 - E795. [Abstract] [Full Text] [PDF]

				N. Dzamko, J. D. Schertzer, J. G. Ryall, R. Steel, S. L. Macaulay, S. Wee, Z.-P. Chen, B. J. Michell, J. S. Oakhill, M. J. Watt, et al. AMPK-independent pathways regulate skeletal muscle fatty acid oxidation J. Physiol., December 1, 2008; 586(23): 5819 - 5831. [Abstract] [Full Text] [PDF]

				J. R. Ussher and G. D. Lopaschuk The malonyl CoA axis as a potential target for treating ischaemic heart disease Cardiovasc Res, July 15, 2008; 79(2): 259 - 268. [Abstract] [Full Text] [PDF]

				K. Bouzakri, R. Austin, A. Rune, M. E. Lassman, P. M. Garcia-Roves, J. P. Berger, A. Krook, A. V. Chibalin, B. B. Zhang, and J. R. Zierath Malonyl CoenzymeA Decarboxylase Regulates Lipid and Glucose Metabolism in Human Skeletal Muscle Diabetes, June 1, 2008; 57(6): 1508 - 1516. [Abstract] [Full Text] [PDF]

				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]

				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]

				M. A. Mayes, M. F. Laforest, C. Guillemette, R. B. Gilchrist, and F. J. Richard Adenosine 5'-Monophosphate Kinase-Activated Protein Kinase (PRKA) Activators Delay Meiotic Resumption in Porcine Oocytes Biol Reprod, April 1, 2007; 76(4): 589 - 597. [Abstract] [Full Text] [PDF]

				C. D.L. Folmes and G. D. Lopaschuk Role of malonyl-CoA in heart disease and the hypothalamic control of obesity Cardiovasc Res, January 15, 2007; 73(2): 278 - 287. [Abstract] [Full Text] [PDF]

				N. B. Ruderman, C. Keller, A.-M. Richard, A. K. Saha, Z. Luo, X. Xiang, M. Giralt, V. B. Ritov, E. V. Menshikova, D. E. Kelley, et al. Interleukin-6 Regulation of AMP-Activated Protein Kinase: Potential Role in the Systemic Response to Exercise and Prevention of the Metabolic Syndrome Diabetes, December 1, 2006; 55(Supplement_2): S48 - S54. [Abstract] [Full Text] [PDF]

				M. J. Yoon, G. Y. Lee, J.-J. Chung, Y. H. Ahn, S. H. Hong, and J. B. Kim Adiponectin Increases Fatty Acid Oxidation in Skeletal Muscle Cells by Sequential Activation of AMP-Activated Protein Kinase, p38 Mitogen-Activated Protein Kinase, and Peroxisome Proliferator-Activated Receptor {alpha} Diabetes, September 1, 2006; 55(9): 2562 - 2570. [Abstract] [Full Text] [PDF]

				J. R. B. Dyck and G. D. Lopaschuk AMPK alterations in cardiac physiology and pathology: enemy or ally? J. Physiol., July 1, 2006; 574(1): 95 - 112. [Abstract] [Full Text] [PDF]

				S. B. Jorgensen, E. A. Richter, and J. F. P. Wojtaszewski Role of AMPK in skeletal muscle metabolic regulation and adaptation in relation to exercise J. Physiol., July 1, 2006; 574(1): 17 - 31. [Abstract] [Full Text] [PDF]

				J. E. Kuhl, N. B. Ruderman, N. Musi, L. J. Goodyear, M. E. Patti, S. Crunkhorn, D. Dronamraju, A. Thorell, J. Nygren, O. Ljungkvist, et al. Exercise training decreases the concentration of malonyl-CoA and increases the expression and activity of malonyl-CoA decarboxylase in human muscle Am J Physiol Endocrinol Metab, June 1, 2006; 290(6): E1296 - E1303. [Abstract] [Full Text] [PDF]

				B. Kiens Skeletal Muscle Lipid Metabolism in Exercise and Insulin Resistance Physiol Rev, January 1, 2006; 86(1): 205 - 243. [Abstract] [Full Text] [PDF]

				M. M. Assifi, G. Suchankova, S. Constant, M. Prentki, A. K. Saha, and N. B. Ruderman AMP-activated protein kinase and coordination of hepatic fatty acid metabolism of starved/carbohydrate-refed rats Am J Physiol Endocrinol Metab, November 1, 2005; 289(5): E794 - E800. [Abstract] [Full Text] [PDF]

				W. C. Stanley, F. A. Recchia, and G. D. Lopaschuk Myocardial Substrate Metabolism in the Normal and Failing Heart Physiol Rev, July 1, 2005; 85(3): 1093 - 1129. [Abstract] [Full Text] [PDF]

				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]

				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]

				M. A. Raney, A. J. Yee, M. K. Todd, and L. P. Turcotte AMPK activation is not critical in the regulation of muscle FA uptake and oxidation during low-intensity muscle contraction Am J Physiol Endocrinol Metab, March 1, 2005; 288(3): E592 - E598. [Abstract] [Full Text] [PDF]

				C. Roepstorff, N. Halberg, T. Hillig, A. K. Saha, N. B. Ruderman, J. F. P. Wojtaszewski, E. A. Richter, and B. Kiens Malonyl-CoA and carnitine in regulation of fat oxidation in human skeletal muscle during exercise Am J Physiol Endocrinol Metab, January 1, 2005; 288(1): E133 - E142. [Abstract] [Full Text] [PDF]

				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]

				R. J. Sigal, G. P. Kenny, D. H. Wasserman, and C. Castaneda-Sceppa Physical Activity/Exercise and Type 2 Diabetes Diabetes Care, October 1, 2004; 27(10): 2518 - 2539. [Full Text] [PDF]

				M. A. Iglesias, S. M. Furler, G. J. Cooney, E. W. Kraegen, and J.-M. Ye AMP-Activated Protein Kinase Activation by AICAR Increases Both Muscle Fatty Acid and Glucose Uptake in White Muscle of Insulin-Resistant Rats In Vivo Diabetes, July 1, 2004; 53(7): 1649 - 1654. [Abstract] [Full Text] [PDF]

				J. Shearer, P. T. Fueger, B. Vorndick, D. P. Bracy, J. N. Rottman, J. A. Clanton, and D. H. Wasserman AMP Kinase-Induced Skeletal Muscle Glucose But Not Long-Chain Fatty Acid Uptake Is Dependent on Nitric Oxide Diabetes, June 1, 2004; 53(6): 1429 - 1435. [Abstract] [Full Text] [PDF]

				R. Roduit, C. Nolan, C. Alarcon, P. Moore, A. Barbeau, V. Delghingaro-Augusto, E. Przybykowski, J. Morin, F. Masse, B. Massie, et al. A Role for the Malonyl-CoA/Long-Chain Acyl-CoA Pathway of Lipid Signaling in the Regulation of Insulin Secretion in Response to Both Fuel and Nonfuel Stimuli Diabetes, April 1, 2004; 53(4): 1007 - 1019. [Abstract] [Full Text] [PDF]

				N. B. Ruderman, A. K. Saha, and E. W. Kraegen Minireview: Malonyl CoA, AMP-Activated Protein Kinase, and Adiposity Endocrinology, December 1, 2003; 144(12): 5166 - 5171. [Abstract] [Full Text] [PDF]

				M.-H. Zou, X.-Y. Hou, C.-M. Shi, S. Kirkpatick, F. Liu, M. H. Goldman, and R. A. Cohen Activation of 5'-AMP-activated Kinase Is Mediated through c-Src and Phosphoinositide 3-Kinase Activity during Hypoxia-Reoxygenation of Bovine Aortic Endothelial Cells: ROLE OF PEROXYNITRITE J. Biol. Chem., September 5, 2003; 278(36): 34003 - 34010. [Abstract] [Full Text] [PDF]

				L. Abu-Elheiga, W. Oh, P. Kordari, and S. J. Wakil Acetyl-CoA carboxylase 2 mutant mice are protected against obesity and diabetes induced by high-fat/high-carbohydrate diets PNAS, September 2, 2003; 100(18): 10207 - 10212. [Abstract] [Full Text] [PDF]

				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]

				S. Hardy, W. El-Assaad, E. Przybytkowski, E. Joly, M. Prentki, and Y. Langelier Saturated Fatty Acid-induced Apoptosis in MDA-MB-231 Breast Cancer Cells: A ROLE FOR CARDIOLIPIN J. Biol. Chem., August 22, 2003; 278(34): 31861 - 31870. [Abstract] [Full Text] [PDF]

				S. I. Itani, A. K. Saha, T. G. Kurowski, H. R. Coffin, K. Tornheim, and N. B. Ruderman Glucose Autoregulates Its Uptake in Skeletal Muscle: Involvement of AMP-Activated Protein Kinase Diabetes, July 1, 2003; 52(7): 1635 - 1640. [Abstract] [Full Text] [PDF]

				G. R. Steinberg, J. W. E. Rush, and D. J. Dyck AMPK expression and phosphorylation are increased in rodent muscle after chronic leptin treatment Am J Physiol Endocrinol Metab, March 1, 2003; 284(3): E648 - E654. [Abstract] [Full Text] [PDF]

				A. O. Besikci, F. M. Campbell, T. A. Hopkins, J. R. B. Dyck, and G. D. Lopaschuk Relative importance of malonyl CoA and carnitine in maturation of fatty acid oxidation in newborn rabbit heart Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H283 - H289. [Abstract] [Full Text] [PDF]

				M. A. Iglesias, J.-M. Ye, G. Frangioudakis, A. K. Saha, E. Tomas, N. B. Ruderman, G. J. Cooney, and E. W. Kraegen AICAR Administration Causes an Apparent Enhancement of Muscle and Liver Insulin Action in Insulin-Resistant High-Fat-Fed Rats Diabetes, October 1, 2002; 51(10): 2886 - 2894. [Abstract] [Full Text] [PDF]

				M. Poirier, G. Vincent, A. E. Reszko, B. Bouchard, J. K. Kelleher, H. Brunengraber, and C. Des Rosiers Probing the link between citrate and malonyl-CoA in perfused rat hearts Am J Physiol Heart Circ Physiol, October 1, 2002; 283(4): H1379 - H1386. [Abstract] [Full Text] [PDF]

				H. Park, V. K. Kaushik, S. Constant, M. Prentki, E. Przybytkowski, N. B. Ruderman, and A. K. Saha Coordinate Regulation of Malonyl-CoA Decarboxylase, sn-Glycerol-3-phosphate Acyltransferase, and Acetyl-CoA Carboxylase by AMP-activated Protein Kinase in Rat Tissues in Response to Exercise J. Biol. Chem., August 30, 2002; 277(36): 32571 - 32577. [Abstract] [Full Text] [PDF]

				K. Sakamoto and L. J. Goodyear Exercise Effects on Muscle Insulin Signaling and Action: Invited Review: Intracellular signaling in contracting skeletal muscle J Appl Physiol, July 1, 2002; 93(1): 369 - 383. [Abstract] [Full Text] [PDF]

				E. S. Buhl, N. Jessen, R. Pold, T. Ledet, A. Flyvbjerg, S. B. Pedersen, O. Pedersen, O. Schmitz, and S. Lund Long-Term AICAR Administration Reduces Metabolic Disturbances and Lowers Blood Pressure in Rats Displaying Features of the Insulin Resistance Syndrome Diabetes, July 1, 2002; 51(7): 2199 - 2206. [Abstract] [Full Text] [PDF]

				S. H. Park, S. R. Gammon, J. D. Knippers, S. R. Paulsen, D. S. Rubink, and W. W. Winder Phosphorylation-activity relationships of AMPK and acetyl-CoA carboxylase in muscle J Appl Physiol, June 1, 2002; 92(6): 2475 - 2482. [Abstract] [Full Text] [PDF]

				H. Sakoda, T. Ogihara, M. Anai, M. Fujishiro, H. Ono, Y. Onishi, H. Katagiri, M. Abe, Y. Fukushima, N. Shojima, et al. Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes Am J Physiol Endocrinol Metab, June 1, 2002; 282(6): E1239 - E1244. [Abstract] [Full Text] [PDF]

				M. E. Young, P. McNulty, and H. Taegtmeyer Adaptation and Maladaptation of the Heart in Diabetes: Part II: Potential Mechanisms Circulation, April 16, 2002; 105(15): 1861 - 1870. [Full Text] [PDF]

				X. X. YU, D. A. LEWIN, W. FORREST, and S. H. ADAMS Cold elicits the simultaneous induction of fatty acid synthesis and {beta}-oxidation in murine brown adipose tissue: prediction from differential gene expression and confirmation in vivo FASEB J, February 1, 2002; 16(2): 155 - 168. [Abstract] [Full Text] [PDF]

				F. M. Campbell, R. Kozak, A. Wagner, J. Y. Altarejos, J. R. B. Dyck, D. D. Belke, D. L. Severson, D. P. Kelly, and G. D. Lopaschuk A Role for Peroxisome Proliferator-activated Receptor alpha (PPARalpha ) in the Control of Cardiac Malonyl-CoA Levels. REDUCED FATTY ACID OXIDATION RATES AND INCREASED GLUCOSE OXIDATION RATES IN THE HEARTS OF MICE LACKING PPARalpha ARE ASSOCIATED WITH HIGHER CONCENTRATIONS OF MALONYL-CoA AND REDUCED EXPRESSION OF MALONYL-CoA DECARBOXYLASE J. Biol. Chem., February 1, 2002; 277(6): 4098 - 4103. [Abstract] [Full Text] [PDF]

				J. D. McGarry Banting Lecture 2001: Dysregulation of Fatty Acid Metabolism in the Etiology of Type 2 Diabetes Diabetes, January 1, 2002; 51(1): 7 - 18. [Full Text] [PDF]

				W. W. Winder Energy-sensing and signaling by AMP-activated protein kinase in skeletal muscle J Appl Physiol, September 1, 2001; 91(3): 1017 - 1028. [Abstract] [Full Text] [PDF]

				V. K. Kaushik, M. E. Young, D. J. Dean, T. G. Kurowski, A. K. Saha, and N. B. Ruderman Regulation of fatty acid oxidation and glucose metabolism in rat soleus muscle: effects of AICAR Am J Physiol Endocrinol Metab, August 1, 2001; 281(2): E335 - E340. [Abstract] [Full Text] [PDF]

				M. E. Young, G. W. Goodwin, J. Ying, P. Guthrie, C. R. Wilson, F. A. Laws, and H. Taegtmeyer Regulation of cardiac and skeletal muscle malonyl-CoA decarboxylase by fatty acids Am J Physiol Endocrinol Metab, March 1, 2001; 280(3): E471 - E479. [Abstract] [Full Text] [PDF]

				Z.-P. Chen, G. K. McConell, B. J. Michell, R. J. Snow, B. J. Canny, and B. E. Kemp AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation Am J Physiol Endocrinol Metab, November 1, 2000; 279(5): E1202 - E1206. [Abstract] [Full Text] [PDF]

				T. J. Stephens, Z.-P. Chen, B. J. Canny, B. J. Michell, B. E. Kemp, and G. K. McConell Progressive increase in human skeletal muscle AMPKalpha 2 activity and ACC phosphorylation during exercise Am J Physiol Endocrinol Metab, March 1, 2002; 282(3): E688 - E694. [Abstract] [Full Text] [PDF]

				Z. Dagher, N. Ruderman, K. Tornheim, and Y. Ido Acute Regulation of Fatty Acid Oxidation and AMP-Activated Protein Kinase in Human Umbilical Vein Endothelial Cells Circ. Res., June 22, 2001; 88(12): 1276 - 1282. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 275/32/24279    most recent C000291200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Saha, A. K. Articles by Ruderman, N. B. Search for Related Content PubMed PubMed Citation Articles by Saha, A. K. Articles by Ruderman, N. B. Social Bookmarking                      What's this?