RAPID COMMUNICATION
Regulation of fatty acid oxidation of the heart by MCD and ACC during contractile stimulation

Gary W. Goodwin and Heinrich Taegtmeyer

Division of Cardiology, Department of Internal Medicine, University of Texas-Houston Medical School, Houston, Texas 77030


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypothesis that the level of malonyl-CoA, as well as the corresponding rate of total fatty acid oxidation of the heart, is regulated by the opposing actions of acetyl-CoA carboxylase (ACC) and malonyl-CoA decarboxylase (MCD). We used isolated working rat hearts perfused under physiological conditions. MCD in heart homogenates was measured specifically by 14CO2 production from [3-14C]malonyl-CoA, and ACC was measured specifically based on the portion of total carboxylase that is citrate sensitive. Increased heart work (1 µM epinephrine + 40% increase in afterload) elicited a 40% increase in total beta -oxidation of exogenous plus endogenous lipids, accompanied by a 33% decrease in malonyl-CoA. The basal activity and citrate sensitivity of ACC (reflecting its phosphorylation state) and citrate content were unchanged. AMP levels were also unchanged. MCD activity, when measured at a subsaturating concentration of malonyl-CoA (50 µM), was increased by 55%. We conclude that physiological increments in AMP during the work transition are insufficient to promote ACC phosphorylation by AMP-stimulated protein kinase. Rather, increased fatty acid oxidation results from increased malonyl-CoA degradation by MCD.

malonyl-CoA; citrate; acetyl-CoA carboxylase; malonyl-CoA decarboxylase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

FATTY ACIDS are the principal respiratory substrate for the heart (18, 24), and the rate of total fatty acid oxidation is increased by contractile stimulation (7). Both exogenous and endogenous (triglycerides within heart myocytes) fats are utilized for mitochondrial beta -oxidation, the latter by way of turnover of the triglyceride pool (7, 20). The flux-generating step, as well as a regulated step for triglyceride utilization, is lipolysis (9, 16, 25). Flux for total fatty acid oxidation is regulated at the level of entry of long-chain fatty acyl-CoA esters into mitochondria at carnitine palmitoyltransferase I (CPT-I, reviewed in Ref. 14).

CPT-I is inhibited by malonyl-CoA, which acts as a downstream signaling molecule for an elaborate scheme of cellular regulation designed to adjust rates of fatty acid oxidation to diverse signals for metabolic demand in addition to signals for the availability of respiratory substrates. The paradigm that is developing for skeletal muscle is that metabolic demand is signaled by a kinase cascade (reviewed in Ref. 8), whereas substrate availability is signaled by allosteric regulation of acetyl-CoA carboxylase (19), which is responsible for malonyl-CoA synthesis in the cytosol.

The kinase cascade (AMP-stimulated protein kinase and its upstream kinase) senses the status of AMP and phosphocreatine (8, 17). Stimulation of AMP kinase (AMPK) and AMPK kinase by AMP or relief of inhibition by phosphocreatine causes phosphorylation and inactivation of acetyl-CoA carboxylase (ACC), reduced malonyl-CoA levels, and increased flux through CPT-I. The other signaling pathway that feeds into malonyl-CoA, which senses the availability of carbohydrates for respiration, is allosteric stimulation of ACC by citrate (21, 27). Citrate is also a precursor for acetyl-CoA synthesis in the cytosol, providing substrate for ACC.

The aforementioned regulatory pathways feed into malonyl-CoA synthesis by ACC. Little is known about the opposing pathways responsible for the removal of malonyl-CoA. In muscle, which exhibits little or no de novo lipogenesis (3), malonyl-CoA decarboxylase (MCD) seems to be a major pathway for malonyl-CoA disposal (2) and is therefore a likely candidate for regulation of beta -oxidation.

The objective of the present study was to define the pathways responsible for regulation of malonyl-CoA levels and, hence, of fatty acid oxidation of the heart in relation to contractile state. Specifically, we tested for regulation of ACC by the aforementioned signaling pathways and also tested for regulation of MCD. Regulation of ACC in relation to contractile state has not previously been examined in heart to our knowledge. These are the first measurements of MCD in relation to contractile state in any tissue to our knowledge. We postulate reciprocal regulation of opposing pathways for malonyl-CoA synthesis and degradation.

We included two improvements over existing studies of regulation of heart fatty acid oxidation. First, we postulate that CPT-I regulates flux for total beta -oxidation of exogenous plus endogenous (triglyceride derived) fatty acids. We measured exogenous oleate oxidation by a conventional method (3H2O production from [9,10-3H]oleate) and also determined total beta -oxidation by an independent method. The latter values are probably more meaningful in terms of regulation of CPT-I. Second, the measurement of ACC in heart homogenates is complicated by the presence of large amounts of nonspecific carboxylase activity (27, 28). Pyruvate carboxylase and/or propionyl-CoA carboxylase are biotin enzymes that contribute to carboxylation of acetyl-CoA in vitro and do not depend on citrate for activity. To overcome this limitation, we expressed ACC as the portion of total carboxylase that is citrate dependent. We believe that this is the first specific measurement of ACC in the heart. We also examined a range of citrate concentrations to define the sensitivity to activation by citrate, which is an important feature of the regulation of the enzyme by phosphorylation (21, 27, 28).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Sources of materials. [3-14C]malonyl-CoA (not commercially available) was synthesized enzymatically from acetyl-CoA, ATP, [14C]NaHCO3, and purified ACC, essentially as described by Kolattukudy et al. (10). After overnight incubation at room temperature, glucose (10 mM) and yeast hexokinase (desalted, 1 U) were added to remove unreacted ATP, which cochromatographs with malonyl-CoA, and then the reaction was adjusted to pH 3 with HCl to remove unreacted 14CO2. The product was purified on DEAE-cellulose (1 × 10 cm) equilibrated with 3 mM HCl, as described by Kolattukudy et al. The column was developed with a gradient of LiCl (0-0.2 M in 3 mM HCl) over a 5-column volume. [3-14C]malonyl-CoA eluted as the only peak of radioactivity, coinciding with a sharp, well-resolved peak of absorbance at 260 nM. The specific activity (7 Ci/µmol calculated from the A260) was the same as that of the [14C]NaHCO3 starting material (6.8 Ci/µmol). The yield of [3-14C]malonyl-CoA from the limiting reagent (acetyl-CoA) was 20% (3 µCi). ACC used for this synthesis (also unavailable commercially) was purified from fresh rat liver after the enzyme was induced (4 rats were fasted for 2 days and then fed bread for 3 days), with the first two steps of the "alternate procedure" described by Ahmad and Ahmad (1), followed by chromatography on DEAE-cellulose, as described by Tanabe et al. (26). We obtained 12 U (specific activity 2.1 U/mg protein). The sources of other materials were given previously (7) or were from Sigma (St. Louis, MO) or Boehringer Mannheim (Indianapolis, IN).

Heart perfusions and perfusion protocol. We used hearts from perfusions of our previous study (7). Procedures for the working heart perfusions and for metabolic flux determination were described in detail in that study (7). The perfusate was Krebs-Henseleit buffer with 5 mM glucose, 40 µU/ml insulin, 0.5 mM lactate, and 0.4 mM oleate prebound to 3% albumin. The free Ca2+ concentration was 1.4 mM. Rat hearts were freeze-clamped on the cannula with aluminum tongs cooled in liquid N2. "Unstimulated" refers to hearts freeze-clamped just before stimulation of contractile activity. "Stimulation" refers to hearts freeze-clamped after 20 min of contractile stimulation, resulting from raising the afterload from 100 to 140 cmH2O and by adding of 1 µM epinephrine to the perfusate.

Analytical procedures. Frozen hearts, stored at -70°C, were weighed and ground to a fine powder under liquid N2, and a portion was taken for dry weight determination. Metabolites were measured in freshly prepared 6% perchloric acid extracts of heart, adjusted to pH 5 with buffered KOH. AMP and citrate were measured with established enzymatic methods (5). Malonyl-CoA was measured radiochemically with purified rat liver fatty acid synthase (1.1 U/mg protein) and [acetyl-3H]acetyl-CoA (Sigma). We included a malonyl-CoA internal standard for each determination, as described by McGarry et al. (15).

ACC assays. We initially measured carboxylase activity after fractionating heart homogenates with polyethylene glycol 8000 (fraction precipitating between 2.5 and 6%) as described by Kudo et al. (11). This procedure did not reduce non-citrate-dependent carboxylase activity (see also Fig. 3 of Ref. 11) and was abandoned. The procedure we used was adapted from Witters et al. (29). The frozen, powdered tissue (50 mg) was dispersed into 9 vol (wt/vol) of cold homogenization buffer, and the icy slurry was mixed with a glass rod. Phenylmethylsulfonyl fluoride (0.1 mM) was added to the slurry from a 0.1 M solution in methanol. The slurry was further homogenized with several passes of a tight-fitting Teflon Potter until thawed and then was centrifuged at 40,000 g for 30 min. The supernatant was dialyzed overnight against 100 vol of cold homogenization buffer consisting of 50 mM KH2PO4, 50 mM KF, 5 mM Na4-pyrophosphate, 1 mM EDTA, and 1 mg/ml BSA (fatty acid free), adjusted to pH 7.0 with KOH. The buffer was freshly supplemented with 1 mM dithiothreitol, 1 mM benzamidine, 1 µM leupeptin, 1 µM pepstatin-A, and 1 µg/ml aprotinin. ACC assays were based on acetyl-CoA-dependent 14CO2 fixation. The assay buffer was 50 mM HEPES, 2.1 mM ATP, 5 mM creatine phosphate, 1 mg/ml BSA (fatty acid free), and 10 mM Mg-acetate, adjusted to pH 7.5 at 30°C with KOH. The buffer was freshly supplemented with 0.3 mM acetyl-CoA, 1 mM dithiothreitol, and 0.1 mg/ml creatine kinase (Boehringer). Assays were performed without (in duplicate) or with the indicated additions of Mg-citrate, with a stock solution containing equimolar K3-citrate and Mg-acetate, pH 7.5. The reaction volume was 50 µl, and the sample volume was 10 µl. After thermal equilibration to 30°C, reactions were started by the addition of [14C]NaHCO3 (1 µCi, final concentration of 10 mM) and stopped after 10 min by the addition of 10 µl of 30% perchloric acid (fume hood). The reaction mix was centrifuged for 1 min (Microfuge), and 50 µl were spotted onto Whatman 31 ET filter paper (2 × 2 cm) and dried overnight in a vacuum over KOH pellets. The paper was taken for scintillation counting after the addition of 2 ml of H2O and 10 ml of scintillation mixture (Ultima Gold, Packard, Meriden, CT). Values were corrected for blanks performed without added acetyl-CoA (~100 dpm). ACC was calculated by subtracting the activity measured in the absence of added citrate from the activity in the presence of the indicated concentrations of citrate. Values were normalized to protein determined by the method of Lowry et al. (13), with BSA as the standard.

MCD assays. The activity of MCD in heart homogenates was too low to measure spectrophotometrically. We therefore used a sensitive and specific radiochemical assay based on 14CO2 production from [3-14C]malonyl-CoA, adapted from Kolattukudy et al. (10). Homogenates of frozen, powdered heart (5% wt/vol) were prepared as described previously for the ACC assay. After centrifugation, the supernatant was taken directly for the MCD assay. The homogenization buffer and assay buffer were the same and included proteinase inhibitors, phosphatase inhibitors (phosphate and fluoride), and EDTA, in case MCD is regulated by phosphorylation. The buffer was 0.1 M Tris, 50 mM KF, 50 mM KPi, and 1 mM EDTA, adjusted to pH 8.0 at 30°C with HCl. It was freshly supplemented with 2 mM dithiothreitol, 1 mM benzamidine, 1 µM leupeptin, 1 µM pepstatin-A, and 1 µg/ml aprotinin. The reaction volume was 200 µl, and the sample volume was 20 µl. After thermal equilibration to 30°C, reactions were initiated with malonyl-CoA (10,000 dpm, final concentration of 0.05 or 0.2 mM), the reaction tube was placed in a glass scintillation vial containing 1 ml of hyamine hydroxide (1 M in methanol), and the vial was fitted with a serum cap. Reactions were stopped after 10 min by injection of 0.2 ml of 6% perchloric acid. The vials were shaken overnight to collect 14CO2 and then were taken for scintillation counting after the addition of 10 ml of scintillation mixture. Values were corrected for blanks with homogenization buffer in place of tissue homogenate. Blanks were equivalent to background radioactivity (20 dpm). We established that 14CO2 production was linear with respect to time and amount of tissue extract used in the assays.

Data are expressed as means ± SE. Statistical comparison was by Student's t-test for unpaired data. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Malonyl-CoA levels and the regulation of total beta -oxidation. Table 1 gives rates of exogenous oleate oxidation (3H2O from [9,10-3H]oleate), total fatty acid oxidation of exogenous plus endogenous lipids, and the level of malonyl-CoA in the hearts before and after stimulation of heart contractile activity. These are the values that we reported previously (7). Values for total beta -oxidation were calculated from oxygen consumption and total carbohydrate oxidation and are completely independent of the reported measures for oleate oxidation. Contractile stimulation caused a 75% increase in hydraulic power (7) and a 33% decrease in the tissue content of malonyl-CoA. The decrease in malonyl-CoA caused a 40% increase in total fatty acid oxidation. The increase in exogenous oleate oxidation (20%) was not significant. These results are consistent with our hypothesis that CPT-I regulates flux for total beta -oxidation of exogenous plus endogenous lipids. An increase in the contribution of endogenous lipids to total fatty acid oxidation is to be expected based on the stimulation of hormone-sensitive lipase within heart myocytes by phosphorylation by protein kinase A (9, 16, 25).

                              
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Table 1.   Fatty acid oxidation (exogenous and total) and content of malonyl-CoA before and after contractile stimulation

The decrease in malonyl-CoA does not result from phosphorylation of ACC. To explain the decrease in malonyl-CoA, we examined regulation of ACC. For this, homogenates of the whole heart were prepared under conditions that protect the phosphorylation state of ACC (we included fluoride, phosphate, and pyrophosphate to inhibit phosphatases and EDTA to inhibit kinases in case any ATP remains in the homogenate). We also dialyzed the preparation to remove endogenous citrate. Most of total carboxylase activity in the hearts at physiological levels of citrate (roughly 0.2 mM) was not citrate dependent. The activity without added citrate was 0.59 ± 0.17 nmol · min-1 · mg protein-1 in the unstimulated state and 0.87 ± 0.07 nmol · min-1 · mg protein-1 in the stimulated state. This is the expected result. Carboxylation of acetyl-CoA in the absence of citrate results from the actions of propionyl-CoA carboxylase and/or pyruvate carboxylase, which are both active in heart (27, 28). To measure the activity of ACC specifically, we calculated the citrate-dependent portion of total carboxylase activity by subtracting the activity measured without citrate from the activity measured with added Mg-citrate. Figure 1 shows ACC activity over a range of concentrations of Mg-citrate added to the assay. The basal activity at a given citrate concentration and the citrate sensitivity of the enzyme, which reflect the phosphorylation state of ACC by AMPK, were not different between the two groups. Therefore, the phosphorylation state of ACC does not explain the decrease in malonyl-CoA levels of the heart resulting from contractile stimulation.


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Fig. 1.   Acteyl-CoA carboxylase (ACC) activity vs. Mg-citrate added to assay. Values are means ± SE; n = 5/group. Hearts were freeze-clamped before or after stimulation of contractile activity, and then heart homogenates were prepared for ACC assay. open circle , unstimulated contractile activity; , stimulated contractile activity.

The lack of change in phosphorylation state of ACC by AMPK reflects the fact that, for normoxic heart, AMP does not increase sufficiently during contractile stimulation to activate the AMP-kinase cascade. The level of AMP in the hearts was 1.49 ± 0.33 µmol/g dry wt before stimulation (n = 5) and 1.44 ± 0.15 µmol/g dry wt after stimulation (n = 15).

Another explanation for the decrease in malonyl-CoA could be that the content of citrate in the cytosol decreased, because cytosolic citrate stimulates ACC. The content of citrate specifically in the cytosolic compartment is difficult to determine because citrate partitions between mitochondria and cytosol. However, the whole tissue content of citrate did not decrease [it actually increased slightly from 2.05 ± 0.19 µmol/g dry wt before stimulation (n = 5) to 2.42 ± 0.09 µmol/g dry wt after stimulation (n = 15)]. This suggests, but does not prove, that the decrease in malonyl-CoA did not occur because the content of citrate in the cytosol decreased.

The decrease in malonyl-CoA with contractile stimulation could result from stimulation of malonyl-CoA degradation by MCD. Another potential level of regulation of malonyl-CoA is by the rate of degradation back to acetyl-CoA, catalyzed by MCD. Table 2 gives activities for MCD in whole heart homogenates measured at two concentrations of malonyl-CoA: 0.2 mM and 50 µM. The latter concentration is the Michealis-Menten constant (Km) of purified rat liver mitochondrial MCD (10) and is a subsaturating concentration in our heart homogenates, giving roughly one-half of the activity obtained with 0.2 mM substrate. The rationale for measuring the activity at high and low concentrations is because the ambient concentration of malonyl-CoA in heart cytosol (a few µM) is well below the Km of MCD. Under this condition, the flux for MCD is proportional to the ratio of maximum velocity (Vmax) to Km. Acute regulation of MCD flux could be achieved, in principle, equally by way of the Km or the Vmax. Regulation by way of the Km would not be detected based on measurements with a high concentration of malonyl-CoA.

                              
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Table 2.   Malonyl-CoA decarboxylase activity before and after contractile stimulation

There was no change in the activity of MCD on contractile stimulation when measured with 0.2 mM malonyl-CoA. There was a 55% increase in MCD activity on contractile stimulation when measured with 50 µM malonyl-CoA (Table 2). This suggests that the apparent Km of the enzyme for malonyl-CoA decreased. We then calculated values for apparent Km and Vmax of MCD in the tissue homogenates, assuming hyperbolic kinetics (Table 2). The apparent Km was decreased 77% after stimulation of contractile activity. Taken together, our data suggest that increased MCD flux at an ambient concentration of malonyl-CoA contributed to the observed decrease in malonyl-CoA levels and the increase in total fatty acid oxidation.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Our data suggest that increased degradation of malonyl-CoA by acute stimulation of MCD is responsible for the observed decrease in the tissue content of malonyl-CoA and, hence, an increase in total fatty acid oxidation of the heart on stimulation of contractile activity. This is based on the observation that the decrease in malonyl-CoA was correlated with the change in the activity of MCD but not of ACC. We could not rule out the possibility that reduced cytosolic citrate contributed to diminished synthesis of malonyl-CoA by ACC, although the slight increase in total tissue citrate suggests that this was not the case.

We postulated reciprocal regulation of ACC and MCD. Contrary to the hypothesis, we did not find inactivation of ACC during contractile stimulation. Inactivation of ACC in heart was anticipated from observations in skeletal muscle. Contractile stimulation of skeletal muscle causes phosphorylation and inactivation of ACC because AMPK becomes stimulated by the rise in AMP (28). We propose that the difference between heart and skeletal muscle in this respect is because the dynamic range of the contractile state of the heart is not as large as that of skeletal muscle. Unlike skeletal muscle, the heart exhibits high basal contractile activity and did not exhibit increased AMP during stimulation in our study. Presumably, the large dynamic range of the contractile state of skeletal muscle will contribute to larger transient shifts in high-energy phosphates, contributing to increased beta -oxidation resulting from stimulation of AMPK.

We interpreted ACC activity as the portion of total carboxylase activity that is citrate dependent. This was necessary because most of the activity contributing to acetyl-CoA carboxylation in heart homogenates is not relevant to the regulation of malonyl-CoA levels in the cytosol. Propionyl-CoA carboxylase and/or pyruvate carboxylase contributes to acetyl-CoA carboxylation in vitro, is abundant in heart mitochondria, and is not readily separated from ACC without a specific antibody (21, 27, 28). Nonspecific carboxylation explains high citrate-independent acetyl-CoA carboxylation in a previous study of heart ACC (11) because ACCbeta (the 280-kDa isoform that predominates in heart and skeletal muscle) has nearly complete dependence on citrate for activity. This is based on observations with purified enzyme (27) and from Vavvas et al. (28), who recently measured ACC specifically in skeletal muscle by immunoprecipitating the enzyme in a catalytically active form. Our interpretation suggests that the decrease in carboxylation of acetyl-CoA in reperfused hearts, reported by Kudo et al. (11), did not result from a specific decrease in ACC (it appears that the citrate-dependent portion of total carboxylase actually increased slightly). Rather, the absence of a change in ACC during reperfusion in that study seems more consistent with their observation that fatty acid oxidation was also unchanged during reperfusion, when measured in absolute terms (11).

We observed an increase in the activity of MCD when measured at a low, but not a high, concentration of malonyl-CoA. This suggests that the enzyme was regulated by way of the Km. Because the ambient concentration of malonyl-CoA in the heart (a few µM) is much less than the Km of MCD for malonyl-CoA (~50 µM), enzyme activity measured at low concentrations of substrate is more relevant to flux through the enzyme under ambient conditions and the Km becomes a valid parameter for enzyme regulation. Changes in enzyme expression are expected to change the Vmax as well, but the duration of our experiment (20 min between unstimulated and stimulated hearts) was probably too brief for differences in enzyme expression to manifest.

We did not discern the subcellular localization of MCD in this study (we measured the activity in the whole heart). MCD is thought to exist in both cytosolic and mitochondrial compartments in skeletal muscle (2) and heart (12). Dyck et al. (6) recently reported MCD activity in rat heart mitochondria, but it is not clear if mitochondrial MCD could regulate malonyl-CoA levels in the cytosol. On the basis that malonyl-CoA is not a substrate for carnitine acetyltransferase, Scholte (22) concluded that mitochondrial MCD cannot affect extramitochondrial malonyl-CoA. MCD appears to exist in mitochondria to prevent inhibition of pyruvate carboxylase (23) and methylmalonyl-CoA mutase (4) by malonyl-CoA synthesized by pyruvate carboxylase and/or propionyl-CoA carboxylase. This role is not necessarily inconsistent with the regulation of cytosolic levels of malonyl-CoA by mitochondrial MCD. Nevertheless, it seems likely that cytosolic MCD is relevant to regulation of fatty acid oxidation.

In summary, our results suggest that acute regulation of total fatty acid oxidation of the heart during contractile stimulation is regulated by the rate of disposal of malonyl-CoA catalyzed by MCD. We did not find evidence for regulation of ACC in this circumstance. Changes in AMP during contractile stimulation of normoxic heart are insufficient to affect malonyl-CoA levels through the AMP-kinase cascade.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grant RO1-43133 and a Grant-in-Aid from the American Heart Association, Texas Affiliate (97G-329 and 9960099Y).


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. W. Goodwin, Univ. of Texas-Houston Medical School, 6431 Fannin, MSB 1.246, Houston, TX 77030 (E-mail: ggoodwin{at}heart.med.uth.tmc.edu).

Received 19 March 1999; accepted in final form 1 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahmad, F., and P. M. Ahmad. Acetyl-CoA carboxylase from rat mammary gland. Methods Enzymol. 71: 16-26, 1981[Medline].

2.   Alam, N., and A. D. Saggerson. Malonyl-CoA and the regulation of fatty acid oxidation in soleus muscle. Biochem. J. 324: 233-241, 1998.

3.   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[Medline].

4.   Babior, B. M., A. D. Woodams, and J. D. Brodie. Cleavage of coenzyme B12 by methylmalonyl coenzyme A mutase. J. Biol. Chem. 248: 1445-1450, 1973[Abstract/Free Full Text].

5.   Bergmeyer, H. U. Methods of Enzymatic Analysis. New York: Academic, 1974.

6.   Dyck, J. R. B., A. J. Barr, R. L. Barr, P. E. Kolattukudy, and G. D. Lopaschuk. Characterization of cardiac malonyl-CoA decarboxylase and its putative role in regulating fatty acid oxidation. Am. J. Physiol. 275 (Heart Circ. Physiol. 44): H2122-H2129, 1998[Abstract/Free Full Text].

7.   Goodwin, G. W., C. S. Taylor, and H. Taegtmeyer. Regulation of energy metabolism of the heart during acute increase in heart work. J. Biol. Chem. 273: 29530-29539, 1998[Abstract/Free Full Text].

8.   Hardie, D. G., and D. Carling. The AMP-activated protein kinase. Fuel gauge of the mammalian cell? Eur. J. Biochem. 246: 259-273, 1997[Abstract].

9.   Heathers, G. P., N. Al-Muhtaseb, and R. V. Brunt. The effect of adrenergic agents on the activities of glycerol 3-phosphate acyltransferase and triglyceride lipase in the isolated perfused rat heart. J. Mol. Cell. Cardiol. 17: 785-796, 1985[Medline].

10.   Kolattukudy, P. E., A. J. Poulose, and Y. S. Kim. Malonyl-CoA decarboxylase from avian, mammalian, and microbial sources. Methods Enzymol. 71: 150-163, 1981[Medline].

11.   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].

12.   Landriscina, C., G. V. Gnoni, and E. Quagliariello. Properties of malonyl-CoA decarboxylase and its relation with malonyl-CoA incorporation into fatty acids by rat liver mitochondria. Eur. J. Biochem. 19: 573-580, 1971[Medline].

13.   Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951[Free Full Text].

14.   McGarry, J. D., and N. F. Brown. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur. J. Biochem. 244: 1-14, 1997[Abstract].

15.   McGarry, J. D., M. J. Stark, and D. W. Foster. Hepatic malonyl-CoA levels of fed, fasted, and diabetic rats as measured using a simple radioisotopic assay. J. Biol. Chem. 253: 8291-8293, 1978[Abstract].

16.   Nilsson, N. Ö, P. Strålfors, G. Fredrikson, and P. Belfrage. Regulation of adipose tissue lipolysis: effects of noradrenaline and insulin on phosphorylation of hormone-sensitive lipase and on lipolysis in intact rat adipocytes. FEBS Lett. 111: 125-130, 1980[Medline].

17.   Ponticos, M., Q. L. Lu, J. E. Morgan, D. G. Hardie, T. A. Partridge, and D. Carling. Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle. EMBO J. 17: 1688-1699, 1998[Abstract/Free Full Text].

18.   Rothlin, M. E., and R. J. Bing. Extraction and release of individual free fatty acids by the heart and fat depots. J. Clin. Invest. 40: 1380-1386, 1961.

19.   Ruderman, N. B., A. K. Saha, D. Vavvas, and L. A. Witters. Malonyl-CoA, fuel sensing, and insulin resistance. Am. J. Physiol. 276 (Endocrinol. Metab. 39): E1-E18, 1999[Abstract/Free Full Text].

20.   Saddik, M., and G. D. Lopaschuk. Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat heart. J. Biol. Chem. 266: 8162-8170, 1991[Abstract/Free Full Text].

21.   Saha, A. K., D. Vavvas, T. G. Kurowski, A. Apazidis, L. A. Witters, S. Eleazar, and N. B. Ruderman. Malonyl-CoA regulation in skeletal muscle: its link to cell citrate and the glucose-fatty acid cycle. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E641-E648, 1997[Abstract/Free Full Text].

22.   Scholte, H. R. Liver malonyl-CoA decarboxylase. Biochim. Biophys. Acta 309: 457-465, 1973[Medline].

23.   Scrutton, M. C., and M. F. Utter. Pyruvate carboxylase. J. Biol. Chem. 242: 1723-1735, 1967[Abstract/Free Full Text].

24.   Shipp, J. C., L. H. Opie, and D. Challoner. Fatty acid and glucose metabolism in the perfused heart. Nature 189: 1018-1019, 1961.

25.   Small, C. A., A. J. Garton, and S. J. Yeaman. The presence and role of hormone-sensitive lipase in heart muscle. Biochem. J. 258: 67-72, 1989[Medline].

26.   Tanabe, T., S. Nakanishi, T. Hashimoto, H. Ogiwara, J. Nikawa, and S. Numa. Acetyl-CoA carboxylase from rat liver. Methods Enzymol. 71: 5-15, 1981[Medline].

27.   Thampy, K. G. Formation of malonyl-CoA in rat heart. Identification and purification of an isozyme of acetyl-CoA carboxylase from rat heart. J. Biol. Chem. 264: 17631-17634, 1989[Abstract/Free Full Text].

28.   Vavvas, D., A. Apazidis, A. K. Saha, J. Gamble, A. Patel, B. E. Kemp, L. A. Witters, and N. B. Ruderman. Contraction-induced changes in acetyl-CoA carboxylase and 5'-AMP-activated kinase in skeletal muscle. J. Biol. Chem. 272: 13255-13261, 1997[Abstract/Free Full Text].

29.   Witters, L. A., T. D. Watts, D. L. Daniels, and J. L. Evans. Insulin stimulates the dephosphorylation and activation of acetyl-CoA carboxylase. Proc. Natl. Acad. Sci. USA 85: 5473-5477, 1988[Abstract].


Am J Physiol Endocrinol Metab 277(4):E772-E777
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