Division of Cardiology, Department of Internal Medicine, University of Texas-Houston Medical School, Houston, Texas 77030
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ABSTRACT |
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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 -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
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INTRODUCTION |
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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 -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 -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 -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
-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).
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METHODS |
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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.
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RESULTS |
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Malonyl-CoA levels and the regulation of total
-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
-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
-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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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 · min1 · 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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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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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.
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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
-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 ACC (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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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