Departments of 1 Biochemistry and 2 Nutrition, University of Montreal, Montreal, Quebec, Canada H3C 3J7
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ABSTRACT |
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Cytosolic citrate is proposed to play a crucial role in substrate fuel selection in the heart. However, little is known about factors regulating the transfer of citrate from the mitochondria, where it is synthesized, to the cytosol. Further to our observation that rat hearts perfused under normoxia release citrate whose 13C labeling pattern reflects that of mitochondrial citrate (B. Comte, G. Vincent, B. Bouchard, and C. Des Rosiers. J. Biol. Chem. 272: 26117-26124, 1997), we report here data indicating that this citrate release is a specific process reflecting the mitochondrial efflux of citrate, a process referred to as cataplerosis. Indeed, measured rates of citrate release, which vary between 2 and 21 nmol/min, are modulated by the nature and concentration of exogenous substrates feeding acetyl-CoA (fatty acid) and oxaloacetate (lactate plus pyruvate) for the mitochondrial citrate synthase reaction. Such release rates that represent at most 2% of the citric acid cycle flux are in agreement with the activity of the mitochondrial tricarboxylate transporter whose participation is also substantiated by 1) parallel variations in citrate release rates and tissue levels of citrate plus malate, the antiporter, and 2) a lowering of the citrate release rate by 1,2,3-benzenetricarboxylic acid, a specific inhibitor of the transporter. Taken together, the results from the present study indicate that citrate cataplerosis is modulated by substrate supply, in agreement with the role of cytosolic citrate in fuel partitioning, and occurs, at least in part, through the mitochondrial tricarboxylate transporter.
energy metabolism; citric acid cycle; 13C mass isotopomer analysis; mitochondrial tricarboxylic acid transporter
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INTRODUCTION |
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AS THE FIRST INTERMEDIATE committed to the citric acid
cycle (CAC), citrate plays a crucial role in cardiac
energy metabolism. However, its metabolic role may not be limited to
the mitochondria. Indeed, cytosolic citrate is proposed to also play a
central role in substrate fuel partitioning (for a recent review see
Ref. 28). An increase in cytosolic citrate, after its efflux from the
mitochondria where it is synthesized, could restrict 1) glucose
utilization by inhibiting glycolysis at the level of
phosphofructokinase (PFK; see Refs. 11 and 8 for review) and/or 2)
long-chain fatty acid (LCFA) -oxidation after its conversion to
malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I (CPT-I;
see Refs. 1, 19, 20, 21, 31). The regulatory role of cytosolic citrate
has long been recognized in lipogenic tissues such as the liver and
adipose tissues, where it also supplies acetyl units for de novo fatty
acid synthesis. In nonlipogenic tissues, a generalized theory of fuel
sensing was formulated recently on the basis of experimental evidence
obtained in
-cells (26) and skeletal muscle (28, 30, 31) whereby
cytosolic citrate would act as a signal to cells that they have an
excess of fuel for their immediate needs. Further investigations are,
however, required to support such a mechanistic scheme in the heart.
In cardiac cells, a regulatory role for cytosolic citrate as an inhibitor of glycolysis was first proposed in 1963 by Garland et al. (11). However, the significance of mitochondrial citrate efflux, or cataplerosis, was questioned because of the low activity of the mitochondrial tricarboxylate transporter (5, 32). Nevertheless, a mitochondrial citrate efflux was demonstrated in isolated heart mitochondria (5, 14, 27). This efflux, which occurred in response to added malate, was favored at a high NADH-to-NAD+ concentration ratio or state 4 respiration (14) and was restricted by 1,2,3-benzenetricarboxylic acid (BTC), a specific inhibitor of the mitochondrial tricarboxylate transporter (5, 23, 27). To the best of our knowledge, mitochondrial citrate cataplerosis has not been examined in the intact heart. However, cardiac citrate release has been documented in vivo in humans (33, 34) and ex vivo in the perfused rat heart (6, 7). Of potential clinical relevance, myocardial citrate release was increased in patients with coronary artery disease in a proportion that correlated with disease severity (33, 34). Furthermore, a relationship between cardiac citrate release and mitochondrial citrate cataplerosis was suggested by our finding (6, 7) that rat hearts perfused with 13C-labeled substrates release citrate whose 13C labeling pattern reflects that of mitochondrial citrate. This finding, and the possibility that a dysregulated myocardial citrate release represents a specific chronic alteration of energy metabolism in patients with ischemic disease, prompted us to further examine this process.
The purpose of the present study was to elucidate factors modulating citrate release by the perfused rat heart. Citrate release rates were quantitated by isotope dilution gas chromatography-mass spectrometry (GC-MS) and flow rate measurements in hearts perfused under normoxia with various substrate mixtures, including physiological LCFA. In view of the crucial role of mitochondrial citrate in the CAC, we hypothesized that, if citrate release reflects mitochondrial citrate cataplerosis, it should be specifically modulated by 1) the energy demand, which is a determinant of citrate utilization by the CAC and 2) the supply of substrates for citrate synthesis, namely acetyl-CoA and anaplerotic oxaloacetate (OAA). Furthermore, the release of citrate should not be correlated with that of 1) lactate dehydrogenase (LDH), an index of membrane damage, or 2) the CAC intermediate succinate, an index of oxygen deprivation (16). Finally, provided that citrate release involves the mitochondrial tricarboxylate transporter, it should be inhibited by 1,2,3-BTC but not by its inactive chemical analog 1,2,4-BTC.
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EXPERIMENTAL PROCEDURES |
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Chemicals
Chemicals, organic solvents, enzymes, coenzymes, insulin, and BSA (fraction V) were purchased from Boehringer Mannheim (Laval, Quebec), Fisher Scientific (Montreal, Quebec), Sigma-Aldrich Chemicals (Milwaukee, WI), Anachemia (Dorval, Quebec), and ICN Canada (Montreal, Quebec). [2,2,3,3-2H4]succinate, [2,2,4,4-2H4]citrate, [U-13C3]lactate (99%), [U-13C3]pyruvate (99%), [1-13C]oleic acid, and [1,2-13C2]octanoic acid (99%) were obtained from Isotec (Miamisburg, OH), Cambridge Isotopes Laboratories (Woburn, MA), and CDN Isotopes (Pointe-Claire, Quebec). RS-3-hydroxy-[2,2,3,4,4,4-2H6]butyrate (RS-[2H6]BHB) and [1,2,3,6-13C4]citrate were synthesized as described previously (9, 15). The derivatization agent N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide was supplied by Regis Chemical (Morton Grove, IL). All aqueous solutions were made with water purified by a "Milli-Q" system (Millipore, St-Laurent, Quebec). The albumin solution [BSA, fraction V, fatty acid poor (Bayer, Kankakee, IL): 1.2 kg in 8 liters of Krebs buffer without glucose] was dialyzed at 4°C against 25 liters of the same buffer for 8 h. The buffer was changed six times (for a total of 150 liters of Krebs in 48 h) to reduce the background citrate concentration to the low micromolar range (1.2 ± 0.1 µM). The dialyzed solution was stored atHeart Perfusions
Animal experiments were approved by the local animal care committee in compliance with the guidelines of the Canadian Council on Animal Care. After opening the chest and inserting a cannula in the aorta, hearts were excised and transferred to a Langendorff perfusion setup, as described previously (6, 7, 16). Briefly, the hearts of fed male Sprague-Dawley rats (180-240 g; Charles-River, St-Constant, Quebec) were anesthetized by intraperitoneal injection of pentobarbital sodium (65 mg/kg) and then were perfused retrogradely through the aorta at a constant pressure of 70 mmHg with a nonrecirculating modified Krebs-Ringer bicarbonate buffer containing (in mM) 119 NaCl, 4.8 KCl, 1.3 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3 (pH 7.4) and various additives (substrates, hormones, or drugs), as indicated. The perfusate was gassed directly with 5% CO2-95% O2 at 37°C. For heart perfusions in the presence of albumin, the buffer contained in a stirred reservoir was pumped through a filter at a rate of 80 ml/min at the top of a jacketed glass oxygenator with a large inner surface area exposed to 5% CO2-95% O2. The oxygenated buffer was delivered to the heart through the aortic cannula by a pump whose rate was adjusted to keep the perfusion pressure at 70 mmHg (between 10 and 15 ml/min). Excess oxygenated buffer was returned to the buffer reservoir through an overflow outlet.The following functional parameters were monitored continuously through instruments linked to a microcomputer: 1) coronary flow, using an electromagnetic flow probe (model FM501; Carolina Medical Electronics, King, NC), 2) temperature, using a thermocouple (Yellow Springs Instrument, Yellow Springs, OH), and 3) contractile function (heart rate, systolic and diastolic pressures, dP/dtmax), using a saline-filled latex balloon inserted in the left ventricular cavity, inflated to achieve an initial end-diastolic pressure of 5 mmHg, and connected to a pressure transducer (Digi-Med Heart Performance Analyzer, Micro-Med, Louisville, KY). The hearts were allowed to beat spontaneously throughout the experiments.
Experimental Design
All heart perfusion experiments included a 20-min stabilization period followed by a 30- to 90-min perfusion period (Fig. 1). Three groups of perfusion experiments were conducted. Under these conditions, we quantitated citrate release rates and documented the following parameters: 1) contractile activity (from continuous monitoring of dP/dtmax), an index of the energy demand, 2) 13C labeling of citrate isolated from the effluent perfusate and tissue, to assess the origin of the effluent citrate, 3) 13C enrichment of the acetyl (C4+5) and OAA (C1+2+3+6) moiety of effluent citrate, to probe the origin and supply of substrates for the citrate synthase reaction, 4) the release rate of ketone bodies (KB), an index of acetyl-CoA spillover from the CAC (6, 7), 5) the release rate of succinate, a CAC intermediate whose release rate is increased by oxygen deprivation (16), 6) the release of LDH, an index of cellular necrosis, and 7) tissue levels of citrate and malate, an antiporter for citrate efflux from the mitochondria (5, 14, 27).
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For the addition of 13C substrate(s), the unlabeled
substrate(s) was replaced by the corresponding labeled substrate(s),
either [1,2-13C2]octanoate or
[1-13C]oleate ± [U-13C3](lactate + pyruvate). We
reported previously that plateau enrichment of effluent citrate is
reached within 20 min (7). All of these 13C-labeled
substrates are metabolized to mitochondrial citrate by different
pathways: [1-13C]oleate and
[1,2-13C2]octanoate are
-oxidized to M1 and M2 isotopomers of acetyl-CoA, respectively, and
[U-13C3](lactate + pyruvate) are
decarboxylated to M2 acetyl-CoA and carboxylated to M3 OAA.
Group 1: Perfusion in the presence of the medium-chain fatty acid
octanoate.
As a follow-up to our previous work (6, 7), citrate release rates were
assessed initially in isolated rat hearts perfused with 11 mM glucose,
1 mM lactate, 0.2 mM pyruvate, and 0.2 mM octanoate. Compared with this
substrate mixture, referred to as the control condition, other
interventions were designed to 1) accelerate the rate of
citrate utilization by the CAC, by increasing energy demand with the
-adrenergic agonist isoproterenol or 2) limit its rate of
synthesis by lowering the availability of substrates for the citrate
synthase reaction, either acetyl-CoA or OAA. Isoproterenol was infused
at a final concentration of 1 µM. Acetyl-CoA supply was reduced by
decreasing exogenous octanoate concentration from 0.2 to 0.02 mM.
Anaplerotic OAA supply was lowered by the removal of lactate and
pyruvate from the perfusion buffer (6). Note that octanoate is a
nonphysiological fatty acid whose
-oxidation is not regulated by
CPT-I, and its concentration in the perfusion buffer sets the rate of
acetyl-CoA generation. At 0.2 mM, it leads to a maximal
-oxidative
rate (17). The formation of acetyl-CoA in excess of citrate synthesis
is reflected by a release of KB, i.e., acetoacetate and
-hydroxybutyrate (6, 7). Although the heart is normally a net
consumer of KB, pseudoketogenesis can occur via reversal of the
succinyl-CoA transferase reaction (10). Thus we interpret the KB
release as reflecting acetyl-CoA spillover from the CAC.
Group 2: Perfusion with physiological substrate mixtures. In addition to the results obtained in the first group of perfusion experiments, we perfused hearts with substrate mixtures mimicking physiological conditions. To mimic the fed state, hearts were perfused with 5.5 mM glucose, 8 nM insulin, and two different concentrations of lactate and pyruvate. To mimic the fasted state, hearts were perfused in the additional presence of an LCFA, 0.4 mM oleate (complexed to 4% albumin).
Group 3: Effect of 1,2,3-BTC, an inhibitor of the mitochondrial citrate transporter. To evaluate the origin and specificity of citrate release, the effect of 1,2,3-BTC, the only known inhibitor of the mitochondrial citrate carrier, was documented and compared with its inactive chemical analog 1,2,4-BTC (5, 27). The entire perfusion protocol lasted 90 min and was divided into three 30-min periods. It was designed to document citrate release before, during, and after treatment with 10 mM 1,2,3- or 1,2,4-BTC. This concentration was chosen on the basis of other studies with intact cells (23).
Sample Processing
Samples of effluent perfusate were collected on ice and processed as follows: 1) 10 ml remained untreated, and 2) 10 ml were treated with 1 M sodium borohydride (NaBH4; 500 µl/10 ml) to reduce OAA to malate and acetoacetate to BHB. For the analysis of effluent citrate, samples (10 ml) were concentrated to 4 ml under a stream of air at 50°C. For the analysis of effluent citrate in albumin-containing buffer, samples of perfusate were first treated with saturated sulfosalicylic acid (250 µl) and centrifuged for 10 min at 10,000 rpm. Next, after pH adjustment (between 4 and 7), the supernatants were concentrated to 4 ml and centrifuged through an Ultrafree-4 Millipore filter at 6,000 rpm for 30 min. All samples were stored atAnalytical Procedures
GC-MS.
Methods to determine 13C mass isotopomer distribution and
concentrations of the following metabolites isolated from effluent perfusates or tissues have been previously described in detail (6, 7,
16): citrate (C16) as well as its acetyl (C4+5) and OAA (C1+2+3+6) moieties, BHB,
succinate, fumarate, and pyruvate. The internal standards used for
quantification of succinate, BHB, and citrate releases were
[2,2,3,3-2H4]succinate,
RS-[2,2,3,4,4,4-2H6]BHB,
and [1,2,3,6-13C4]- or
[2,2,4,4-2H4]citrate, respectively.
Other assays. The concentrations of tissue citrate and malate were determined using standard enzymatic assays (2) on a Roche Cobas Fara spectrophotometer. LDH release was assessed as described previously (3). Analyses of total and ionized calcium levels in perfusion buffer samples were conducted by the Clinical Biochemistry Laboratory of Notre-Dame Hospital.
Calculations
13C labeling of metabolites.
The absolute molar percent enrichment (MPE) of individual
13C-labeled mass isotopomers (Mi) of a given
metabolite was calculated as follows
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(1) |
Release rates of metabolites. Calculations of the concentration of a metabolite, using the corresponding heavy labeled internal standard, have been described previously (16). Total release rates of the metabolites were obtained by multiplying their perfusate concentrations (nmol/ml) by the coronary flow rate (ml/min). For all metabolites (citrate, KB, or succinate), a solution of Krebs buffer containing the internal standards was always processed and analyzed in parallel to correct for the background signal.
Flux parameters.
The development of equations to calculate the relative contributions of
octanoate and pyruvate to citrate formation, from the 13C
labeling pattern of citrate released by hearts perfused with [1-13C]octanoate or
[U-13C3](pyruvate + lactate), have
been described in detail previously (7). In the present study, we
report the following flux ratios: 1) the contribution of
octanoate or oleate to the acetyl moiety of citrate; 2) the
contribution of pyruvate to the acetyl and OAA moieties of citrate, and
3) the contribution of the unlabeled pyruvate arising from
glucose or glycogen to tissue pyruvate. With
[1,2-13C2]octanoate as the only
labeled substrate, we measured the mole fraction (MF) in M2 of the
acetyl moiety of citrate [instead of the MF in M1 for
[1-13C]octanoate, as in our previous study
(6)]. The MF in a given mass isotopomer (Mi) of a
metabolite is equivalent to MPE/100. Then, the equation to calculate
the flux ratio (octanoate oxidation/citrate synthesis) becomes
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(2) |
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(3) |
Statistical Analysis
Individual enrichments are averages of two to five GC-MS injections. The data are expressed as means ± SE of n heart perfusions. The following tests were applied for statistical evaluation of the data: paired or unpaired t-test, one-way ANOVA followed by a Bonferroni multiple-comparison post test, one-way ANOVA for repeated measures followed by a Dunnett multiple comparison test, linear regression analysis, or Spearman's correlation. A probability of P < 0.05 was considered to be significant. ![]() |
RESULTS |
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Origin and Magnitude of Citrate Release by Perfused Rat Hearts
Isolated rat hearts perfused under normoxia released citrate in the effluent perfusate under all conditions examined. The measured citrate release rates varied between 2 and 21 nmol/min, depending on the nature of the substrate mixture supplied to the heart, but remained constant under a given perfusion condition for up to 90 min. Such release rates represent at most 2% of the total CAC flux rate (estimated at 2.0 ± 0.3 µmol/min in the Langendorff-perfused rat heart; see Ref. 7). Citrate release rates were highest in hearts perfused with a mixture of substrates feeding both acetyl-CoA (fatty acid) and anaplerotic OAA (lactate and pyruvate) to the citrate synthase reaction (see sections on Modulation of Citrate Release for details). They were lowest, but still measurable with precision, when the hearts were perfused with 11 mM glucose alone (2.4 ± 0.5 nmol/min, n = 4). As shown in Fig. 2, the 13C mass isotopomer distribution of citrate isolated from the effluent reflected that of tissue citrate. The 13C substrates used in this study are known to label citrate through the mitochondrial citrate synthase reaction, after their conversion to acetyl-CoA (M1 or M2 isotopomers) and/or anaplerotic OAA (M3 isotopomers). Therefore, we concluded that effluent citrate is likely to be of mitochondrial origin. The results presented below demonstrate the modulation of citrate release by substrate supply and provide evidence for the specificity of the process.
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Modulation of Citrate Release: Experiments with Octanoate (Group 1)
Functional status. Hearts perfused under normoxia for up to 70 min with 11 mM glucose, 1 mM lactate, 0.2 mM pyruvate, and 0.2 mM octanoate (control condition) maintained constant spontaneous beating at 280 ± 4 beats/min, a coronary flow rate of 10.4 ± 0.3 ml/min, a rate pressure product of (23.6 ± 0.7) × 103 mmHg × beats/min (not shown), and a contractile activity (dP/dtmax) of 2,379 ± 121 mmHg/s (data not shown). The contractile activity of the heart was not modified significantly by lowering the exogenous octanoate concentration from 0.2 to 0.02 mM or by removing lactate and pyruvate. However, it was increased significantly by the infusion of 1 µM isoproterenol to 146 ± 13% of the control value (P < 0.05, n = 5; paired t-test). Thus, from the contractile activity of the heart, which is the main determinant of its energy demand, we concluded that the rate of citrate utilization by the CAC was similar under all conditions, except with isoproterenol with which it was augmented.
Metabolic status: Availability and source of substrates for citrate
synthesis.
Let us examine how the availability of the two substrates for the
citrate synthase reaction varied under the various perfusion conditions
of group 1. Although the availability of anaplerotic OAA for
citrate synthesis was not assessed directly in this group of
perfusions, we predicted from previous studies (6, 7) that removal of
lactate plus pyruvate from the buffer would lower the supply of
anaplerotic OAA. The availability and sources of acetyl-CoA for citrate
synthesis are inferred from 1) the flux ratio (octanoate
oxidation/citrate synthesis) evaluated from the M2 enrichment values of
the C4+5 of effluent citrate using Eq. 21 (Fig.
3A) and 2)
the KB release rates (Fig. 3B). The observed variations in
these two parameters are compatible with the known metabolism of
octanoate. Indeed, because octanoate -oxidation is not regulated at
CPT-I, its concentration in the perfusion buffer sets the rate of
acetyl-CoA generation. Accordingly, whenever octanoate was supplied at
0.2 mM, a concentration leading to maximal
-oxidative rates (17),
the acetyl-CoA of citrate was predominantly supplied by octanoate
-oxidation [>80% as indicated by the flux ratios (octanoate
oxidation/citrate synthesis); Fig. 3A] at a rate
exceeding citrate synthesis (as indicated by KB release; Fig.
3B). Note that, under control conditions, the KB release rate
represents as much as 25% of the rate of 1) octanoate uptake or 2) acetyl-CoA oxidation in the CAC (6). When octanoate was supplied at 0.02 mM, only 30% of the acetyl-CoA of citrate arose from
-oxidation, and the KB release rate was close to the detection level, in agreement with a decreased supply of acetyl-CoA.
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Citrate release and tissue levels of citrate and malate. Hearts perfused under control conditions for 70 min released citrate at a constant rate of 21 ± 1 nmol/min (Fig. 3C). This rate was decreased significantly by 1) accelerating citrate utilization by the CAC through the addition of 1 µM isoproterenol or by 2) limiting the supply of substrates for citrate synthesis, either acetyl-CoA, by the lowering of exogenous octanoate concentration from 0.2 to 0.02 mM, or anaplerotic OAA, by the removal of exogenous lactate and pyruvate. The similar decrease in citrate release rates under conditions where the delivery of either acetyl-CoA or OAA was limited emphasizes the requirement of both substrates for the synthesis of excess citrate relative to the CAC flux. It is noteworthy that, although the citrate release rate was decreased under limited OAA supply, the KB release rate was increased, suggesting a greater acetyl-CoA spillover from the CAC.
Comparison of Fig. 3, C and D, reveals that citrate release rates varied in parallel with tissue levels of citrate plus malate, the antiporter for the mitochondrial tricarboxylate transporter. Note the closer relationship between citrate release rates and tissue levels of citrate plus malate than with tissue citrate alone.Modulation of Citrate Release: Experiments with Physiological Substrate Mixtures (Group 2)
In addition to the results from the first group of perfusion experiments, we documented citrate release in hearts perfused with substrate mixtures mimicking the physiological state, either the fed (5.5 mM glucose, 0.5 or 1 mM lactate, 0.05 or 0.2 mM pyruvate, and 8 nM insulin) or the fasted state (5.5 mM glucose, 1 mM lactate, 0.2 mM pyruvate, 8 nM insulin and 0.4 mM LCFA oleate).Functional status. Hearts perfused under normoxia with 5.5 mM glucose, 0.5 or 1 mM lactate, 0.05 or 0.2 mM pyruvate, and 8 nM insulin maintained contractile activity (dP/dtmax) of 3,264 ± 95 mmHg/s. In the presence of oleate, dP/dtmax values were 2,256 ± 164 mmHg/s (n = 6; P < 0.05 with vs. without oleate).
Citrate release.
Hearts perfused with 5.5 mM glucose, 0.5 mM lactate, 0.05 mM pyruvate,
and 8 nM insulin released citrate at a rate of 6.0 ± 0.6 nmol/min
(Fig. 4). This release rate was increased
significantly by 1) raising the concentrations of lactate plus
pyruvate to 1 and 0.2 mM, respectively, and by 2) adding the
LCFA oleate (0.4 mM). It is noteworthy that the citrate release rates
measured in the presence of LCFA oleate were not significantly
different from those measured with the medium-chain fatty acid (MCFA)
octanoate.
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Metabolic status: availability and source of substrates for citrate
synthesis.
In an attempt to explain the similar citrate release rates in hearts
perfused with octanoate and oleate, we determined the relative
contribution of pyruvate and oleate to the formation of acetyl-CoA and
anaplerotic OAA. This was done by perfusing hearts with a mixture of
13C-labeled substrates (0.4 mM
[1-13C]oleate, 1 mM
[U-13C3]lactate, and 0.2 mM
[U-13C3]pyruvate) and determining the
13C labeling of effluent citrate and of tissue pyruvate,
succinate, and fumarate, as described previously (6, 7). Note that KB
release by hearts perfused with oleate was low (15.5 ± 1.7 nmol/min,
n = 6, not shown), in agreement with a regulated -oxidation rate (Ref. 22 and Tables 1 and
2).
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Specificity of Citrate Release (Groups 1-3)
Effect of 1,2,3- and 1,2,4-BTC (group 3). FUNCTIONAL STATUS. The addition of 10 mM 1,2,3-BTC or 1,2,4-BTC to hearts perfused with 5.5 mM glucose, 1 mM lactate, 0.2 mM pyruvate, and 8 nM insulin resulted in a similar decrease in dP/dtmax values from 3,203 ± 122 to 2,099 ± 190 mmHg/s (P < 0.05, n = 4; one-way ANOVA for repeated measures followed by a Dunnett multiple comparison test). Upon removal of these agents, dP/dtmax returned within 10 min to initial values, indicating a reversible effect (P < 0.05). Both agents also had a reversible influence on coronary flow rates (a decrease from 10.2 to 8.2 ml/min, P < 0.05, n = 8). These effects of 1,2,3-BTC and 1,2,4-BTC may be explained by calcium chelation. Indeed, the addition of these two agents to the perfusion buffer lowered the concentration of ionized calcium from 0.94 ± 0.01 to 0.59 ± 0.07 mM (P < 0.05, n = 4; unpaired t-test), whereas the total calcium concentration was similar at 1.25 ± 0.02 mM (n = 4; data not shown).
CITRATE RELEASE. In contrast to their similar effects on contractile activity, there was a differential effect of 1,2,3-BTC and 1,2,4-BTC on citrate release, in agreement with the participation of the mitochondrial tricarboxylate transporter. Indeed, the addition of 10 mM 1,2,3-BTC but not 1,2,4-BTC resulted in a significant reversible 25% decrease of the citrate release rate (Fig. 5).
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LDH release (groups 1-3).
Under all conditions examined, LDH release rates were 20- to 50-fold
lower than those measured in hearts reperfused after 90 min of low-flow
ischemia or 40 min of hypoxia (3, 16). Furthermore, the LDH
release rates did not correlate with the citrate release rates
(r = 0.1, P = 0.95, not significant). A significant increase in LDH release was observed during isoproterenol infusion (from 18 ± 2 to 62 ± 14 mU/min, P < 0.05, n = 4; paired t-test). Similarly, LDH release was
increased upon removal of 1,2,3-BTC (from 19 ± 9 to 74 ± 20 mU/min,
P < 0.05, n = 4; one-way ANOVA for repeated
measures followed by a Dunnett multiple comparison test) but not of
1,2,4-BTC, thus raising the possibility of a detrimental effect
associated with the inhibition of citrate transport from the
mitochondria to the cytosol.
Succinate release (groups 1-3). Under all conditions, succinate release remained between 2 and 20 nmol/min, which is three- to sixfold lower than rates reported for oxygen-deprived hearts (16). There was no correlation between citrate and succinate release rates (r = 0.48, P = 0.2), indicating that these two cataplerotic reactions are regulated independently (data not shown).
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DISCUSSION |
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Results from this study demonstrate that citrate release by rat hearts
perfused ex vivo under normoxia is specifically modulated by various
perfusion interventions. The specificity of this process is also
supported by the lack of correlation between the release rates of
citrate and those of LDH, an index of cellular membrane damage, or of
succinate, another CAC intermediate whose release increases with oxygen
deprivation (16). Furthermore, 1) the identical 13C
labeling of tissue and effluent citrate resulting from
13C-labeled substrates feeding acetyl-CoA and OAA to the
mitochondrial citrate synthase reaction and 2) a modulation of
citrate release rates by the nature and concentrations of these
substrates indicate that citrate release reflects mitochondrial citrate
cataplerosis. Altogether, the results from the present study provide a
basis for discussing the following aspects relevant to mitochondrial cataplerosis: 1) the participation of the tricarboxylate
transporter relative to other transporters, 2) its modulation
by metabolic conditions, and 3) its link to the regulatory role
of cytosolic citrate. A schematized overview of the metabolic processes
relevant to this discussion is presented in Fig.
6.
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In the heart, the existence of mitochondrial citrate cataplerosis has
often been questioned because of the low activity of the mitochondrial
tricarboxylate transporter. The activity of this transporter may be low
when compared with lipogenic tissue such as the liver. Still this low
activity is compatible with the 1) citrate release rates
reported in the present study and 2) a role of cytosolic
citrate as a "signaling molecule" in nonlipogenic tissues (28).
The participation of this transporter in mitochondrial citrate
cataplerosis in the intact heart is substantiated by the following
results from our investigation. First, citrate release by the heart was
inhibited by 1,2,3-BTC, the only known specific inhibitor of the
mitochondrial tricarboxylate transporter, but not by 1,2,4-BTC, the
inactive chemical analog. Second, citrate release rates varied in
parallel with the tissue levels of citrate plus malate, the antiporter.
A closer relationship between citrate release rates and tissue levels
of citrate plus malate than with tissue citrate supports the suggestion
of Ruderman et al. (28) that cytosolic citrate would be reflected more
closely in the sum of whole cell concentration of citrate plus malate
than citrate alone. Our data indicate that the tissue ratio
citrate/malate varies with the availability and/or source of acetyl-CoA
for citrate synthesis. Indeed, this ratio was close to 1 for hearts
perfused with 0.2 mM octanoate but was only for hearts perfused with 0.02 mM octanoate.
Regarding the relatively low inhibition of citrate release (25%) by
1,2,3-BTC, there is a possibility that, due to steric hindrance,
1,2,3-BTC does not easily reach the extramitochondrial space. Thus a
greater concentration of exogenous 1,2,3-BTC would be necessary for
maximal inhibition of the mitochondrial transporter. In guinea pig
heart mitochondria, 50 mM 1,2,3-BTC achieved maximal inhibition of the
activity of the transporter (27), whereas, in rat heart mitochondria,
the same concentration resulted in only 25% inhibition (5). We did not
attempt to use such a high concentration of 1,2,3-BTC or 1,2,4-BTC in
the intact beating heart since a decrease in contractile function,
possibly due to calcium chelation, was already observed at a
concentration of 10 mM. An alternative explanation for the low
inhibition by 1,2,3-BTC would be the participation of the following
additional mechanism for the transfer of citrate from the mitochondria
to the cytosol (13): the conversion of citrate to -ketoglutarate in
the mitochondria through normal operation of the CAC, followed by the
transport of
-ketoglutarate from the mitochondria to the cytosol by
the dicarboxylate transporter, and the conversion of
-ketoglutarate to citrate by reversal of the cytosolic NADP-linked isocitrate dehydrogenase (ICDH) reaction. In the heart, the activity of the mitochondrial dicarboxylate transporter is severalfold higher than that
of the tricarboxylate transporter (12). Although the occurrence of this
alternative pathway was not tested specifically in the present study,
the following data from previous heart perfusion experiments with
[U-13C5]glutamate suggest that it
is unlikely to be involved (6). Under normoxia, the maximal
contribution of reversal of the NADP-ICDH reaction to citrate formation
was evaluated at most at 5%, with the remaining 95% being formed
through the citrate synthase reaction. Because the activity of
cytosolic NADP-ICDH is at most 2% of total ICDH activity (25), this
sets its maximal contribution at 0.1% of the CAC, which is 20-fold
lower than the citrate release rate, i.e., 2% of the CAC.
Therefore, based on the above considerations, it is likely that the mitochondrial tricarboxylate transporter participates in the transfer of citrate from the mitochondria to the cytosol under our conditions. Besides, our observation of an increased LDH release associated with the addition of 1,2,3-BTC, but not 1,2,4-BTC, suggests that the activity of this transporter, and thus mitochondrial citrate cataplerosis, could be important for the integrity of myocardial cells. At present, we do not have an explanation for this unexpected finding. Mitochondrial transmembrane carrier deficiencies have recently been considered as potential causes of mitochondriopathies (12). It is proposed that defects in these carriers may induce imperfect energy metabolism, probably as a result of osmotic disturbances within the mitochondria. So far, little is known about the heart mitochondrial citrate transporter aside from its mRNA expression in the heart (12). This subject is, however, beyond the scope of the present study.
What then triggers mitochondrial citrate cataplerosis in the heart? How
does cardiac mitochondrial citrate cataplerosis integrate itself with a
role of cytosolic citrate as a signaling molecule? Before attempting to
answer these questions, let us first summarize the proposed role of
cytosolic citrate in fuel signaling in nonlipogenic tissues, such as
the heart (28). Cytosolic citrate would act as a signal to the cell
that it has an excess of fuel for its immediate needs. Thus an increase
of cytosolic citrate could restrain the use of both fatty acid (via
malonyl-CoA CPT-I inhibition) and glucose (via PFK inhibition) for
energy production. In the heart, the regulatory role of cytosolic
citrate on glycolysis was first proposed in 1963 by Garland et
al. (11) who reported a correlation between citrate tissue
levels and PFK activity. As for a link between cytosolic citrate and
malonyl-CoA synthesis, it remains to be clarified. Malonyl-CoA is
synthesized from acetyl-CoA by acetyl-CoA carboxylase (ACC). In
skeletal muscle, experimental evidence supports a role for cytosolic
citrate both as 1) a precursor of acetyl-CoA, after citrate
cleavage by the ATP citrate lyase, and 2) an allosteric
activator of ACC (28). However, the kinetic and regulatory properties
of the cardiac ACC, predominantly the ACC isoform, are such that its
activity appears to depend more on the acetyl-CoA supply than on
citrate activation (19, 29). Therefore, could cytosolic citrate be a
precursor of malonyl-CoA in the heart? To the best of our knowledge,
this possibility has not been examined. The activity of the cytosolic
ATP citrate lyase in the heart is low [0.2 µmol/min × g
wet wt (1)], but probably sufficient to maintain the very small
pool of malonyl-CoA [nmol/g wet wt (1, 19, 21)].
In light of the above considerations, let us now examine how our data
on citrate release rates, reflecting mitochondrial cataplerosis, are in
agreement with the proposed regulatory role of cytosolic citrate. Our
series of heart perfusions with the MCFA octanoate indicate that
citrate release rates are modulated by 1) the energy demand of
the heart and 2) the availability of substrates for the citrate
synthase reaction. Although the effect of energy demand should be
substantiated by studies in the working rat heart perfused at different
workloads, these data support a role for cytosolic citrate as a signal
to the cell that the fuels available are in excess for its immediate
needs. For hearts perfused with octanoate and physiological
concentrations of lactate and pyruvate, we proposed previously that the
high production of mitochondrial acetyl-CoA and NADH from octanoate
-oxidation, which results in state 4 respiration (18),
inhibits pyruvate decarboxylation and favors anaplerotic pyruvate
carboxylation (6, 7). This sets up conditions for citrate accumulation
and efflux from the mitochondria to the cytosol. We emphasized that a
surge of anaplerotic pyruvate carboxylation was required to balance the
cataplerotic efflux of citrate. In view of the proposed regulatory role
of cytosolic citrate, such a link between pyruvate anaplerosis and
citrate cataplerosis would support a role for these reactions in
metabolic signaling in the heart.
However, other factors appear to contribute to the high rate of citrate
release by hearts perfused with glucose, lactate, pyruvate, and the
LCFA oleate. Indeed, under these conditions, the relative substrate
flux through pyruvate carboxylation and decarboxylation differs
markedly from that measured in hearts perfused with glucose, lactate,
pyruvate, and the MCFA octanoate (from the flux ratio pyruvate
carboxylation-to-pyruvate decarboxylation reported in Table 2 and Ref.
6: 0.25 ± 0.03 vs. 2.8 ± 0.7, respectively). We can only speculate
on the nature of the other factor(s) involved. We exclude 1) a
difference in the rate of citrate utilization by the CAC, since hearts
perfused with octanoate and oleate showed similar
dP/dtmax values and 2) the participation of
another anaplerotic reaction aside from pyruvate carboxylation, for
example, at the level of -ketoglutarate or succinate, since there is
little tracer dilution between tissue citrate and succinate. However,
there is a possibility that citrate release in the effluent underestimates mitochondrial citrate cataplerosis because of 1) citrate metabolism in the cytosol by the ATP citrate lyase and 2) the presence of a regulated transport system for citrate in the plasma membrane. Little is known about these two processes in the
heart, although a sodium-dependent transporter was characterized in
tissues such as the kidney and liver (24). As for the ATP citrate
lyase, a role for this enzyme in the removal of citrate from the
cytosol was suggested (5) where its activity would be limited by the
low level of cytosolic CoASH resulting from LCFA activation. In
contrast, the activation of MCFA such as octanoate occurs in the
mitochondria. Thus, in hearts perfused in the absence of LCFA, citrate
release may represent a minimal estimate of mitochondrial citrate
cataplerosis due to higher activity of the ATP citrate lyase.
Despite these uncertainties regarding the magnitude of mitochondrial
citrate cataplerosis, as reflected by citrate release, the observed
variations in citrate release rates in hearts perfused with the various
substrate mixtures appear nevertheless compatible with a regulatory
role of cytosolic citrate in glycolysis and fatty acid oxidation.
Indeed, this dual role of citrate requires its presence in the cytosol
under all metabolic conditions, although its concentration should be
higher when glycolysis is inhibited. Accordingly, citrate release was
observed under all conditions examined, even in the presence of glucose
as sole substrate. Furthermore, citrate release rates were highest in
hearts perfused with physiological concentrations of glucose, lactate,
pyruvate, and a fatty acid, either the LCFA oleate or the MCFA
octanoate. Under these conditions, we documented active -oxidation
and a low contribution of glycolysis to tissue pyruvate formation (data
from Table 2 and Ref. 6). Similarly, in vivo studies with normal human
subjects showed that myocardial citrate release correlated positively
with LCFA uptake but negatively with glucose uptake (33, 34).
Interestingly, these correlations were not observed in patients with
coronary artery disease, suggesting dysregulation. As for a possible
link between citrate cataplerosis and malonyl-CoA, although only
circumstantial, the results from heart perfusion with glucose, insulin,
lactate, and pyruvate appear more compatible with a role of citrate as a precursor, rather than an activator, of malonyl-CoA formation. Indeed, under these conditions, where tissue malonyl-CoA and the glycolytic rate were expected to be greater than in hearts perfused in
the additional presence of LCFA (1, 19), citrate release rates were
lower. Still, they were modulated by physiological concentrations of
exogenous lactate and pyruvate.
In conclusion, the results from this study demonstrate that citrate release by rat hearts perfused ex vivo under normoxia is a specific process, reflecting citrate efflux from the mitochondria to the cytosol or cataplerosis. They indicate that citrate cataplerosis is modulated by energy demand and by the nature and concentrations of substrates feeding the citrate synthase reaction, either acetyl-CoA or anaplerotic OAA. These changes are in agreement with the crucial roles of citrate 1) in the mitochondria for energy production and 2) in the cytosol as a signal molecule regulating substrate fuel selection. However, under some conditions, such as in the absence of LCFA, the measured citrate release rates may underestimate the magnitude of mitochondrial citrate cataplerosis due to citrate metabolism in the cytosol by the ATP citrate lyase. The participation of this enzyme, which is essential to the role of citrate as a precursor of malonyl-CoA, remains to be demonstrated. Finally, our results indicate that mitochondrial citrate cataplerosis occurs at least in part through the tricarboxylate transporter and suggest a potential physiological significance of this transporter for the integrity of myocardial cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. John C. Chatham for helpful comments and Dr. Henri Brunengraber for generously supplying the dialyzed albumin solution. Thanks are also due to Ovid Da Silva of the Research Support Office, Centre Hospitalier de l'Université de Montréal, for editing this text.
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FOOTNOTES |
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The study was supported by Medical Research Council of Canada (Grants MA-9575 and MT-10920 to C. Des Rosiers and a studentship to G. Vincent).
Part of this work was presented at the Experimental Biology Meeting in New Orleans, LA, in May 1997 and in Washington, DC, in April 1999 and at the XVI World Congress of the International Society for Heart Research in Greece in May 1998.
Present address for B. Comte: Division of Nutrition, Mount Sinaï Medical Center, One Mount Sinaï Dr., Cleveland, OH 44106-4198.
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.
1
The acetyl moiety of effluent citrate
(C4+5) labeled from
[1,2-13C2]octanoate was enriched
solely in M2 isotopomers (data not shown). M1 enrichment of the acetyl
moiety was low and imprecise (not significant, when tested against the
null hypothesis, not shown), indicating negligible recycling of
13C label through malate pyruvate
acetyl-CoA reactions.
Address for reprint requests and other correspondence: C. Des Rosiers, Laboratoire du métabolisme intermédiaire, Y-3616, Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, 1560 Sherbrooke St. East, Montréal, Québec, Canada H2L 4M1 (E-mail: desrosiers{at}sympatico.ca).
Received 13 August 1999; accepted in final form 29 November 1999.
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