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Am J Physiol Heart Circ Physiol 294: H1571-H1580, 2008. First published January 25, 2008; doi:10.1152/ajpheart.01340.2007
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Alterations in carbohydrate metabolism and its regulation in PPAR{alpha} null mouse hearts

Roselle Gélinas,1 François Labarthe,2 Bertrand Bouchard,1 Janie Mc Duff,1 Guy Charron,1 Martin E. Young,3 and Christine Des Rosiers1

1Department of Nutrition, Université de Montréal and Montreal Heart Institute, Montreal, Quebec, Canada; 2Centre Hospitalier Régional et Universitaire de Tours, Université François Rabelais, Institut National de la Santé et de la Recherche Médicale E211, Tours, France; and 3Department of Pediatrics, Baylor College of Medicine, Houston, Texas

Submitted 14 November 2007 ; accepted in final form 24 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although a shift from fatty acids (FAs) to carbohydrates (CHOs) is considered beneficial for the diseased heart, it is unclear why subjects with FA β-oxidation defects are prone to cardiac decompensation under stress conditions. The present study investigated potential alterations in the myocardial utilization of CHOs for energy production and anaplerosis in 12-wk-old peroxisome proliferator-activating receptor-{alpha} (PPAR{alpha}) null mice (a model of FA β-oxidation defects). Carbon-13 methodology was used to assess substrate flux through energy-yielding pathways in hearts perfused ex vivo at two workloads with a physiological substrate mixture mimicking the fed state, and real-time RT-quantitative polymerase chain reaction was used to document the expression of selected metabolic genes. When compared with that from control C57BL/6 mice, isolated working hearts from PPAR{alpha} null mice displayed an impaired capacity to withstand a rise in preload (mimicking an increased venous return as it occurs during exercise) as reflected by a 20% decline in the aortic flow rate. At the metabolic level, beyond the expected shift from FA (5-fold down) to CHO (1.5-fold up; P < 0.001) at both preloads, PPAR{alpha} null hearts also displayed 1) a significantly greater contribution of exogenous lactate and glucose and/or glycogen (2-fold up) to endogenous pyruvate formation, whereas that of exogenous pyruvate remained unchanged and 2) marginal alterations in citric acid cycle-related parameters. The lactate production rate was the only measured parameter that was affected differently by preloads in control and PPAR{alpha} null mouse hearts, suggesting a restricted reserve for the latter hearts to enhance glycolysis when the energy demand is increased. Alterations in the expression of some glycolysis-related genes suggest potential mechanisms involved in this defective CHO metabolism. Collectively, our data highlight the importance of metabolic alterations in CHO metabolism associated with FA oxidation defects as a factor that may predispose the heart to decompensation under stress conditions even in the fed state.

isolated working mouse heart perfusion; citric acid cycle; 13C isotopomer analysis; glycolysis; ATP-citrate lyase; peroxisome proliferator-activated receptor-{alpha}

IN THE HEALTHY adult heart, the concerted regulation of long-chain fatty acid (LCFA) and carbohydrate (CHO) metabolism ensures optimal energy production, in which LCFAs are often considered to be the predominant energy substrate under most conditions. This contrasts with the hypertrophied and/or failing heart, which displays a shift from LCFAs toward CHOs utilization that has been attributed, at least in part, to the deactivation of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) (5, 12, 28, 36, 41). This nuclear transcription factor, which is highly expressed in the heart, is known to be activated by fatty acids (FAs) and to regulate the expression of genes encoding for LCFA uptake and oxidation (7). Although this shift from LCFAs to CHOs for energy production has been shown to be beneficial for the ischemic and failing heart (26, 40), and reactivation of PPAR{alpha} in the hypertrophied heart was shown to result in contractile dysfunction (50), there are also potential detrimental consequences associated with decreased LCFA oxidation. These include intracellular lipid accumulation and its associated lipotoxic sequelae (26) as well as energy starvation (22, 28). This is best illustrated by patients with inherited LCFA oxidation disorders, who are known to be prone to cardiac decompensation under stress and fasting conditions (6, 39). Additional studies in humans and animals also emphasize the importance of LCFAs as a determinant of contractile function, structural remodeling, and mitochondrial energy metabolism in the failing, postinfarct, or hypertrophied heart (29, 33, 46). Hence, a better understanding of the potential consequences of a shift from LCFAs to CHOs appears relevant to the development of metabolic therapies for both patients with heart failure or with inherited LCFA oxidation defects.

Over the past decade, one animal model that has greatly contributed toward improving our understanding of the role of LCFA metabolism in the heart, as well as at the whole body level, is the PPAR{alpha} null mice model (2, 23, 49). Similar to patients with inherited LCFA β-oxidation defects, these mice were shown to display hypoglycemia and hypoketonemia under fasting conditions (24, 35). Furthermore, with the use of the ex vivo perfused heart model, a number of studies have substantiated their lower capacity to oxidize LCFAs, which is associated with increased CHO oxidation and glycolysis, a metabolic phenotype that resembles the hypertrophied and failing heart. This increased glucose utilization appears to improve the recovery of PPAR{alpha} null mouse hearts following ischemia (30) but limit energy production when these hearts are subjected to increased workload (induced by raising the calcium concentration from 2 to 4 mM) (27). This effect was shown to be corrected by overexpressing the glucose transporter GLUT1, suggesting a potential avenue for metabolic therapy via increased CHO utilization. However, these studies were performed in Langendorff-perfused hearts in the presence of a low (5 mM) glucose concentration, as opposed to working hearts perfused in the presence of 11 mM glucose (i.e., conditions of greater similarity to the mouse in vivo). Dysregulation of CHO metabolism has been documented in the liver of PPAR{alpha} null mice (43), although little is known about the acute and chronic regulation of energy substrates beyond LCFAs in the hearts from these mice (9). In addition, these hearts may display alterations in CHO-related pathways such as anaplerotic pyruvate carboxylation (PC), a process that supplies oxaloacetate (OAA) for citrate synthesis (CS) and was previously shown to be essential for the maintenance of contractile function (13). The latter aspect appears worth considering given that the nutritional interventions with anaplerotic substrates were shown to improve the cardiomyopathy of one patient with genetic LCFA oxidation deficiency (34).

The purpose of the present study was to investigate whether PPAR{alpha} null mice display alterations in the utilization and/or regulation of CHOs for energy production or anaplerosis, which may impair their capacity to respond to an increased workload even in the fed state. To test this hypothesis, we used 10–12-wk-old control and PPAR{alpha} null mice and assessed substrate fluxes through energy-yielding and anaplerotic pathways in hearts perfused ex vivo with a physiological substrate mixture mimicking the fed state through carbon-13 (13C)-labeling methodology (19). We documented the impact of an increase in preload, mimicking a raise in venous return as it occurs during exercise. In parallel, indexes of cardiac performance and of membrane integrity were continuously monitored. Finally, following our novel observation that the lactate production rate was the only metabolic parameter that may explain the impaired capacity of PPAR{alpha} null mouse hearts to maintain their function following an increase in workload, we investigated potential sites of altered regulation of CHO metabolism by documenting the impact of a 24-h fast on the expression of selected metabolic genes and metabolites involved in CHO metabolism and its regulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

Sources of chemicals, biological products, and 13C-labeled substrates, as well as the procedures for the dialysis of BSA (BSA fraction V; Intergen), have been reported previously (47).

Heart Perfusion in a Semirecirculating Mode

Animal experiments were approved by the local Animal Care Committee in agreement with the guidelines of the Canadian Council on Animal Care. Male PPAR{alpha} null mice on a C57BL/6 genetic background (Taconic) and their control, C57BL/6NTac (C57BL/6, Taconic), which were 10–12 wk old (26.4 ± 0.4 and 24.4 ± 0.4 g, respectively, P < 0.05) and had free access to water and food, were anesthetized (1 µl/g ip) with a mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml) and heparinized (5,000 U/kg sc) 15 min before surgery. The procedure for mouse heart isolation and its ex vivo perfusion in a working mode have been previously described in detail (19). The only modification was a change in the preload pressure as described in Perfusion Protocols. The composition of the Krebs-Henseleit perfusion buffer, which was maintained at 37.5°C and gassed with 95% O2-5% CO2, contained (in mM) 110 NaCl, 4.7 KCl, 2.1 CaCl2, 0.24 KH2PO4, 0.48 K2HPO4, 0.48 Na2HPO4, 1.2 MgSO4, 25 NaHCO3, and 0.1 EDTA. The free calcium concentration was 1.55 ± 0.3 mM. Functional and physiological parameters were monitored throughout the perfusion period as described previously (19). Myocardial oxygen consumption (MVO2), intracellular pH, rate pressure product, cardiac power, and cardiac efficiency were calculated from equations reported previously (19).

Perfusion Protocols

Working mouse hearts were perfused for 30 min with a semirecirculating Krebs-Henseleit buffer containing the following substrates, cofactors, and hormones: 11 mM glucose, 1.5 mM lactate, 0.2 mM pyruvate, 0.4 mM oleate complexed to 3% albumin, 0.8 nM insulin, 50 µM L-carnitine, and 0.5 nM epinephrine. The rationale for this choice of substrate and hormone concentrations, which are within the range of reported values of mouse plasma concentrations in the nonfasting state, was previously reported (19). It is noteworthy that we chose a concentration of 1.5 mM lactate, because in blood drawn by cardiac puncture in a syringe containing sulfosalicylic acid to precipitate proteins and prevent anaerobic glycolysis, we evaluated lactate concentration to be 1.8 ± 0.3 mM (19). Two different perfusion protocols were used to document the impact of an increase in the preload pressure, which remained within the physiological range.

Protocol 1. Hearts from C57BL/6 (n = 13) and PPAR{alpha} null (n = 12) mice were perfused for 30 min at a preload and afterload pressure of 12 and 50 mmHg, respectively.

Protocol 2. Hearts from PPAR{alpha} null and C57BL/6 (n = 7 for both groups) were perfused for 30 min with an afterload pressure of 50 mmHg as in protocol 1. However, the preload pressure was kept at 12 mmHg for the first 10 min and increased to 15 mmHg for the remaining 20 min. For any given perfusion, one or two of the unlabeled substrates were replaced by its corresponding labeled substrate(s). Three different 13C-labeled substrate mixtures used were 1) [U-13C18]oleate [initial molar percent enrichment (MPE), 25%], 2) [U-13C3]lactate-[U-13C3]pyruvate (initial MPE, 99%), and 3) [U-13C3]pyruvate (initial MPE, 50%)-[3-13C]pyruvate (initial MPE, 50%)-[U-13C3]lactate (initial MPE, 99%). All three substrate mixtures were used for protocol 1, but only mixtures 2 and 3 were used for protocol 2.

As previously described (19), throughout the perfusion, influent and effluent samples were collected to 1) control PO2, PCO2, pH, Ca2+, and other ion concentrations; 2) assess lactate dehydrogenase (LDH) release, an index of membrane integrity; and 3) document the lactate and citrate release rates. At the end of the perfusion period, hearts were freeze clamped with metal tongs chilled in liquid nitrogen, weighed, and stored at –80°C for further analyses.

Effect of a 24-h Fast

Male C57BL/6 and PPAR{alpha} null mice (10–12 wk old; Taconic), which were kept in individual cages in the same room, were fed ad libitum (n = 5 in each group) or fasted for 24 h (n = 10 in each group) and had free access to water. Fasting was initiated at 9:00 AM, and all mice were euthanized 24 h later under anesthesia induced by an injection of a solution (1 µl/g ip) of ketamine (100 mg/ml) and xylazine (20 mg/ml). Mean body weights of fed and fasted PPAR{alpha} null mice (26.6 ± 1.0 and 23.5 ± 0.5 g, respectively, P < 0.05) were different from C57BL/6 mice (21.9 ± 0.4 and 20.0 ± 0.5 g, respectively, P < 0.01), but the weight loss due to fasting was similar in both groups (~17.5%). Hearts were rapidly excised, freeze clamped with metal tongs chilled in liquid nitrogen, and stored in liquid nitrogen until further analyses. Concomitantly, blood samples were collected from the abdominal cavity using a syringe containing 10.8 mg EDTA and immediately centrifuged for 10 min at 15,000 g, and plasma samples were then stored at –80°C until further analyses.

Analytical Procedures

A previously published study from our laboratory (19) provides detailed descriptions for the following: 1) measurements of the 13C enrichment and concentrations of citric acid cycle (CAC) intermediates (citrate, succinate, fumarate, and malate) by gas chromatography-mass spectrometry (GC-MS; Agilent 6890N gas chromatograph equipped with a HP-5 column coupled to a 5973N mass spectrometer) and 2) activities of selected CAC enzymes CS, aconitase, and NADP+-isocitrate dehydrogenase by spectrophotometry. Plasma concentrations of glucose and lactate were quantified using enzymatic assays and ketone bodies by GC-MS as previously described (19, 48).

Flux Parameters

Previously published studies from our laboratory (19, 47) provide definitions of the 13C terminology and a detailed description for the calculations of 1) flux ratios relevant to substrate selection for CS from 13C enrichment of the acetyl (4C and 5C) and OAA (1C, 2C, 3C, and 6C) moiety of citrate, 2) efflux rates of unlabeled lactate and pyruvate reflecting glycolysis from exogenous glucose and/or glycogen (for perfusions with 13C-labeled lactate and pyruvate and mixtures 2 and 3), as well as 3) absolute flux rates for pyruvate decarboxylation (PDC) and ATP production from the stoichiometric relationship between oxygen consumption and citrate formation from the various substrates as assessed from the determined flux ratios. In this study, we also report the fractional contribution (FC) of individual CHO to pyruvate formation, which was calculated from the MPE M1 and M3 of tissue pyruvate measured in hearts perfused with [U-13C3]pyruvate (initial MPE, 50%)-[3-13C]pyruvate (initial MPE, 50%)-[U-13C3]lactate (initial MPE, 99%) using the following equations: 1) FC of exogenous pyruvate = FCexogenous pyruvate = MPE M1 tissue pyruvate/MPE M1 exogenous pyruvate, 2) FC of exogenous lactate = FCexogenous lactate = [MPE M3 – MPE M1] tissue pyruvate/MPE M3 exogenous lactate, and 3) FC of other CHOs such as exogenous glucose and/or glycogen = FCother CHO = 1 – FCexogenous pyruvate – FCexogenous lactate. Furthermore, we estimated the absolute flux rates for acetyl-CoA formation for CS coming specifically from exogenous glucose and/or endogenous glycogen, which was calculated by multiplying the absolute PDC flux rates by other CHO FC (FCother CHO).

Reverse Transcription-Quantitative Polymerase Chain Reaction Gene Expression Analysis.

The following metabolic genes were selected: 1) PPAR{alpha}-regulated genes, medium-chain acyl-CoA dehydrogenase (Acadm), and uncoupling protein 3 (Ucp3); 2) genes related to CHO metabolism or its regulation such as glucose transporter-4 (Slc2a4), phosphofructokinase-1 (Pfkm), and pyruvate dehydrogenase kinase 4 (Pdk4); 3) genes related to anaplerosis such as pyruvate carboxylase (Pcx), propionyl-CoA carboxylase B (Pccb), and cytosolic NADP+-linked malic enzyme (Me2); and 4) a gene related to cytosolic citrate metabolism, ATP-citrate lyase (Acly). All gene primer pairs shown in Table 1 have been designed with the Beacon Designer v5.0 program using mouse sequences available in GenBank. We used amplicons of 126–219 bp overlapping the exon-exon boundary. Total RNA was isolated from pulverized frozen heart tissue using an RNeasy mini kit for fibrous tissue following the manufacturer's standard protocol (Qiagen). The purity, integrity, and quantity of RNA were evaluated using a BioAanalyzer 2100 (Agilent Technologies). Total RNA (2.5 µg) for each sample, which was run in duplicates, has been reverse transcribed at 55°C using 15 units of cloned avian myeloblastosis virus reverse transcriptase (Invitrogen Life Technologies) and 8 pmol of antisense specific primers (Integrated DNA Technologies Corporate). Only primer pairs giving ≥90% efficiency over a six-log serial dilution of a control RT reaction from a sample highly expressing the specific target gene of interest were used. Total RT products (3–5 ng equivalent mRNA) were analyzed by quantitative PCR using the 2x Platinum SYBR Green qPCR Supermix-UDG according to the manufacturer's specifications (Invitrogen Life Technologies). Cycling was achieved in a MX3005p cycler (Stratagene; conditions, 95°C for 10 min and 40 cycles of 95°C for 30 s, 50°C for 45 s, and 72°C for 45 s). At the end of each run, the absence of primer-dimer formation and the presence of a unique amplicon were confirmed using the dissociation curves. Quantification was accomplished with MxPro software v3 (Stratagene) using internal reference dye normalization and the number of PCR cycles required for the fluorescent signal to reach a detection threshold ({Delta}{Delta}CT) correction (25). Levels of selected gene transcripts for each samples were averaged and normalized to the housekeeping gene, namely glyceraldehyde-3-phosphate dehydrogenase (Gapdh). This gene was selected because among all tested housekeeping genes (Gapdh, Hprt1, and Actb), it displayed the lowest variations between the various experimental conditions. To validate the method, we independently analyzed two genes (Pdk4 and Ucp3) using TaqMan quantitative RT-PCR, as described previously (51, 52). Furthermore, Me2 and Pcx were also analyzed using TaqMan assays, as described previously (13). Briefly, standard RNA was made for these assays by the T7 polymerase method (Ambion, Austin, TX), using total RNA isolated from mouse hearts; the use of standard RNA allows an absolute quantification of gene expression. The correlation between the CT and the amount of standard was linear over at least a five-log range of RNA for all assays (data not shown). Quantitative RT-PCR data are represented as mRNA molecules relative to those of cyclophilin A (Ppia).


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  		Table 1. Primer and probe sequences used in comparative real-time RT-quantitative polymerase chain reaction

 
Statistical Analyses

Data are expressed as means ± SE; n = 4–12 heart or plasma samples. Statistical significance was reached at P < 0.05 using an unpaired t-test or a two-way ANOVA, followed by the Bonferroni selected-comparison test.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Isolated Working PPAR{alpha} Null Hearts Displayed an Impaired Capacity to Withstand a Physiological Raise in Preload

We used our isolated working mouse heart model (19) to compare the metabolic and functional responses of PPAR{alpha} null and control mice with a physiological raise in preload from 12 to 15 mmHg, which mimics an increased venous return as it occurs during exercise. Upon ex vivo perfusion at a physiological afterload of 50 mmHg with a buffer containing a substrate mixture mimicking the fed state (19), control hearts maintained constant values for the various functional and physiological parameters shown in Table 2 during the entire 30-min perfusion at the two preload pressures. At a preload of 12 mmHg, PPAR{alpha} null hearts maintained functional parameters similar to control hearts for all parameters, except for a significantly 20% lower maximum value for the first derivative of left ventricular pressure (dP/dtmax) value (Table 2). In contrast, at 15 mmHg these hearts showed additional impairments in left ventricular function (Table 2). This was evidenced by a 20% decline of aortic flow and a 40% increase in coronary flow, which correlated with a 25% higher MVO2 (r2 = 0.86; P < 0.001) and resulted in a 22% lower cardiac efficiency. Finally, there was also a greater rate of LDH release from PPAR{alpha} null hearts, reflecting diminished membrane integrity. Altogether, the aforementioned results illustrate the impaired capacity for young PPAR{alpha} null mouse hearts to maintain normal functions when energy demand is increased by a small raise in preload.


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  		Table 2. Functional and physiological parameters of control C57BL/6 and PPAR{alpha} null mouse hearts perfused ex vivo in the working mode at two preloads

 
Isolated Working PPAR{alpha} Null Mouse Hearts Display Alterations in CHO Utilization That are Affected by Preload

When compared with controls, PPAR{alpha} null hearts depicted a fivefold (P < 0.001) lower contribution of exogenous LCFA [exogenous oleate oxidation (OLE)/CS] to energy production associated with a ~52% increase in that of CHOs via PDC/CS (74 ± 3% vs. 48 ± 5%, P < 0.001; Fig. 1). The higher contribution of CHO was attributed to a twofold increase in the contribution of exogenous lactate and glucose and/or endogenous glycogen, whereas that of exogenous pyruvate remained unchanged. It is noteworthy that in both groups of mice, similar PDC-to-CS flux ratios were estimated from either 1) M1 tissue pyruvate and M1 acetyl moiety of citrate (coming from [3-13C]pyruvate) or 2) M3 tissue pyruvate and M2 acetyl moiety of citrate (coming from [U-13C3]lactate; data not shown), suggesting that exogenous lactate and pyruvate are incorporated into the same intracellular pyruvate pool. However, the percent contribution of the various CHOs to intracellular pyruvate formation also differed between the two groups of mice. Specifically, in PPAR{alpha} null hearts, the percent contribution of lactate (33 ± 2% vs. 23 ± 2%, P < 0.05) and glucose and/or endogenous glycogen (48 ± 1% vs. 42 ± 1%, P < 0.05) was higher, whereas that of pyruvate was lower (19 ± 1% vs. 35 ± 3%, P < 0.003) compared with that of controls. Similar metabolic alterations were observed at the higher preload (data not shown). The lactate production rate, which reflects the partitioning of pyruvate produced from glycolysis between cytosolic reduction by LDH and mitochondrial uptake-oxidation, was the only metabolic parameter that differed at the two preloads between the two groups of mice. Specifically, PPAR{alpha} null mouse hearts displayed a significantly threefold-higher lactate release rate at the lower preload (Fig. 2A). However, although a raise in preload significantly increased the lactate production rate in C57BL/6 mouse hearts, it had no effect on that of hearts from PPAR{alpha} null mice. This contrasts with the estimated rate of formation of glycolytically derived acetyl-CoA from glucose and/or glycogen (Fig. 2B), which was approximately twofold higher in PPAR{alpha} null mouse hearts at the two preload pressures. A similar pattern was also observed for the absolute PDC flux rate (lower preload, PPAR{alpha}, 1.65 ± 0.13 vs. C57BL/6, 0.95 ± 0.13 µmol·min–1·g wet wt–1, P < 0.001; and higher preload, PPAR{alpha}, 1.87 ± 0.25 vs. C57BL/6, 1.14 ± 0.15 nmol·min–1·g wet wt–1, P < 0.05). Hence, collectively, these results emphasize the importance of CHO utilization and specifically of the glycolytic flux in PPAR{alpha} null hearts under the condition of increased energy demand even in the fed state. Specifically, they suggest that in PPAR{alpha} null mouse hearts, lactate release rates are already maximal at the lower preload and cannot increase further following a rise in workload even when glucose is supplied at 11 mM.


Figure 1
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  		Fig. 1. Relative contribution of various substrates to mitochondrial acetyl-CoA in working hearts from control C57BL/6 and peroxisome proliferator-activating receptor-{alpha} (PPAR{alpha}) null mice. Data are means ± SE of 5–9 perfusion experiments with [U-13C18]oleate, [U-13C3]lactate-[U-13C3]pyruvate, or [U-13C3]pyruvate-[3-13C]pyruvate-[U-13C3]lactate. The contribution of 1) carbohydrates exogenous pyruvate (Pyr), exogenous lactate (Lac), and glucose and/or glycogen (Glc) via pyruvate decarboxylation (white bars); 2) fatty acids via β-oxidation [exogenous oleate oxidation (OLE); black bars]; and 3) other substrates (OS; gray bars) to acetyl-CoA formation are expressed relative to citrate synthesis (CS) and calculated from the measured tissue mass isotopomer distribution of 1) citrate and its oxaloacetate (OAA) moiety from which we extrapolate the acetyl moiety of citrate and 2) succinate. ***P < 0.001, PPAR{alpha} null vs. C57BL/6 mouse hearts.

 

Figure 2
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  		Fig. 2. Lactate production (A) and estimated production of glycolytically derived acetyl-CoA (B) in working hearts of control C57BL/6 and PPAR{alpha} null mice perfused at 2 preloads. Data are means ± SE of 7–9 heart perfusion experiments using C57BL/6 (black bars) and PPAR{alpha} null mice (white bars). Lactate production rates were calculated from the product of coronary flow rates and lactate concentration differences between influent and effluent perfusates determined by gas chromatography-mass spectrometry (GC-MS) and enzymatic assays. Glycolytically derived acetyl-CoA (Glc/CS) was estimated from the fractional contribution of glucose and/or glycogen to pyruvate formation, the pyruvate-to-decarboxylation flux ratio, and the citric acid cycle (CAC) flux rate reported in Table 3. Results are expressed related to heart weight [in gram wet wt (gww)]. *P < 0.05, **P < 0.005, and ***P < 0.001, PPAR{alpha} null vs. C57BL/6 mouse hearts; $P < 0.05, 15 vs. 12 mmHg.

 
Isolated Working PPAR{alpha} Null Mouse Hearts Display Modest Changes in CAC-Related Parameters at Both Preloads

To substantiate the potential consequences of a metabolic shift from LCFAs to CHOs in hearts from PPAR{alpha} null mice, we compared CAC-related parameters of hearts freeze clamped after 30 min of ex vivo perfusion (Table 3). The levels of CAC intermediates, which are crucial for the functioning of the CAC and hence energy production, are regulated by two metabolic processes, namely the refueling of CAC catalytic carbons by anaplerosis and mitochondrial CAC intermediate efflux (8). They may also be affected by CAC enzymatic activities. Table 3 reports data from hearts perfused at 12 mmHg, but similar results were obtained at 15 mmHg (data not shown).


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  		Table 3. CAC-related parameters measured in working control C57BL/6 and PPAR{alpha} null mouse hearts perfused at 12 mmHg

 
Despite their metabolic shift from LCFAs to CHOs, PPAR{alpha} null mouse hearts showed values similar to control hearts for the following parameters: 1) the relative and absolute PC/CS flux rate, which represents the anaplerosis flux via pyruvate carboxylase and/or malic enzyme; 2) tissue levels of individual and total CAC intermediates (i.e., citrate, succinate, fumarate, and malate); and 3) the absolute CAC flux rate calculated from the measured MVO2 value and the stoichiometric relationships between oxygen consumption and citrate formation from CHOs and FAs. Similar values for the estimated CAC flux rate imply that in PPAR{alpha} null mouse hearts, the decrease in acetyl-CoA production from LCFAs (1,102 ± 100 nmol·min–1·g wet wt–1) appears to be compensated by an increased production from CHO (906 ± 146 nmol·min–1·g wet wt–1, P = 0.33).

However, PPAR{alpha} null mouse hearts depicted some changes in other CAC-related parameters compared with control hearts. Indeed, we found that when perfused at 12 mmHg, PPAR{alpha} null mouse hearts displayed approximately threefold greater citrate release rates compared with those of controls (P < 0.05; Table 3). This measurement has previously been used as a surrogate of cytosolic levels of citrate (19, 22, 47), which is difficult to assess with precisions given that it represents <5% of the total cellular pool of citrate. Hence, a greater citrate release rate in PPAR{alpha} null mouse hearts suggests a higher cytosolic citrate level. However, the presence of a significant concentration of citrate in the albumin solution (~10 µM) resulted in some imprecision in the measured values at 12 mmHg and prevented the precise measurements of citrate in influent and effluent perfusates of hearts perfused at 15 mmHg. Finally, we also found that the maximal enzyme activity of NADP+-linked isocitrate dehydrogenase was a ~25% increase in PPAR{alpha} null mouse hearts (Table 3), although that of CS (P = 0.17) and aconitase (P = 0.30) was similar to controls.

Taken together, those results indicate that PPAR{alpha} null mouse hearts show marginal changes in CAC-related parameters compared with control hearts, except for increased citrate release and NADP+-linked isocitrate dehydrogenase activity. However, the latter changes were not affected by the preload pressure.

PPAR{alpha} Null Mouse Hearts Show Marked Differences in Myocardial CAC Intermediates and Expression of Genes Related to CHO Metabolism and its Regulation, Which are Affected by Nutritional Status

To identify potential sites of altered regulation of CHO metabolism, particularly glycolysis, we documented plasma levels of energy substrates as well as the myocardial CAC intermediate levels and the expression of selected metabolic genes involved in CHO metabolism and its regulation in the fed state as well as following a 24-h fasting period. The latter condition, which can induce cardiac decompensation in subjects with LCFA oxidation, was chosen because it is known to upregulate PPAR{alpha}-regulated gene expression. Hence, changes in gene expression in fed versus fasted control hearts provided us with information as to whether a specific gene is regulated by PPAR{alpha}.

Circulating and Tissue Levels of Energy-Related Metabolites

In the fed state, a condition corresponding to our heart perfusion experiments, only minor differences in circulating substrate levels were observed between the two groups of mice, except for lactate (Fig. 3, A–E). Note that in both groups of mice, values measured for ketone body concentrations, particularly acetoacetate, were lower than those previously reported for C57BL/6 mice by Stowe et al. (42). In contrast, measured values for plasma metabolites following a 24-h fast, which concur and expand on previously published studies (18, 20, 27), demonstrate drastically lower levels of plasma glucose, lactate, and the ketone body β-hydroxybutyrate for PPAR{alpha} null mice than for control mice. Hence, one would expect that this shortage of substrate supply to the heart during fasting would only exacerbate the aforementioned alterations in CHO metabolism, and the associated contractile dysfunction, in PPAR{alpha} null mouse hearts. In fact, at the myocardial level, our results demonstrate that the total pool of CAC intermediates, which is known to vary with substrate availability and to increase with fasting (53), was significantly decreased in PPAR{alpha} null mice compared with controls, but only in the fasted state (Fig. 3F). The difference in total CAC levels between the two groups in the fasted state is mainly explained by a lower level of succinate in the PPAR{alpha} null compared with C57BL/6 hearts (271 ± 57 vs. 630 ± 42 nmol/g wet wt, respectively; data not shown).


Figure 3
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  		Fig. 3. Impact of nutritional state on plasma and myocardial metabolite levels in control C57BL/6 and PPAR{alpha} null mice. Data are means ± SE of 4 to 5 fed and 10 24-h fasted C57BL/6 (black bars) and PPAR{alpha} null (white bars) mice. Plasma levels of glucose (A) and lactate (B) were quantified by enzymatic assays. Plasma levels of the ketone bodies, β-hydroxybutyrate (BHB; C), and acetoacetate (AcAc; D), as well as plasma (E) and tissue (F) levels of CAC intermediates, were quantified by GC-MS. *P < 0.05 and **P < 0.01, PPAR{alpha} null vs. C57BL/6 mouse hearts; $$P < 0.01 and $$$P < 0.001 fasted vs. fed.

 
Metabolic Gene Expression Profiling

As expected, PPAR{alpha} null hearts depict markedly lower mRNA levels for known PPAR{alpha}-regulated metabolic genes Acadm and Ucp3, both in the fed and fasted state (Fig. 4, A and B). In addition, the effect of fasting, which greatly increased the expression of these PPAR{alpha} target genes in control C57BL/6 mouse hearts, concurring with literature data (24), was greatly attenuated in PPAR{alpha} null mouse hearts. The mRNA levels of most selected genes implicated in glucose metabolism and its regulation also differed between control and PPAR{alpha} null mice. Concurrent with literature data (17), the mRNA levels of Pdk4 showed a pattern similar to other PPAR{alpha}-regulated metabolic genes (Fig. 4C). None of the other genes showed such a pattern of expression (Fig. 4, D–I). However, in the fed state, the following genes depicted markedly lower levels of transcripts in PPAR{alpha} null mouse hearts: 1) Acly (70%), which encodes a protein that may determine levels of cytosolic citrate, which is an inhibitor of glycolytic phosphofructokinase-1; and 2) Me2 (50%), in which the role in the heart remains to be clarified although it has been proposed to be involved in anaplerosis (44). As for the other transcripts measured, the following genes showed the greatest increases in fasted PPAR{alpha} null mouse hearts: 1) the glucose transporter Slc2a4 (55%), 2) Pfkm (30%), and 3) the anaplerotic enzyme Pcx (50%). Note that the our finding of an enhanced expression of gene encoding for GLUT4 in fasted PPAR{alpha} null mouse hearts concurs with the higher GLUT4 protein expression and glucose uptake reported in these hearts by Panagia et al. (30).


Figure 4
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  		Fig. 4. Impact of nutritional state on myocardial messenger RNA (mRNA) levels of selected metabolic genes in control C57BL/6 and PPAR{alpha} null mice. Data are means ± SE of 4–10 nonperfused freeze-clamped hearts from fed or 24-h fasted C57BL/6 (black bars) and PPAR{alpha} null (white bars) mice. mRNA levels of uncoupling protein 3 (Ucp3; A), medium-chain acyl-CoA dehydrogenase (Acadm; B), pyruvate dehydrogenase kinase-4 (Pdk4; C), glucose transporter-4 (Slc2a4; D), phosphofructokinase-1 (Pfkm; E), ATP-citrate lyase (Acly; F), and propionyl-CoA carboxylase B (Pccb; H) are normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase, whereas those of pyruvate carboxylase (Pcx; G) and of NADP+-linked malic enzyme (Me2; I) are normalized to cyclophilin (Ppia). *P < 0.05, **P < 0.01, and ***P < 0.001, PPAR{alpha} null vs. C57BL/6 mouse hearts; $P < 0.05, $$P < 0.01, and $$$P < 0.001, fasted vs. fed.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Before this study, there had been only a few studies that had investigated substrate fuel utilization and its regulation in PPAR{alpha} null mouse hearts (3, 9, 27, 30, 37), none of which had specifically examined the utilization and/or regulation of CHOs for energy production or anaplerosis. Using our ex vivo working heart model, we documented the impact of an increase in workload induced by raising the preload from 12 to 15 mmHg, which remains in the physiological range. This mimics an increased venous return as it occurs during exercise. While at the lower preload, PPAR{alpha} null mouse hearts maintain cardiac function similar to that of controls, except for a ~20% lower dP/dtmax, these hearts displayed a markedly impaired capacity to withstand a rise in preload, despite providing what are generally accepted to be physiological levels of calcium, hormones (insulin), cofactors (carnitine), and nutrients, including concentrations of glucose mimicking the fed state (11 mM).

At the metabolic level, beyond the expected decrease (~80%) in the contribution of exogenous oleate β-oxidation and increase (~52%) in that of CHOs to acetyl-CoA production for CS (Fig. 1), which concur with previous studies (9, 27), our metabolic data highlight the following additional metabolic alterations in PPAR{alpha} null hearts. First, these hearts depict a modified contribution of the various CHOs to acetyl-CoA formation, namely an increased contribution of lactate, glucose, and/or glycogen, whereas that of pyruvate remained unchanged. The lack of increase in the contribution of exogenous pyruvate to acetyl-CoA formation in PPAR{alpha} null mouse hearts appears to result from a lower contribution to intracellular pyruvate formation (2-fold), suggesting that exogenous pyruvate uptake cannot be increased in these hearts. Another potential explanation would be a compartmentation of cytosolic pyruvate, as suggested by evidence in the literature (10, 19). However, the latter explanation is not supported by our finding of similar PDC-to-CS flux ratios calculated from exogenous [3-13C]pyruvate and [U-13C3]lactate, suggesting their incorporation into the same intracellular pyruvate pool. Although this issue remains to be further investigated, the increased contribution of lactate, but not of pyruvate, to acetyl-CoA formation in PPAR{alpha} null mouse hearts may be viewed as an adaptive mechanism aimed at optimizing energy production. Indeed, with the use of a revised estimate for the stoichiometry of oxidative phosphorylation (16), the ATP-to-O2 ratio calculated for the complete oxidation of glycogen (5.7), glucose (5.2), and lactate (4.8) is higher than that for pyruvate (4.1). Incidentally, the estimated CAC flux rates and, hence, the calculated ATP production rates were similar in control and PPAR{alpha} null mouse hearts (PPAR{alpha}, 39.2 ± 0.8 vs. C57BL/6, 37.7 ± 0.8 µmol·min–1·g wet wt–1, P = 0.18 at the lower preload). However, in the latter hearts, ATP is produced almost exclusively from CHO metabolism (97 ± 7%) compared with 49 ± 5% in controls. Second, perfused PPAR{alpha} null mouse hearts displayed an approximately threefold increase in citrate efflux and a 22% higher activity of NADP+-isocitrate dehydrogenase, which reflects predominantly the mitochondrial isoform (32), although other measured CAC-related parameters were similar to those of controls, including the anaplerotic PC-to-CS flux ratio and tissue levels of CAC intermediates.

The aforementioned metabolic alterations were documented at both preloads and cannot explain the altered response of PPAR{alpha} null hearts to a raise in workload. In fact, the rate of lactate production from exogenous glucose and/or endogenous glycogen was the only measured metabolic parameter that differed between perfused PPAR{alpha} null and control mouse hearts and was modified by preload. Specifically, our data indicate that in PPAR{alpha} null mouse hearts, lactate production rates are already maximal at the lower preload, being threefold higher than those of controls, and cannot increase further following a rise in workload even when glucose is supplied at 11 mM. In fact, at the lower preload, the contribution of CHOs to ATP production appears to be already maximal in PPAR{alpha} null mouse hearts (97 ± 7% compared with 49 ± 5% in controls). This may suggest a restricted reserve for these hearts to further enhance glycolysis when the energy demand is increased, which may lead to a state of energy starvation.

Potential sites of dysregulation of CHO metabolism were also investigated by conducting experiments in which we documented the impact of nutritional status (fed or 24 h fasted) on the levels of plasma and tissue metabolites and the gene expression of selected metabolic genes. The use of a 24-h fast challenge enables one to discriminate PPAR{alpha}-regulated genes. For example, gene expression performed in the fed condition demonstrated the predictable decreased expression of PPAR{alpha}-regulated genes coding for medium-chain acyl-CoA dehydrogenase and uncoupling protein 3 in PPAR{alpha} null mouse hearts. Furthermore, the expression of these genes was increased severalfold by fasting in control hearts, an effect that was attenuated in PPAR{alpha} null mice (20). Nevertheless, these mice did show a residual expression of these genes, which is likely to be linked to additional transcription factors (e.g., PPARβ/{delta}) (14). Among other measured transcripts, Pdk4 was the only other gene for which we observed a pattern of response that suggested an involvement of PPAR{alpha}, which concurs with data in the current literature (17).

However, more importantly, we also report data on the expression of genes that had not been previously reported in fed PPAR{alpha} null mouse hearts. First, regarding the genes coding for selected key protein or enzymes implicated in the utilization of CHOs, we found that these hearts displayed an enhanced expression of gene encoding for GLUT4, a mechanism that can favor glucose uptake and utilization. In contrast, gene expression of glycolytic phosphofructokinase-1 was unchanged, although an increased expression may have been expected to compensate the decreased expression of genes related to FA utilization. Second, the transcript levels for the anaplerotic genes Pcx and Pccb in fed PPAR{alpha} null mouse hearts showed marginal differences compared with those in controls, concurring with the absence of significant differences for the measured rates of anaplerotic PC between these two groups of hearts. Finally, we observed markedly reduced mRNA levels for cytosolic Acly (~70%) and Me2 (50%). It is noteworthy that in the liver, these genes have been shown to be regulated by the transcription factor sterol regulatory element-binding protein 1 (1, 38), and PPAR{alpha} was found to play a permissive role (31).

Our findings of a lower Acly transcript level in the fed state together with the higher citrate release rate documented in perfused PPAR{alpha} null mouse hearts suggest one potential mechanism contributing to the restricted capacity to enhance glycolysis at the higher preload, namely, the higher cytosolic citrate level in these hearts. The cytosolic ATP-citrate lyase, in which the activity in the heart is evaluated to be ~0.2 µmol·min–1·g wet wt–1 (4), cleaves citrate to acetyl-CoA and OAA. Hence, this activity may be a determinant for the cytosolic level of citrate (47) and for glycolysis, given that citrate is a potent allosteric inhibitor of phosphofructokinase-1 (see Ref. 41 for review).

Additional potential mechanisms may be responsible for the restricted capacity of PPAR{alpha} null mouse hearts to enhance its lactate production rates in response to a raise in preload and should also be considered. These include the increased production of nitric oxide, which is known to negatively impact on myocardial glucose uptake and utilization (11, 45). This explanation would be compatible with the higher coronary flow of PPAR{alpha} null mouse hearts at the higher preload. Incidentally, a recent study by Guellich et al. (15) demonstrated oxidative damage to contractile proteins, specifically tyrosine nitration of myosin, in these hearts, suggesting an increased production of free radicals and nitric oxide. This may also offer an explanation for the change in activity of NADP+-isocitrate dehydrogenase, an enzyme that is affected by oxidative stress-related molecules. It is noteworthy, however, that an enhanced free radical production in PPAR{alpha} null mouse heart may result in an overestimation of the reported absolute CAC flux rates, which are calculated from MVO2 values. In fact, we cannot evaluate the percentage of oxygen leak associated with free radical production.

In conclusion, results from this study support the notion that beyond their low capacity to oxidize LCFAs, young PPAR{alpha} null mice display alterations in the myocardial metabolism and regulation of CHOs, particularly glucose and glycogen, which may impair their capacity to withstand an increase in energy demand induced by a physiological raise in workload even in the fed state. In fact, ex vivo perfusion experiments revealed that PPAR{alpha} null mouse hearts were unable to increase their glycolytic rate following a raise in preload, which mimics an increased venous return as it occurs during exercise, despite providing what are generally accepted to be physiological levels of calcium, hormones (insulin), cofactors (carnitine), and nutrients, including concentrations of glucose mimicking the fed state (11 mM). These results suggest that a dysregulation of CHO metabolism may also impact energy production and the contractility of hearts that display low LCFA oxidation due to either inherited gene defects or subsequent to the deactivation of PPAR{alpha}, such as it may occur in hypertrophied and failing hearts. Based on our findings, we suggest that one type of metabolic intervention that could benefit the latter hearts would be medium-chain FAs, particularly odd-carbon medium-chain FAs, which are succinate precursors (34). These FAs are more readily β-oxidized, and their hepatic metabolism would favor gluconeogenesis while forming ketone bodies that will become available to the heart for energy production (21).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Canadian Institutes of Health Research Grant 9575 (to C. Des Rosiers), National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-069752, and studentships to R. Gélinas (Department of Nutrition at the Université de Montréal).


   ACKNOWLEDGMENTS
 
This work was presented at the Experimental Biology meeting held in Washington, DC, in April 2007, at the Society Heart and Vascular Metabolism meeting held in Semiahmoo, WA, in September 2006, in Maastricht, The Netherlands, in June 2007, and at the World Congress of the International Society of Heart Research held in Bologna, Italy, in June 2007.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Des Rosiers, Laboratory of Intermediary Metabolism, Montreal Heart Inst. Research Center, 5000 Bélanger East St., Rm. 5350, Montreal, Quebec, Canada H1T 1C8 (e-mail: christine.des.rosiers{at}umontreal.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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