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Am J Physiol Heart Circ Physiol 295: H256-H265, 2008. First published May 16, 2008; doi:10.1152/ajpheart.91489.2007
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Reduced heart size and increased myocardial fuel substrate oxidation in ACC2 mutant mice

M. Faadiel Essop,1,2 Heidi S. Camp,3 Cheol Soo Choi,4 Saumya Sharma,5 Ryan M. Fryer,6 Glenn A. Reinhart,6 Patrick H. Guthrie,5 Assia Bentebibel,7,8 Zeiwei Gu,7 Gerald I. Shulman,4 Heinrich Taegtmeyer,5 Salih J. Wakil,7 and Lutfi Abu-Elheiga7

1Department of Physiological Sciences, Stellenbosch University, Stellenbosch; 2Hatter Heart Research Institute, University of Cape Town Faculty of Health Sciences, Cape Town, South Africa; 3Metabolic Disease Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park; 4Departments of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut; 5Department of Internal Medicine, Division of Cardiology, University of Texas-Houston Medical School, Houston; 6Department of Integrative Pharmacology, Abbott Laboratories, Abbott Park, Illinois; 7Verna and Mars McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas; and 8Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain

Submitted 18 December 2007 ; accepted in final form 28 April 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC2) is a key regulator of mitochondrial fatty acid (FA) uptake via carnitine palmitoyltransferase 1 (CPT1). To test the hypothesis that oxidative metabolism is upregulated in hearts from animals lacking ACC2 (employing a transgenic Acc2-mutant mouse), we assessed cardiac function in vivo and determined rates of myocardial substrate oxidation ex vivo. When examined by echocardiography, there was no difference in systolic function, but left ventricular mass of the Acc2-mutant (MUT) mouse was significantly reduced (~25%) compared with wild-types (WT). Reduced activation of the mammalian target of rapamycin (mTOR) and its downstream target p70S6K was found in MUT hearts. Exogenous oxidation rates of oleate were increased ~22%, and, unexpectedly, exogenous glucose oxidation rates were also increased in MUT hearts. Using a hyperinsulinemic-euglycemic clamp, we found that glucose uptake in MUT hearts was increased by ~83%. Myocardial triglyceride levels were significantly reduced in MUT vs. WT while glycogen content was the same. In parallel, transcript levels of PPAR{alpha} and its target genes, pyruvate dehydrogenase kinase-4 (PDK-4), malonyl-CoA decarboxylase (MCD), and mCPT1, were downregulated in MUT mice. In summary, we report that 1) Acc2-mutant hearts exhibit a marked preference for the oxidation of both glucose and FAs coupled with greater utilization of endogenous fuel substrates (triglycerides), 2) attenuated mTOR signaling may result in reduced heart sizes observed in Acc2-mutant mice, and 3) Acc2-mutant hearts displayed normal functional parameters despite a significant decrease in size.

acetyl-CoA carboxylase; fatty acid β-oxidation; glucose oxidation; PPAR{alpha}

CARNITINE PALMITOYLTRANSFERASE 1 (CPT1), which catalyzes the rate-limiting step of mitochondrial fatty acid (FA) uptake, is subject to stringent regulatory mechanisms (24). Malonyl-CoA is a potent allosteric inhibitor of CPT1 and acts as a rheostat for overall mitochondrial FA β-oxidation (FAO) (26). Malonyl-CoA levels are controlled by rates of synthesis [via acetyl-CoA carboxylase (ACC)] and rates of degradation [via malonyl-CoA decarboxylase (MCD)]. Of the two ACC isoforms, ACC1 ({approx}265 kDa) is predominantly expressed in lipogenic tissues such as the liver and adipocytes (1, 23, 36), while ACC2 ({approx}280 kDa) is enriched in tissues with enhanced oxidative capacity for example, heart and skeletal muscle (2, 8, 37). The distinct ACC isoform expression profiles suggest that malonyl-CoA generated by ACC1 and ACC2 may differentially regulate FA synthesis and FAO. In support, we presented evidence for separate ACC subcellular locations, with ACC1 localized to the cytosol and ACC2 associated with mitochondria (3). Moreover, Acc2-mutant mice (lacking functional ACC2) displayed increased FAO rates in skeletal muscle and reduced body weight and fat content (4). On the contrary, a null mutation of ACC1 in mice resulted in embryonic lethality (6).

Recently, we found that Acc2-mutant mice, unlike wild-type controls, did not develop diabetes when fed an obesity-inducing diet (5). Moreover, Debard et al. (13) reported increased ACC2 gene expression levels in skeletal muscle of type 2 diabetic patients. Together, these data suggest that high levels of ACC2 may contribute to the development of the diabetic phenotype and that diminished ACC2 expression in this particular context may hold therapeutic value. In light of these observations, we investigated the effects of in vivo ACC2 lack on the regulation of myocardial fuel substrate oxidation. In this study, we performed Langendorff heart perfusions comparing the previously described Acc2-mutant transgenic mouse strain (4) with age-matched controls and matched substrate oxidation data with expression levels of cardiac metabolic genes and functional data generated by echocardiography. Here, we found that both myocardial oleate and glucose oxidation rates were higher in Acc2-mutant mice, associated with sustained cardiac contractile function, but reduced heart size. Reduced activation of the mammalian target of rapamycin (mTOR) and its downstream target p70S6K was found in MUT hearts and may account for its decreased size. In parallel, expression of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) and its target genes was attenuated in Acc2-mutant mice compared with controls. We propose that this may help sustain normal function of the Acc2-mutant mouse heart.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Generation of the Acc2-mutant transgenic mouse strain has been previously described (3). Male mutant and wild-type strains were housed under controlled conditions (12:12-h light-dark cycle; 25°C) in the Animal Care Center at Baylor College of Medicine and had ad libitum access to standard laboratory chow (Purina Mills, Richmond, IN) and water. All experiments were performed using the 129/Sv background strain except for the glucose uptake and echocardiographic studies where the C57/129/Sv background strain (mixed background) was employed. Animal experiments were approved by the Animal Care and Use Commitee at Baylor College of Medicine and conducted in accordance with the Baylor College of Medicine's Animal Care and Use Guidelines. All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Measurement of myocardial malonyl-CoA levels. Heart tissue was harvested, snap-frozen in liquid nitrogen, and then homogenized in a 1:10 volume (wt/vol) of ice-cold 5% sulfosalicylic acid containing 50 µM dithioerythritol. Homogenates were centrifuged at 15,000 g for 60 min at 2°C and the supernatants were filtered through a 0.22-µm filter (Ultrafree-MC, Millipore, Bedford, MA). Samples were stored at –80°C before liquid chromatography mass spectrometry analysis. HPLC was run in binary mode [A: 5 mM dimethylbutylamine and 6 mM HOAc; B: 0.1% formic acid in CH3CN (EMD Chemicals, Gibbstown, NJ)] at 200 ml/min with the third pump to deliver postcolumn mixing solvent CH3CN. The data were acquired on an Applied Biosystem Pulsar I quadrapole-TOF mass spectrometer (Foster City, CA) using positive ion TOFMS mode.

Measurement of myocardial triglyceride levels. Heart triglyceride levels were measured as described by Chandler et al. (11). Hearts (n = 5) were excised, washed with PBS, and stored at –80°C before being processed. Individual hearts were homogenized using a Kinematica Polytron mechanical tissue blender for 30 s (medium speed), followed by extraction with 2 vol of chloroform:methanol (1:1). The organic fraction was evaporated using nitrogen, and the pellet was dissolved in 1% Triton X-100 (diluted in ethanol). Samples were diluted 1:5 and triglyceride levels were measured using the Infinity Triglyceride Kit (Thermo Electron, Rugby, UK) that was adapted for colorimetric analysis in 96-well plate format.

Measurement of myocardial glycogen levels. Wild-type and Acc2-mutant mice (n = 5) were killed by decapitation, and hearts were rapidly isolated and immediately frozen in liquid nitrogen. Myocardial glycogen levels were determined using the method of Hassid and Abraham (19). Frozen hearts were placed in test tubes and dissolved in 30% KOH (wt/vol) whereafter tubes were immersed in boiling water and glycogen precipitated with ethanol. Glycogen was converted to monosaccharides by boiling the pellet in 1 ml of 3.79 M H2SO4 for 3 h followed by the addition of 0.1 ml 0.33 M MOPS. The solution was thereafter neutralized to pH 7 using 10 M KOH. The final volume was recorded and glucose concentration was measured using a glucose-determining kit (Sigma, St. Louis, MO). Glycogen content was expressed as milligrams of glucose per gram (wet wt).

Determination of ex vivo cardiac fuel substrate metabolism. Five-month-old male Acc2-mutant and matched wild-type mice were anesthetized with chloral hydrate (0.3 mg/g body wt ip). Hearts were excised and thereafter perfused for 30 min in the Langendorff mode at an afterload of 80 cmH2O with Krebs-Henseleit (K-H) buffer containing 5 mM D-glucose (plus 27 µCi/l [U-14C]glucose), 0.4 mM sodium oleate (plus 40 µCi/l [9,10-3H]oleate) bound to 1% BSA (Cohn fraction V, FA free; Serologicals, Norcross, GA), and insulin (40 µU/ml; Lilly). Throughout the experiment, the K-H buffer was equilibrated with 95% O2-5% CO2. Coronary flow was measured every 5 min. At the end of the perfusions, hearts were freeze-clamped and stored in liquid nitrogen (for dry weight determination). Rates of oleate and glucose oxidation were determined as described previously (17). Myocardial oxygen consumption MVO2 was calculated by measuring the arterio-venous O2 content difference, O2(a–v)–, which was multiplied by the coronary flow.

RNA isolation and quantitative RT-PCR. At 5 mo of age, hearts from male Acc2-mutant and matched wild-type mice were isolated and freeze-clamped in liquid nitrogen for subsequent RNA extraction and gene expression analysis. RNA isolation and quantitative RT-PCR of samples were performed as previously described (14) for 1) FA metabolic genes–PPAR{alpha}, a pivotal transcriptional activator of several FA utilization genes; muscle-type carnitine palmitoyltransferase 1 (mCPT1), the rate-limiting mitochondrial FA transfer enzyme; medium-chain acyl-CoA dehydrogenase (MCAD), a representative FAO enzyme; PPAR-{gamma} coactivator 1{alpha} (PGC-1{alpha}), a transcriptional coactivator controlling mitochondrial biogenesis and cellular energy metabolic pathways; and 2) glucose metabolic genes–cardiac-enriched glucose transporter isoforms GLUT1 and GLUT4; pyruvate dehydrogenase kinase-4 (PDK-4), an indirect inhibitor of glucose oxidation. The nucleotide sequences for primers and probes have been previously published (14, 41). Data were normalized to the number of cyclophilin transcripts, which was not significantly different between the treatment groups (data not shown).

Preparation of mitochondrial fractions. Mitochondria-enriched fractions were obtained from wild-type and mutant mouse hearts as previously described (31), with minor modifications. Hearts (3–4 animals) were homogenized in 250 mM sucrose buffer using an omni mixer and subsequently centrifuged at 1,000 g for 15 min. The pellet was homogenized and centrifuged at 600 g for 10 min. The resulting supernatant was centrifuged at 15,000 g for 15 min, and the pellet was resuspended in 100 µl of a buffer containing 250 mM sucrose and 150 mM KCl. All manipulations were performed at 4°C. Mitochondrial protein concentrations were determined using the Bio-Rad protein assay with bovine albumin as a standard.

To determine steady-state peptide levels of mitochondrial mCPT1, 30 µg of mitochondrial protein were subjected to polyacrylamide gel electrophoresis (4–12% NuPAGE MES gels). Mitochondrial mCPT1 peptide levels were detected using an antibody kindly provided by Dr. V. A. Zammit (Warwick Medical School, Coventry, UK). For equal loading controls, the same blot was stripped, washed, and probed with avidin peroxidase that detects mitochondrial biotinylated proteins such as propionyl-CoA carboxylase (PCC), which was equally expressed in hearts of wild-type and Acc2-mutant mice.

Total protein isolation and immunoblotting. Hearts from wild-type and mutant Acc2 mice were excised, snap-frozen, and ground in liquid nitrogen. This powder was added to a homogenization buffer consisting of 3 ml of RIPA buffer [50 mM Tris, pH 7.5; 150 mM NaCl; 1% NP-40; 0.5% deoxy cholic acid; 0.1% SDS; 1 mM NaVO3; 10 mM NaF; 0.2 mM EDTA; protease inhibitor cocktail (1 mM PMSF and 3 µg/ml of leupeptine, pepstatin, aprotinin, and anti-trypsin)]. The suspension was homogenized using a Polytron for 30 s followed by 3 x 10-s sonication bursts. The lysate was thereafter centrifuged at 27,000 g for 1 h. Protein concentrations were determined and 30 µg of each sample were separated on 4–12% NuPAGE MES gels.

MCD antibodies were kindly provided by Dr. M. Prentki (University of Montreal, Canada). Steady-state peptide levels of ACC, pyruvate carboxylase (PC), and PCC were detected by avidin peroxidase using either ECL detection (Amersham) or the 3,3',5,5'-tetramethylbenzidine (TMB) liquid substrate system for membranes (Sigma). Peptide levels of mTOR, pmTOR Ser2481, Akt, and pAKT Ser473 were determined by Western blot analysis using antibodies from Cell Signaling (Danvers, MA) and visualized by ECL.

Mouse echocardiography. Transthoracic echocardiography was performed using an Acuson Sequoia (Siemens) ultrasonograph with a 15-MHz transducer as detailed previously (18). For acquisition of two-dimensional guided M-mode images at the tips of papillary muscles and Doppler studies, mice were sedated by intraperitoneal administration of 100 mg/kg ketamine and maintained on a heated platform in a left lateral decubitus position. The chest was shaved and prewarmed coupling gel was applied. Transmitral and aortic velocities were measured using Doppler pulse wave imaging. All images were saved to an on-board optical disk.

End diastolic and systolic left ventricular (LV) diameter as well as anterior and posterior wall (AW and PW, respectively) thickness were measured on line from M-mode images using the leading edge-to-leading edge convention. All parameters were measured over at least three consecutive cardiac cycles and averaged. LV fractional shortening was calculated as [(LV diameterdiastole – LV diametersystole)/LV diameterdiastole] x 100 and LV mass was calculated by using the formula {1.05 x [(PWdiastole + AWdiastole + LV diameterdiastole)3 – (LV diameterdiastole)3]}. Relative wall thickness was calculated as 2*PWdiastole/LV diameterdiastole. Heart rate was determined from at least three consecutive intervals from the pulse wave Doppler tracings of the LV outflow tract. Isovolumic relaxation time was measured as the time from the closing of the aortic value to the opening of the mitral value from pulse wave Doppler tracings of the LV outflow tract and mitral inflow region. To measure E and A waves, heart rates were slowed down using additional doses (1/4 to 1/2) of ketamine. However, all other measurements (except E and A waves) were performed at the higher heart rate. For tissue Doppler measures, the smallest possible sample volume was placed at the septal mitral annulus to capture the early diastolic (Ea) and late diastolic (Aa) velocities (34, 38). The same person obtained all images and measures and was blinded to genotype.

In vivo measurement of insulin-stimulated heart glucose uptake. After an overnight fast, a hyperinsulinemic-euglycemic clamp was conducted for 120 min with a primed/continuous infusion of human insulin (126 pmol/kg prime, 18 pmol·kg–1·min–1 infusion; Novo Nordisk, Princeton, NJ) to raise plasma insulin within the physiological range. Plasma glucose was clamped basal concentrations (~6.7 mM). 2-Deoxy-D-[1-14C]glucose (14C-2-DG; Perkin Elmer, Boston, MA) was injected as a bolus at the 75th minute of the clamp to estimate the rate of insulin-stimulated tissue glucose uptake as previously described (32). Blood samples (10 µl) for the measurement of plasma 14C activities were taken during the last 45 min of the clamp. At the end of the clamp, mice were anesthetized with pentobarbital sodium injection and tissues were taken for biochemical measurements within 3 min. For the determination of heart glucose uptake, heart samples were homogenized, and the supernatants were subjected to an ion-exchange column to separate tissue 14C-2-DG-6-phosphate (2-DG-6-P) from 2-DG. Heart glucose uptake was calculated from the area under the curve of the plasma 14C-2-DG profile and muscle 14C-2-DG-6-P content, as previously described (40).

Statistical analysis. The time course data for the heart perfusion studies are presented as means ± SE. Statistical analysis on perfusion data was performed using ANOVA repeated-measures one-way ANOVA. The unpaired Student's t-test was used to determine differences between two groups. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In vivo cardiac functional analysis. Echocardiographic studies were performed and data are summarized in Table 1. There was a ~25% decrease in LV mass and LV mass/body wt, consistent with a smaller heart as assessed by dry weight measurement during the metabolic studies. In Acc2-mutant mice, LV diastolic and LV systolic dimensions decreased by 17 and 27%, respectively, compared with wild-types (P < 0.001). The decrease in both LV dimensions did not cause a significant change in fractional shortening (45 ± 5 and 38 ± 12 in Acc2-mutant mouse and matched controls, respectively; Table 1). Although there was a slight increase in the heart rate of Acc2-mutant mice, this did not reach statistical significance (530 ± 82 and 578 ± 61 beats/min for wild-type and Acc2-mutant mice, respectively). When additional ketamine was administered to measure E and A waves, the heart rate decreased to 316 ± 51 and 325 ± 32 beats/min for wild-type and mutant mice, respectively. All other parameters such as PWd, Awd RWth, E/A, Ea/Aa, and IVRT were similar (Table 1).


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  		Table 1. Echocardiographic parameters in WT and Acc2-mutant mice

 
Heart weight. We also determined the heart dry weight of mice at the end of perfusion studies. Interestingly, heart weight (29 ± 2 vs. 48 ± 4 mg; n = 8, P = 0.001) and the heart/body wt ratio were significantly reduced in mutant vs. wild-type mice (0.94 ± 0.01 vs. 1.27 ± 0.11; n = 8, P < 0.05; Fig. 1). These results are consistent with our echocardiography studies, confirming reduced heart size in Acc2-mutant mice.


Figure 1
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  		Fig. 1. Heart weight measurements. Hearts were collected at the end of perfusion studies, dried in an oven at 65°C, and weighed. A: heart weights (HW; in mg). B: heart-to-body wt (BW) ratios for wild-type vs. mutant mice. Values are depicted as means ± SE. *P < 0.001 and **P < 0.05 vs. wild-type.

 
Determination of myocardial malonyl-CoA and MCD levels. Malonyl-CoA levels in Acc2-mutant hearts were markedly reduced (~70%) compared with wild-types (P = 0.0001; Fig. 2A). These levels are consistent with previous measurements in Acc2-mutant hearts using the biochemical method of McGarry et al. (25). The significant reduction in malonyl-CoA levels in hearts of Acc2-mutant mice further confirms that the ACC2 isoform is the major contributor to the myocardial malonyl-CoA pool. These results are in agreement with earlier studies showing the predominance of ACC2 mRNA/protein levels in heart tissues compared with ACC1 (1, 2, 37). Since MCD is also a major regulator of malonyl-CoA levels in muscle and heart tissues, we also measured MCD expression levels. Here, MCD transcript levels were reduced by ~50% in Acc2-mutant hearts compared with wild-types (data not shown). In agreement, we found that MCD protein levels were also decreased (P = 0.001 vs. wild-types; Fig. 2, B and C). As shown in Fig. 2D, ACC1 is still expressed to a significant degree in Acc2-mutant hearts, suggesting that residual malonyl-CoA is due to both remaining ACC1 and MCD downregulation in mutant hearts. There were no significant changes in the level of two mitochondrial enzymes, i.e., PCC and PC (Fig. 2D).


Figure 2
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  		Fig. 2. Reduced malonyl-CoA, MCD, and ACC1 levels in Acc2-mutant hearts. A: Malonyl-CoA levels were measured (refer to METHODS) in hearts collected from 20-wk-old male mice (n = 12). B: MCD peptide levels were detected using µg of protein isolated from Acc2-mutant mice and compared with wild-types. C: densitometric analysis of MCD peptide levels. *P = 0.0001 and **P = 0.001 vs. wild-type. D: 30 µg protein of isolated mitochondria from heart and Acc2-mutant mice were subjected to electrophoresis gel separation and proteins were detected with avidin peroxidase. The arrows indicate the position of biotinylated proteins, i.e., ACC1; ACC2; PC, pyruvate carboxylase; PCC, propionyl-CoA carboxylase.

 
Determination of myocardial glycogen and triglyceride content. Endogenous triglycerides and glycogen are important myocardial energy sources. Here, we found that glycogen levels were similar in hearts of wild-type and Acc2-mutant mice (0.27 ± 0.04 and 0.29 ± 0.05 mg/g wet wt, respectively; Fig. 3A). However, myocardial triglyceride levels were markedly reduced in mutant hearts vs. wild-types (2.25 ± 0.35 and 1.16 ± 0.41 mg/g wet wt, respectively; P = 0.007; Fig. 3B). These results therefore suggest that Acc2-mutant mice do not spare glucose as glycogen.


Figure 3
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  		Fig. 3. Glycogen and triglyceride levels in wild-type and Acc2-mutant hearts. Glycogen (A) and triglyceride (B) levels were determined (refer to METHODS) in hearts from mutant and wild-type hearts (n = 5). Values are depicted as means ± SE. *P = 0.007 vs. wild-type.

 
Ex vivo cardiac fuel substrate oxidation rates. Myocardial oxygen uptake (MVO2) was not statistically different in Acc2-mutant compared wild-types (30 ± 4.9 and 22 ± 5.6 µmol·min–1·g–1 dry wt, respectively; Fig. 4A). In agreement with lower malonyl-CoA levels, myocardial oleate oxidation was higher in mutant vs. wild-type mice (Fig. 4, B and C). Unexpectedly, myocardial glucose oxidation was also significantly higher in Acc2-mutant mice compared with the wild-type (0.08 ± 0.012 and 0.03 ± 0.01 µmol·min–1·g–1 dry wt, respectively; Fig. 5). Coronary flow was not significantly different in Acc2-mutant vs. wild-types (43.23 ± 5.59 and 36.10 ± 11.61 ml·min–1·g–1 dry wt, respectively).


Figure 4
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  		Fig. 4. Ex vivo cardiac MVO2 and oleate oxidation rates. Substrate oxidation was assessed in Langendorff perfused hearts during 20 min of aerobic perfusion in the presence of 0.4 mM oleate, 5 mM glucose, and 40 µU/ml insulin as substrates. Values depicted are means ± SE (n = 5) for perfusate collected at 5-min intervals. A: myocardial oxygen consumption. B: rate of oleate oxidation. C: average rates of oleate oxidation in B. Statistical analysis was performed using ANOVA.

 

Figure 5
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  		Fig. 5. Ex vivo cardiac glucose oxidation rates. Glucose oxidation was assessed in Langendorff perfused hearts during 30 min of aerobic perfusion in the presence of 0.4 mM oleate, 5 mM glucose, and 40 µU/ml insulin as substrates. A: rate of glucose oxidation. B: average values of rates from A. Values depicted are means ± SE (n = 5) for perfusate collected at 15-min intervals (refer to A). Statistical analysis was performed using ANOVA.

 
Myocardial glucose uptake in vivo. We used hyperinsulinemic-euglycemic clamp conditions to determine glucose uptake in hearts. Consistent with the increase in glucose oxidation in isolated hearts of Acc2-mutant mice, the glucose uptake was ~83% higher in Acc2-mutant hearts compared with the wild-types (Fig. 6).


Figure 6
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  		Fig. 6. Increased glucose uptake in hearts of Acc2-mutant mice. Glucose uptake in hearts was assessed by means of hyperinsulinemic-euglycemic clamps. Acc2 depletion significantly improved heart glucose uptake. Data are expressed as mean values ± SE (n = 9).

 
Expression of genes regulating FA and glucose metabolism. To gain further insight into mechanisms underlying myocardial fuel substrate utilization in the Acc2-mutant mouse strain, we measured transcript levels of the nuclear receptor PPAR{alpha}, a transcription factor that regulates numerous genes involved in the uptake and oxidation of FAs. We found that PPAR{alpha} transcript levels were significantly decreased in mutant vs. wild-type mice (Table 2). Transcript levels of two other PPAR{alpha}-regulated genes, i.e., mCPT1 and MCAD, also displayed downward trends. PGC-1{alpha} transcript levels did not significantly differ between mutant and wild-type strains.


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  		Table 2. Transcript levels of cardiac glucose and fatty acid metabolic genes in WT and Acc2-mutant mice

 
The rate-limiting step for exogenous glucose utilization is its transport into cardiomyocytes via specific carrier-mediated transporters, i.e., GLUT1 and GLUT4. Transcript levels of both transporters did not differ significantly between Acc2-mutant and wild-type mice (Table 2). However, transcript levels of PDK-4, an indirect inhibitor of glucose and lactate oxidation, were markedly downregulated in mutant vs. wild-type mice (n = 4, P < 0.05; Table 2).

Western blot analysis of mitochondrial protein extracts showed that mCPT1 expression was significantly reduced in the Acc2-mutant mouse heart compared with wild-types (Fig. 7). The densitometric analysis shows ~2x higher mCPT1 peptide levels in wild-type vs. mutant hearts (Fig. 7). These results are consistent with lower PPAR{alpha} mRNA in hearts of Acc2-mutant mice shown in Table 2.


Figure 7
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  		Fig. 7. Representative Western blot of mCPT1 peptide levels. Western blot analysis of purified mitochondria and crude extracts (METHODS) were separated by 4–12% NuPAGE MES gels. A: 30 µg protein of isolated mitochondria from heart and Acc2-mutant mice were subjected to electrophoresis gel separation. An antibody raised against rat muscle-type CPT1 was used to detect mouse mCPT1 by ECL (n = 7). B: PCC peptide levels indicated as a control for loading in blot A. C: densitometric analysis was performed on mCPT1 blots using standard laboratory techniques and mCPT1 levels are given as wild-type/mutant ratio.

 
To gain insight into mechanisms that may regulate the reduced heart size of Acc2-mutant mice, we determined peptide levels of members of the mTOR pathway, which is implicated in the control of protein translation. We found that mTOR levels were similar in both wild-type and mutant hearts (Fig. 8A). However, phosphorylation of mTOR on Ser2481 was significantly reduced in mutant hearts (P = 0.01 vs. wild-types), suggesting decreased autophosphorylation and mTOR activity. Reduced mTOR phosphorylation was accompanied by a significant decrease in phosphorylation of its downstream target P70S6K (at Thr389; Fig. 8B). There were no significant changes in Akt/pAKT levels (Fig. 8C), suggesting that downregulation of mTOR signaling in Acc2-mutant hearts does not involve the AKT pathway.


Figure 8
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  		Fig. 8. Representative Western blots of mTOR, P70S6K, and Akt in Acc2-mutant hearts. Western blot analysis of protein extracts (METHODS) was separated by 4–12% NuPAGE MES gels. A: mTOR and pmTOR Ser2481 and corresponding densitometric analysis. B: P70S6K and pP70S6K Thr389 and densitometric analysis for P70S6K; the level of pP70S6K was very low in mutant heart extracts. C: Akt and pAKT Ser473 peptide levels and corresponding densitometric analysis. Ponceau staining shows equal loading. *P = 0.01 vs. wild-type.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of this study are 1) a reduced heart size in Acc2-mutant mice, without impaired cardiac function; 2) increased oxidation rates of both oleate and glucose in Acc2-mutant mouse heart; 3) increased glucose uptake in Acc2-mutant mouse heart; and 4) downregulation of PPAR{alpha} and its target genes in the Acc2-mutant mouse.

Reduced heart size in Acc2-mutant mouse. The heart mass of the Acc2-mutant mouse was significantly reduced compared with wild-types (lower heart wt/body wt ratios). We confirmed this reduction in heart size by measuring the dry weight of hearts at the end of perfusion studies, which exhibited a ~25–30% reduction in size. Moreover, echocardiographic analysis performed showed that LV mass and LV wall diameter were significantly reduced in mutant vs. wild-type mice. However, echocardiography also revealed that contractile function was not altered in the Acc2-mutant mouse heart (Table 1).

It is unclear why the heart size of the mutant mouse is reduced and what the precise regulatory mechanisms are. Here, we propose that high FA oxidation rates by the Acc2-mutant heart deplete myocardial diacylglycerol and phosphatidic acid content, thereby leading to reduced phosphorylation of mTOR and its downstream targets, i.e., p70S6K. This is in agreement with previous work showing that phosphatidic acid binds to the FRB domain of mTOR resulting in its activation (16). Phosphorylation of mTOR on Ser2481 was significantly reduced in mutant hearts, suggesting a decrease in autophosphorylation and decreased activity of mTOR. Reduced mTOR phosphorylation was accompanied by a significant decrease in phosphorylation of its downstream target P70S6K (at Thr389). The autophosphorylation site at Ser2481 is surrounded by bulky hydrophobic residues which are similar to a bulky hydrophobic site found in P70S6K (10, 28). Furthermore, a recent study showed that pretreatment of steridogenic luteal cells with rapamycin abolished phosphorylation at the Thr389 site and only partially reduced phosphorylation on Thr421/Ser424, suggesting a correlation between mTOR activity and phosphorylation of P706SK at the Thr389 site (7). We therefore propose that reduced mTOR signaling/activity will eventually result in the decreased heart size observed in the Acc2-mutant mouse.

Increased oxidation rates of exogenous oleate and glucose and higher glucose uptake in Acc2-mutant mouse hearts. Because we previously reported markedly reduced malonyl-CoA levels in the Acc2-mutant mouse heart (4), we predicted increased myocardial FAO rates. Here, we found that myocardial malonyl-CoA levels were markedly reduced in mutant mice, and cardiac FAO rates accordingly increased compared with wild-types. In agreement, we previously reported elevated FAO rates in isolated soleus muscle, hepatocytes, and adipocytes due to lower malonyl-CoA levels found in the Acc2-mutant mouse (4, 5, 27). Although malonyl-CoA levels were reduced in mutant hearts, a residual amount nonetheless remained. We propose that this may be mainly due to lower MCD levels (also a PPAR{alpha} target gene) and remaining ACC1 expression.

A major concern is the question of the effects of constitutively increased fat oxidation in the heart because more than 70% of cardiac energy production results from fat oxidation (9, 39), and originally fuel competition between fat and carbohydrate was observed in the heart (29). Moreover, it is well known that diabetic heart is characterized by increased fat oxidation and reduced glucose metabolism (21, 22, 30). The Acc2-mutant mice had a normal life span and did not exhibit obvious contractile impairment, which is supported by our current echocardiographic studies showing normal heart function. Intriguingly, we found that myocardial glucose oxidation rates in mutant mice were increased despite higher oleate oxidation rates. This increase in glucose oxidation appears to be mainly due to enhanced glucose uptake (Fig. 6). In accordance, we previously reported that adipose tissues of Acc2-mutant mice also displayed increased rates of glucose oxidation (4, 27). Moreover, our recently published study reported that Acc2-mutant mice displayed marked increases in insulin-stimulated whole body glucose uptake, glycolysis, and skeletal muscle glucose uptake (12). This is unexpected since the glucose-FA cycle predicts that increased FAO rates should result in a corresponding reduction in glucose oxidation rates (29).

The amount of oxygen needed for complete oxidation of one mol of oleate and one mol of glucose is 24 and 6 mol, respectively. The combined oxidation of exogenous glucose and oleate (~600 nmol CO2·min–1·g–1 dry wt) is lower than the value for oxygen consumption (20–30,000 nmol O2·min–1·g–1 dry wt). We propose that this may be due to increased breakdown of endogenous (triglycerides) and/or other exogenous fuel substrates (e.g., lactate, ketone bodies) that make a large contribution to the overall energy metabolism of mutant hearts. In agreement with this concept, we found that triglyceride levels were reduced in hearts of the Acc2-mutant mice. We also previously found that plasma β-hydroxybutyrate levels were significantly increased in the fed and fasted state of Acc2-mutant mice (12). In addition, glycogen levels were similar in wild-type and Acc2-mutant hearts, suggesting that exogenous glucose supplied is oxidized rather than being stored as glycogen. Together, our data suggest that Acc2-mutant hearts preferentially oxidize exogenous oleate and glucose, while endogenous fuel substrates such as triglycerides also make a greater contribution to overall oxidation rates compared with wild-types. However, it is important to note that we are comparing oxidation rates of exogenous fuel substrates (supplied in perfusate) and that this may not necessarily correspond with the absolute myocardial oxidation rates of these substrates. Moreover, serum-free FA concentrations are in the nanomolar range; hence, cardiomyocytes rely on hydrolysis of circulating lipoproteins by lipoprotein lipase as the main source for FA supply.

Reduced expression of PPAR{alpha} and target genes in the Acc2-mutant mouse. To gain further insight into these findings, we determined the expression of several cardiac metabolic genes. We found that myocardial transcript levels of PPAR{alpha}, a well-described FA-sensing transcriptional modulator, were significantly reduced in the Acc2-mutant mouse. Gene expression of two other PPAR{alpha} target genes, i.e., MCAD and mCPT1, displayed downward trends, although this did not reach statistical significance. However, protein expression of mCPT1 was significantly reduced in the Acc2-mutant mouse.

Consistent with our ex vivo glucose oxidation data, transcript levels of PDK-4, a PPAR{alpha} target gene, were markedly reduced in mutant mice. However, p-Akt peptide levels were not significantly altered in Acc2-mutant mice. A number of reasons could potentially explain our finding of increased glucose oxidation in this model. 1) Reduced phosphorylation of the PDH complex by lower PDK-4 levels may override the feedback inhibition of FAO products. 2) The activity of pyruvate dehydrogenase may be upregulated. 3) Other factors that may affect glucose oxidation, for example, the intramitochondrial free Ca2+ concentration and/or pyruvate availability, may be increased. 4) There may be crosstalk between malonyl-CoA and the PDH complex, with chronically lowered malonyl-CoA leading to PDH activation. 5) Although GLUT1 and GLUT4 gene expression levels were not significantly altered, we cannot exclude the possibility of increased GLUT translocation from the sarcolemma to plasma membrane, explaining enhanced myocardial glucose uptake. 6) Since lack of ACC2 promotes FAO, this would lead to decreased intracellular FA metabolites and less inhibition of insulin-mediated glucose uptake. Although we did not observe altered p-Akt phosphorylation in mutant hearts, this does not rule out improved insulin sensitivity in this instance.

We propose that the lower PPAR{alpha} and mCPT1 expression observed may be a compensatory mechanism to prevent excessively high myocardial FAO rates in the Acc2-mutant mouse strain. If such a control mechanism was indeed lacking, we predict that excessively high FAO rates could for example result in increased uncoupling of mitochondrial oxidative phosphorylation and reduced cardiac mitochondrial energy production. In agreement with this concept, a recent study investigating myocardial substrate metabolism of an MCD-deficient mouse found increased levels of PPAR{alpha} gene expression associated with unchanged FA and glucose oxidation rates compared with controls (15). Previously, we found reduced plasma free FA (FFA) levels for Acc2-mutant compared with wild-type mice (4). This should therefore result in attenuated FA-induced activation of PPAR{alpha} and its target genes. Thus by lowering both ligand supply and the expression of FA metabolic genes, we believe that excessively high rates of FAO are prevented in the Acc2-mutant heart (35). In support, Young et al. (42) previously reported the downregulation of PPAR{alpha} gene expression in streptozotocin-induced diabetic rats, suggesting that conditions associated with chronic elevation of cardiac FAO may result in decreased PPAR{alpha} levels. Likewise, reduced PPAR{alpha} mRNA levels in hearts of Zucker diabetic fatty rats were associated with elevated plasma FFA and triglyceride levels (43).

Limitations. The ex vivo glucose oxidation rates reported in this study are lower than what have previously been reported from our laboratory and other studies (20, 33). The perfusion data in the current study roughly translate to glucose oxidation contributing ~20% of the energy production of wild-type hearts. We believe that this may be a strain-specific phenomenon related to the particular mouse strain we employed in this study. Also, the perfusion data were generated using a nonworking heart system with low mechanic force and therefore the metabolic profile obtained using this model may not necessarily reflect what occurs in vivo. Thus, although we carried out in vivo analyses of cardiac function, our findings would have been further strengthened by the inclusion of ex vivo heart functional analyses.

In conclusion, we report increased exogenous myocardial energy substrate utilization and lower levels of endogenous fuels (triglycerides) in Acc2-mutant hearts, associated with a reduction in heart size but normal cardiac function. We propose that attenuated mTOR signaling may be responsible for reduced heart size in mutant mice. The fact that mutant hearts functioned normally despite the lack of a pivotal metabolic regulator (i.e., ACC2) is intriguing, although underlying mechanisms are still unknown at this stage. Nonetheless, the study shows that ACC2 inhibition remains an attractive therapeutic target, despite continuously high myocardial FAO rates.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in parts by grants from the Hefni Technical Training Foundation and the National Institutes of Health Grant GM-63115 (to S. J. Wakil); National Heart, Lung, and Blood Institute Grants RO1-HL-073162-01 and T32-HL-07591 to H. Taegtmeyer; the South African MRC, National Research Foundation, Fulbright Commission and Ernest Oppenheimer Memorial Trust to M. F. Essop; and R01-DK-40936, U24-DK-76169, and a Distinguished Clinical Investigator Award from the American Diabetes Association (to G. I. Shulman).


   ACKNOWLEDGMENTS
 
We thank R. Salazar, P. Kordari, and S. Sen for technical assistance, C. R. Wilson and M. Burgmaier for advice, and R. A. Tate for editorial help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Abu-Elheiga, Dept. of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, MS BCM-125, Houston, TX 77030 (e-mail: lutfia{at}bcm.tmc.edu)

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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