Regulation of Malonyl-CoA Concentration and Turnover in the Normal Heart

The goal of this study was to test the relationship between malonyl-CoA concentration and its turnover measured in isolated rat hearts perfused with NaH13CO3. This turnover is a direct measurement of the flux of acetyl-CoA carboxylation in the intact heart. It also reflects the rate of malonyl-CoA decarboxylation, i.e. the only known fate of malonyl-CoA in the heart. Conditions were selected to result in stable malonyl-CoA concentrations ranging from 1.5 to 5 nmol·g wet weight–1. The malonyl-CoA concentration was directly correlated with the turnover of malonyl-CoA, ranging from 0.7 to 4.2 nmol·min–1·g wet weight–1 (slope = 0.98, r2 = 0.94). The Vmax activities of acetyl-CoA carboxylase and of malonyl-CoA decarboxylase exceeded the rate of malonyl-CoA turnover by 2 orders of magnitude and did not correlate with either concentration or turnover of malonyl-CoA. However, conditions of perfusion that increased acetyl-CoA supply resulted in higher turnover and concentration, demonstrating that malonyl-CoA turnover is regulated by the supply of acetyl-CoA. The only condition where the activity of malonyl-CoA decarboxylase regulated malonyl-CoA kinetics was when the enzyme was pharmacologically inhibited, resulting in increased malonyl-CoA concentration and decreased turnover. Our data show that, in the absence of enzyme inhibitors, the rate of acetyl-CoA carboxylation is the main determinant of the malonyl-CoA concentration in the heart. The goal of this study was to test the relationship between malonyl-CoA concentration and its turnover measured in isolated rat hearts perfused with NaH13CO3. This turnover is a direct measurement of the flux of acetyl-CoA carboxylation in the intact heart. It also reflects the rate of malonyl-CoA decarboxylation, i.e. the only known fate of malonyl-CoA in the heart. Conditions were selected to result in stable malonyl-CoA concentrations ranging from 1.5 to 5 nmol·g wet weight–1. The malonyl-CoA concentration was directly correlated with the turnover of malonyl-CoA, ranging from 0.7 to 4.2 nmol·min–1·g wet weight–1 (slope = 0.98, r2 = 0.94). The Vmax activities of acetyl-CoA carboxylase and of malonyl-CoA decarboxylase exceeded the rate of malonyl-CoA turnover by 2 orders of magnitude and did not correlate with either concentration or turnover of malonyl-CoA. However, conditions of perfusion that increased acetyl-CoA supply resulted in higher turnover and concentration, demonstrating that malonyl-CoA turnover is regulated by the supply of acetyl-CoA. The only condition where the activity of malonyl-CoA decarboxylase regulated malonyl-CoA kinetics was when the enzyme was pharmacologically inhibited, resulting in increased malonyl-CoA concentration and decreased turnover. Our data show that, in the absence of enzyme inhibitors, the rate of acetyl-CoA carboxylation is the main determinant of the malonyl-CoA concentration in the heart. Malonyl-CoA is a key intermediate of fatty acid synthesis in lipogenic organs. It is also an inhibitor of carnitine palmitoyltransferase I, a regulator of fatty acid oxidation in most tissues (1McGarry J.D. Mannaerts G.P. Foster D.W. J. Clin. Invest. 1977; 60: 265-270Crossref PubMed Scopus (507) Google Scholar, 2Zammit V.A. Biochem. J. 1999; 343: 505-515Crossref PubMed Scopus (99) Google Scholar, 3Chien D. Dean D. Saha A.K. Flatt J.P. Ruderman N.B. Am. J. Physiol. 2000; 279: E259-E265Crossref PubMed Google Scholar). A number of studies have documented the modulation by malonyl-CoA of fatty acid oxidation in the heart under physiological and pathological conditions, such as maturation (4Lopaschuk G.D. Witters L.A. Itoi T. Barr R. Barr A. J. Biol. Chem. 1994; 269: 25871-25878Abstract Full Text PDF PubMed Google Scholar), diabetes (5Ruderman N.B. Saha A.K. Vavvas D. Witters L.A. Am. J. Physiol. 1999; 276: E1-E18Crossref PubMed Google Scholar), increased cardiac work (6Goodwin G.W. Taegtmeyer H. Am. J. Physiol. 1999; 277: E772-E777Crossref PubMed Google Scholar), and postischemic reperfusion (7Dyck J.R. Lopaschuk G.D. J. Mol. Cell. Cardiol. 2002; 34: 1099-1109Abstract Full Text PDF PubMed Google Scholar). In the heart, malonyl-CoA formed by cytosolic acetyl-CoA carboxylase (ACC) 1The abbreviations used are: ACC, acetyl-CoA carboxylase; MCD, malonyl-CoA decarboxylase. 1The abbreviations used are: ACC, acetyl-CoA carboxylase; MCD, malonyl-CoA decarboxylase. is, as far as is known, disposed only via decarboxylation catalyzed by malonyl-CoA decarboxylase (MCD). Thus, cytosolic acetyl-CoA and malonyl-CoA are the two components of a substrate cycle that regulates carnitine palmitoyltransferase I activity and the rate of mitochondrial fatty acid oxidation in the heart. No information is available on the relationship between the concentration of malonyl-CoA and its turnover, i.e. with the flux of ACC in the intact heart. The rate of turnover of malonyl-CoA is likely to be regulated by the cytosolic acetyl-CoA concentration and by the activities of ACC and MCD. The concentrations of cytosolic acetyl-CoA and malonyl-CoA in the heart are much lower than the Km of the two enzymes for their respective substrates (6Goodwin G.W. Taegtmeyer H. Am. J. Physiol. 1999; 277: E772-E777Crossref PubMed Google Scholar, 8Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar). Thus, it is unlikely that the modulation of ACC and MCD activities exerts a tight control on malonyl-CoA turnover. It was previously shown that the myocardial content of malonyl-CoA is elevated when acetyl-CoA levels are increased by either activation of pyruvate dehydrogenase (8Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar, 9Stanley W.C. Hernandez L.A. Spires D. Bringas J. Wallace S. McCormack J.G. J. Mol. Cell Cardiol. 1996; 28: 905-914Abstract Full Text PDF PubMed Scopus (75) Google Scholar) or by perfusing the heart with octanoate instead of palmitate or oleate (10Longnus S.L. Wambolt R.B. Barr R.L. Lopaschuk G.D. Allard M.F. Am. J. Physiol. 2001; 281: H1561-H1567PubMed Google Scholar, 11Poirier M. Vincent G. Reszko A.E. Bouchard B. Kelleher J.K. Brunengraber H. Des Rosiers C. Am. J. Physiol. 2002; 283: H1379-H1386Crossref PubMed Scopus (30) Google Scholar). The increase in malonyl-CoA content was independent of ACC activity. It is not clear whether the higher concentration of malonyl-CoA in the presence of octanoate reflected (i) an increase in acetyl-CoA carboxylation fueled by an increase in cytosolic acetyl-CoA concentration or/and (ii) a decrease in the flux of malonyl-CoA decarboxylation. The goal of the present investigation was to test the hypothesis that myocardial malonyl-CoA concentration is proportional to the rate of acetyl-CoA carboxylation under physiological conditions. We recently developed a mass spectrometric technique for measuring the malonyl-CoA turnover in isolated organs, based on the time course of its labeling from NaH13CO3 (12Reszko A.E. Kasumov T. Comte B. Pierce B.A. David F. Bederman I.R. Deutsch J. Des Rosiers C. Brunengraber H. Anal. Biochem. 2001; 298: 69-75Crossref PubMed Scopus (42) Google Scholar). The malonyl-CoA concentration can also be assayed in the same samples by isotope dilution using an internal standard of [U-13C3]malonyl-CoA. We used these techniques to study the relationship between malonyl-CoA concentration in rat hearts under physiological conditions and during pharmacological inhibition of MCD. In addition, we measured the malonyl-CoA concentration and turnover in the hearts of anesthetized pigs. Materials—Chemicals and biochemicals were obtained from Sigma. NaH13CO3 and [U-13C3]malonic acid were purchased from Isotec (Miamisburg, OH). A standard of [U-13C3]malonyl-CoA was prepared and purified by high performance liquid chromatography (12Reszko A.E. Kasumov T. Comte B. Pierce B.A. David F. Bederman I.R. Deutsch J. Des Rosiers C. Brunengraber H. Anal. Biochem. 2001; 298: 69-75Crossref PubMed Scopus (42) Google Scholar). Two inhibitors of malonyl-CoA decarboxylase, CBM-300864 and CBM-301940, were kindly provided by Chugai Pharma USA. Organ Perfusion Experiments—Hearts from Sprague-Dawley rats (180–220 g, fed, overnight-fasted, or 2-day fasted) were perfused in the Langendorf mode with non-recirculating bicarbonate buffer containing combinations of substrates (glucose, lactate, pyruvate, oleate, and/or octanoate), which would induce a wide range of malonyl-CoA concentrations (Table I). After 15 min of equilibration, the unlabeled bicarbonate in the perfusate was replaced by 40% enriched [13C]bicarbonate, and the hearts were quick-frozen after 0.5–30 min of tracer infusion. In some groups of experiments, other compounds (inhibitors of MCD, Me2SO) were added to the perfusate as indicated in Table I.Table IPerfusion conditions and malonyl-CoA content of isolated rat heartsGroupRat statusConcentrations of compounds added to the perfusate[Malonyl-CoA][Glucose][Lactate][Pyruvate][Oleate][Other compounds]mmmmmmmmnmol/g1Fed7.510.20.41 μm epinephrine1.45 ± 0.162Fed7.510.211.87 ± 0.1132DS41.22.16 ± 0.154Fed7.510.20.42.21 ± 0.145ONF510.20.32.34 ± 0.126ONF40.50.10.31% Me2SO; control for groups 9-112.35 ± 0.207Fed5.510.2No BSA3.03 ± 0.1482DS40.8 mm octanoate; no BSA5.07 ± 0.259ONF40.50.10.35 μm CBM-300864, 1% Me2SO3.91 ± 0.23ap < 0.01 compared with group 6.10ONF40.50.10.330 μm CBM-300864, 1% Me2SO5.60 ± 0.30ap < 0.01 compared with group 6.11ONF40.50.10.310 μm CBM-301940, 1% Me2SO5.01 ± 0.39ap < 0.01 compared with group 6.a p < 0.01 compared with group 6. Open table in a new tab In Vivo Pig Experiments—Six anesthetized pigs were thoracotomized and fitted with a pump-controlled bypass between the femoral artery and the left anterior descending coronary artery (13Panchal A.R. Comte B. Huang H. Kerwin T. Darvish A. Des Rosiers C. Brunengraber H. Stanley W.C. Am. J. Physiol. 2000; 279: H2390-H2398Crossref PubMed Google Scholar). NaH13CO3 was infused into the bypass at a rate calculated to achieve a 15% enrichment of bicarbonate/CO2 in the infused myocardial territory. The enrichment of bicarbonate/CO2 was measured (12Reszko A.E. Kasumov T. Comte B. Pierce B.A. David F. Bederman I.R. Deutsch J. Des Rosiers C. Brunengraber H. Anal. Biochem. 2001; 298: 69-75Crossref PubMed Scopus (42) Google Scholar) in samples of venous blood from the infused territory just before taking punch biopsies of the myocardium at 1, 10, and 60 min (two pigs at each time point). Analytical Procedures—The concentration and 13C labeling of malonyl-CoA were assayed as described previously (12Reszko A.E. Kasumov T. Comte B. Pierce B.A. David F. Bederman I.R. Deutsch J. Des Rosiers C. Brunengraber H. Anal. Biochem. 2001; 298: 69-75Crossref PubMed Scopus (42) Google Scholar). We developed a gas chromatography-mass spectrometric assay for acetyl-CoA carboxylase activity because in our hands the assay with NaH14CO3 had high blanks. Also, this assay measures non-acid-volatile radioactivity as the product and does not specifically identify malonyl-CoA. Our assay involves (i) incubating the tissue extract with acetyl-CoA and NaH13CO3, (ii) spiking the reaction mixture with an internal standard of [U-13C3]malonyl-CoA, (iii) hydrolyzing malonyl-CoA to malonate, and (iv) isotope ratio gas chromatography-mass spectrometric analysis of the di-tert-butyldimethylsilyl derivative of malonate. The assay is conducted with labeled NaH13CO3 to minimize the background resulting from traces of malonate in the tissue extract. We ran the assay on an 800-g supernatant of a tissue extract prepared by homogenizing 50 mg of powdered heart tissue in 0.15 ml of buffer containing 0.05 m Tris-HCl, pH 7.5, 0.25 m mannitol, 1 mm EDTA, 1 mm EGTA, 50 mm NaF, 5 mm sodium pyrophosphate, 10% glycerol (w/w), 1 mm dithiothreitol, and a 1:1000 dilution of mammalian cell protease inhibitor mixture (Sigma). The 165-μl assay mixture contained 60 mm Tris acetate, pH 7.5, 1 mg/ml dialyzed fatty acid-free bovine serum albumin (Intergen), 2 mm mercaptoethanol, 5 mm magnesium acetate, 1.06 mm acetyl-CoA, and 0 or 10 mm citrate. After 2-min preincubation at 37 °C, the reaction was started by adding an amount of extract containing 25 μg of protein from the supernatant of the whole tissue extract. After 4 min of incubation, the reaction was stopped with 25 μl of 10% perchloric acid, and the sample was spiked with 2 nmol [U-13C3]malonyl-CoA. After centrifugation, the acid extract was brought to pH 12 with 2 m KOH and incubated at 40 °C for 1 h to hydrolyze the CoA esters. After centrifuging KClO4, the supernatant was brought to pH 1 with 1 m HCl and evaporated in a Savant vacuum centrifuge. The residue was treated to prepare di-tert-butyldimethylsilyl malonate, which was assayed by gas chromatography-mass spectrometry (12Reszko A.E. Kasumov T. Comte B. Pierce B.A. David F. Bederman I.R. Deutsch J. Des Rosiers C. Brunengraber H. Anal. Biochem. 2001; 298: 69-75Crossref PubMed Scopus (42) Google Scholar). The amount of M1 malonyl-CoA formed was calculated from the M1/M3 mass isotopomer 2Mass isotopomers are designated as Mn, where n is the number of atomic mass units above the molecular weight of the unlabeled isotopomer M. ratio of malonate and the ACC activity expressed in nmol·min–1·(g wet weight)–1. The activity of MCD in heart tissue was assayed by the method of Kerner and Hoppel (14Kerner J. Hoppel C.L. Anal. Biochem. 2002; 306: 283-289Crossref PubMed Scopus (23) Google Scholar). Since rapid changes in the Km of MCD have been reported in rat heart (6Goodwin G.W. Taegtmeyer H. Am. J. Physiol. 1999; 277: E772-E777Crossref PubMed Google Scholar), we measured MCD activity at a low substrate concentration (0.01 mm malonyl-CoA; ∼5–10% of maximal activity) and under near Vmax conditions (0.3 mm malonyl-CoA). Calculations and Statistics—For each group of perfusions with NaH13CO3, the data of the 13C labeling of malonyl-CoA were fitted to an exponential regression, MPE=MPE(t=∞)[1−e−kt] eq.1 where MPE and MPE(t = ∞) are the molar percent enrichments of malonyl-CoA at any time and at infinite time, respectively. The kinetic constant of the pool, k (min–1), was multiplied by the average malonyl-CoA concentration of the group to yield the turnover of the pool (nmol·min–1·g–1). To test whether the MCD inhibitors significantly affected the kinetic constant of the malonyl-CoA pool, we performed a log rank test (15Daniel W.W. Biostatistics: A Foundation for Analysis in the Health Sciences. John Wiley & Sons, 1999: 633-635Google Scholar) (χ2 distribution with one degree of freedom), assuming that the labeling of malonyl-CoA is described by the above exponential. Adjustment for multiple comparisons was done by the Holm's procedure (16Holm S. Scand. J. Statistics. 1979; 6: 65-70Crossref Google Scholar). In eight groups of rat hearts perfused under the conditions listed in Table I and without inhibitors (groups 1–8), the malonyl-CoA concentrations ranged from 1.5 nmol·g–1 (hearts from fed rats perfused with 1 μm epinephrine) to 5 nmol·g–1 (hearts from 2-day starved rats perfused with 0.8 mm octanoate). Groups 1–8 are listed in Table I in the order of increasing malonyl-CoA concentrations measured in the tissue. Within each group of perfusions, the malonyl-CoA concentration remained stable with the duration of the experiment (coefficient of variation < 16%). The ACC and MCD activities measured in tissue extracts were not different among the groups, thus there was no correlation between malonyl-CoA concentration and enzyme activities (Table II).Table IIActivities of ACC and MCD in perfused rat heartsGroupACC activityMCD activity of [malonyl-CoA]0 mm citrate10 mm citrate0.01 mm0.3 mm1167 ± 5.5203 ± 5.488.1 ± 91,190 ± 702169 ± 9.5222 ± 1680.9 ± 41,280 ± 903123 ± 11176 ± 7.697.1 ± 91,500 ± 1704166 ± 5.2214 ± 9.495.2 ± 6.71,370 ± 1407152 ± 10219 ± 11107 ± 12.31,340 ± 1008164 ± 6.4232 ± 14116 ± 31.51,640 ± 120 Open table in a new tab After 15 min of equilibration, we replaced the unlabeled bicarbonate in the perfusate with 40% enriched NaH13CO3. Fig 1A shows the time course of malonyl-CoA labeling in three of the groups of heart perfusions (groups 1, 2, and 8). The data were fitted to an exponential regression to calculate the kinetic constant and half-life of the malonyl-CoA pool. In the absence of inhibitors (groups 1–8 of Table I), the half-lives of malonyl-CoA labeling ranged from 0.76 to 1.27 min (Fig. 1). The kinetic constant of each group was multiplied by the average malonyl-CoA concentration of the group to yield the turnover of malonyl-CoA in the group. Fig. 2 (solid symbols) shows the linear correlation between the concentration and turnover of malonyl-CoA in groups 1–8 of hearts perfused under various physiological conditions (slope = 0.98; r2 = 0.94).Fig. 2Correlation between the flux of acetyl-CoA carboxylation and malonyl-CoA concentration in perfused rat hearts. Each numbered symbol refers to one group of perfused hearts described in Table I. The linear regression was computed from the data of groups 1–8.View Large Image Figure ViewerDownload (PPT) The effect of two inhibitors of MCD, CBM-300864 and CBM-301940, was tested in hearts perfused with buffer containing 1% Me2SO. In the control group (group 6), 1% Me2SO did not affect the relationship between concentration and turnover of malonyl-CoA (Fig. 2). The two inhibitors increased the concentration of malonyl-CoA (Table I), and 30 μm CBM-300864 significantly decreased the turnover of malonyl-CoA (Fig. 1B and Fig. 2, open symbols). We measured the malonyl-CoA concentration and turnover in the hearts of six anesthetized pigs to get data from an animal species whose metabolic rate per kg is close to that of adult humans. The malonyl-CoA concentration was 1.3 ± 0.3 nmol/g wet weight, and its turnover was 0.15 nmol·min–1·g wet weight–1. Thus, in live pig hearts the malonyl-CoA concentration is comparable with that of the perfused rat heart, but the rate of turnover is about 70–80% lower. This study was conducted to gain insight on the dynamics of the malonyl-CoA pool which plays a major role in controlling fatty acid oxidation in the heart. To date, the regulation of malonyl-CoA metabolism in the heart has been studied (i) by defining the factors that modulate the activities of ACC and MCD and (ii) by correlating malonyl-CoA concentrations with activities of carnitine palmitoyltransferase I and with rates of fatty acid oxidation. Our study was aimed at correlating malonyl-CoA concentrations with rates of acetyl-CoA carboxylation calculated from the kinetics of labeling of malonyl-CoA from NaH13CO3 in intact hearts. The turnover of malonyl-CoA, assayed with NaH13CO3, is a measurement of the rate of acetyl-CoA carboxylation in the intact heart. Fig. 2 shows that the malonyl-CoA concentration in rat hearts perfused in the absence of inhibitors of MCD is directly proportional to the rate of acetyl-CoA carboxylation with a slope of 0.98 (groups 1–8). The activities of ACC and MCD exceeded the rate of malonyl-CoA turnover by 2 orders of magnitude and did not correlate with either concentration or turnover of malonyl-CoA. The only condition where the activity of MCD regulated malonyl-CoA kinetics was when the enzyme was pharmacologically inhibited, which caused an increased malonyl-CoA concentration and decreased turnover. Thus, under a wide range of physiological conditions, the rate of malonyl-CoA decarboxylation appears to adjust to the malonyl-CoA concentration. In the heart, the only known fate of malonyl-CoA is its decarboxylation to acetyl-CoA by MCD. Therefore when the malonyl-CoA concentration is stable (as was the case in our experiments), the isotopic turnover of malonyl-CoA represents not only the flux of acetyl-CoA carboxylation but also the flux of malonyl-CoA decarboxylation. It was recently demonstrated that MCD inhibition with CBM-300863 and CBM-301940 increases malonyl-CoA concentration (17Dyck J.R. Cheng J.F. Stanley W.C. Barr R. Chandler M.P. Brown S. Wallace D. Arrhenius T. Harmon C. Yang G. Nadzan A.M. Lopaschuk G.D. Circ. Res. 2004; 94: e78-e84Crossref PubMed Google Scholar). In the present study we demonstrated that the rise in malonyl-CoA with MCD inhibition was due to a decrease in malonyl-CoA turnover (Figs. 1 and 2, compare groups 9–11 with group 6, the Me2SO control). The data of groups 9–11 no longer followed the relationship between malonyl-CoA concentration and turnover of groups 1–8. The two inhibitors did not affect the activity of ACC assayed in rat heart extract (not shown). It appears that the inhibitors indirectly inhibited acetyl-CoA carboxylation by increasing product inhibition on ACC, presumably without affecting the cytosolic acetyl-CoA concentration. In conclusion, our data demonstrate that, in the absence of inhibitors, the rate of acetyl-CoA carboxylation varies with the mix of substrates offered to the heart but appears to be independent of the ACC and MCD activities measured under Vmax conditions. There is evidence that most of the tissue content of acetyl-CoA is mitochondrial. Therefore, the cytosolic concentration of acetyl-CoA must be in the low μm range. Since the Km of ACC for acetyl-CoA is much higher than the cytosolic concentration of acetyl-CoA, the latter must be a key short-term modulator of the ACC flux, as hypothesized previously (8Saddik M. Gamble J. Witters L.A. Lopaschuk G.D. J. Biol. Chem. 1993; 268: 25836-25845Abstract Full Text PDF PubMed Google Scholar, 9Stanley W.C. Hernandez L.A. Spires D. Bringas J. Wallace S. McCormack J.G. J. Mol. Cell Cardiol. 1996; 28: 905-914Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 10Longnus S.L. Wambolt R.B. Barr R.L. Lopaschuk G.D. Allard M.F. Am. J. Physiol. 2001; 281: H1561-H1567PubMed Google Scholar). Future studies will investigate the respective roles of the various sources of cytosolic acetyl-CoA, which include not only mitochondria but also A.E. Kasumov T. David F. Hoppel C.L. Brunengraber H. Des Rosiers C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). We for with the MCD

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