Probing the link between citrate and malonyl-CoA in perfused rat hearts

Thomas

et al. 2005

Self Cite

We previously showed that a fraction of the acetyls used to synthesize malonyl-CoA in rat heart derives from partial peroxisomal oxidation of very long and long-chain fatty acids. The 13 C labeling ratio (malonylCoA)/(acetyl moiety of citrate) was >1.0 with 13 C-fatty acids, which yields [13 C]acetyl-CoA in both mitochondria and peroxisomes and < 1.0 with substrates, which yields [13 C]acetyl-CoA only in mitochondria. In this study, we tested the influence of 13 C-fatty acid concentration and chain length on the labeling of acetyl-CoA formed in mitochondria and/or peroxisomes. ]acetate, the labeling ratio was <0.7 at all concentrations. Therefore, in rat heart, (i) n-fatty acids shorter than 8 carbons do not undergo peroxisomal oxidation, (ii) octanoate undergoes only one cycle of peroxisomal ␤-oxidation, (iii) there is no detectable transfer to the mitochondria of acetyl-CoA from the cytosol or the peroxisomes, and (iv) the capacity of C 2 -C 18 fatty acids to generate mitochondrial acetyl-CoA decreases with chain length.Malonyl-CoA plays an important role in the regulation of fuel selection by the heart through its potent inhibition of carnitine palmitoyltransferase-I and of mitochondrial fatty acid oxidation (1). Malonyl-CoA is synthesized by acetyl-CoA carboxylase 2, which appears to be attached to the outer membrane of mitochondria facing the cytosol (2, 3). Because the heart is a non-lipogenic organ, the only known fate of malonyl-CoA is its decarboxylation by malonyl-CoA decarboxylase. Thus acetylCoA carboxylase and malonyl-CoA decarboxylase catalyze an acetyl-CoA 3 malonyl-CoA 3 acetyl-CoA substrate cycle that regulates malonyl-CoA concentration and mitochondrial fatty acid oxidation. The regulation of acetyl-CoA carboxylase and malonyl-CoA decarboxylase in the heart has been extensively investigated. For recent reviews, see Refs. 4 -8.Because the cytosolic concentrations of acetyl-CoA and malonyl-CoA are much lower than the K m of acetyl-CoA carboxylase and malonyl-CoA decarboxylase for their corresponding substrates, the concentrations of these substrates may regulate the fluxes through the enzymes. We recently measured the turnover of malonyl-CoA in isolated rat hearts perfused with NaH 13 CO 3 (9). Under conditions where the malonyl-CoA concentration varied over a 5-fold range, this concentration was directly proportional to the acetyl-CoA carboxylase flux, but the total activities of acetyl-CoA carboxylase and malonyl-CoA decarboxylase (measured in tissue extracts) did not vary. This strongly supports the notion that in the heart the cytosolic concentration of acetyl-CoA is a major determinant of the concentration of malonyl-CoA and of the rate of mitochondrial fatty acid oxidation.The acetyl-CoA used by cytosolic acetyl-CoA carboxylase is believed to be transferred from mitochondria either via acetylcarnitine or citrate and ATP-citrate lyase. We recently showed that a fraction of this acetyl-CoA is derived from the partial peroxisomal oxidation of long-chain or very long-chain fatty acids (10). Thi...

Section: Resultssupporting

confidence: 93%

Peroxisomal and Mitochondrial Oxidation of Fatty Acids in the Heart, Assessed from the 13C Labeling of Malonyl-CoA and the Acetyl Moiety of Citrate

Bian

Thomas

et al. 2005

Self Cite

“…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 malonylCoA is elevated when acetyl-CoA levels are increased by either activation of pyruvate dehydrogenase (8,9) or by perfusing the heart with octanoate instead of palmitate or oleate (10,11). The increase in malonyl-CoA content was independent of ACC activity.…”

mentioning

confidence: 96%

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

Reszko¹,

Thomas

et al. 2004

Self Cite

The goal of this study was to test the relationship between malonyl-CoA concentration and its turnover measured in isolated rat hearts perfused with NaH 13 CO 3 . 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 nmol⅐g wet weight؊ 1 . The malonyl-CoA concentration was directly correlated with the turnover of malonyl-CoA, ranging from 0.7 to 4.2 nmol⅐min ؊1 ⅐g wet weight ؊1 (slope ‫؍‬ 0.98, r 2 ‫؍‬ 0.94). The V max activities of acetyl-CoA carboxylase and of malonylCoA 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 acetylCoA 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 (1-3). 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 (4), diabetes (5), increased cardiac work (6), and postischemic reperfusion (7). In the heart, malonyl-CoA formed by cytosolic acetyl-CoA carboxylase (ACC) 1 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 K m of the two enzymes for their respective substrates (6, 8). 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 malonylCoA is elevated when acetyl-CoA levels are increased by either activation of pyruvate dehydrogenase (8, 9) or by perfusing the ...

“…much below the K m of ACC␤ for acetyl-CoA. It has been proposed that mitochondrial acetyl-CoA is transferred to the cytosol either via acetylcarnitine and the carnitine acetyl transferase system (14) or via citrate and ATP-citrate lyase (15,16). Arguing against the role of acetylcarnitine is the reported absence of extramitochondrial carnitine acetyl transferase in the heart (17).…”

mentioning

confidence: 99%

“…Arguing against the role of acetylcarnitine is the reported absence of extramitochondrial carnitine acetyl transferase in the heart (17). Although the activity of ATP-citrate lyase in the heart is low, it could sustain the rates of increase in malonyl-CoA concentration measured in perfused rat hearts following the addition of substrates that raise malonyl-CoA concentration (16). Also, the physiological release of citrate by the heart (18,19) implies that citrate is available to cytosolic ATP-citrate lyase after transport from the mitochondria (20).…”

mentioning

confidence: 99%

“…Also, the physiological release of citrate by the heart (18,19) implies that citrate is available to cytosolic ATP-citrate lyase after transport from the mitochondria (20). Using a new gas chromatography-mass spectrometry technique (21), we showed that [ 13 C]oleate contributes carbon to the acetyl moiety of malonyl-CoA (16). Also, experiments with hydroxycitrate, an inhibitor of ATP-citrate lyase (22), support an at least partial role of citrate as a precursor of the acetyl moiety of malonyl-CoA in the heart (16) and in muscle (15).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Peroxisomal Fatty Acid Oxidation Is a Substantial Source of the Acetyl Moiety of Malonyl-CoA in Rat Heart

Reszko

Jobbins

et al. 2004

Self Cite

Little is known about the sources of acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of mitochondrial fatty acid oxidation in the heart. In perfused rat hearts, we previously showed that malonylCoA is labeled from both carbohydrates and fatty acids. This study was aimed at assessing the mechanisms of incorporation of fatty acid carbons into malonyl-CoA. Rat hearts were perfused with glucose, lactate, pyruvate, and a fatty acid (palmitate, oleate or docosanoate). In each experiment, substrates were 13 C-labeled to yield singly or/and doubly labeled acetyl-CoA. The mass isotopomer distribution of malonyl-CoA was compared with that of the acetyl moiety of citrate, which reflects mitochondrial acetyl-CoA. In the presence of labeled glucose or lactate/pyruvate, the 13 C labeling of malonylCoA was up to 2-fold lower than that of mitochondrial acetyl-CoA. However, in the presence of a fatty acid labeled in its first acetyl moiety, the 13 C labeling of malonyl-CoA was up to 10-fold higher than that of mitochondrial acetyl-CoA. The labeling of malonyl-CoA and of the acetyl moiety of citrate is compatible with peroxisomal ␤-oxidation forming C 12 and C 14 acyl-CoAs and contributing >50% of the fatty acid-derived acetyl groups that end up in malonyl-CoA. This fraction increases with the fatty acid chain length. By supplying acetyl-CoA for malonyl-CoA synthesis, peroxisomal ␤-oxidation may participate in the control of mitochondrial fatty acid oxidation in the heart. In addition, this pathway may supply some acyl groups used in protein acylation, which is increasingly recognized as an important regulatory mechanism for many biochemical processes.Malonyl-CoA is an intermediate of fatty acid synthesis in lipogenic organs. It is also a key regulator of mitochondrial long-chain fatty acid oxidation in most mammalian tissues because it modulates the activity of carnitine palmitoyltransferase-I (1-4). Malonyl-CoA is formed by cytosolic acetyl-CoA carboxylase (ACC) 1 and is disposed off either by lipogenesis or via malonyl-CoA decarboxylase, which reforms acetyl-CoA. Alterations in malonyl-CoA metabolism and regulation have been associated with insulin resistance and obesity (5). Mice lacking ACC␤, the predominant ACC isoform in cardiac and skeletal muscle, show not only decreased malonyl-CoA levels and increased fatty acid oxidation but also major alterations in systemic energy balance with decreased body fat despite increased food intake (6). The above data emphasize the crucial role of malonyl-CoA in fat metabolism and energy balance. In the heart, much work has been conducted on the control of malonyl-CoA metabolism, emphasizing the mechanisms of regulation of ACC␤ and malonyl-CoA decarboxylase. This includes acute changes in activity through phosphorylation via cAMPdependent protein kinase or AMP kinase (for recent reviews, see Refs. 5 and 7-10), as well as chronic regulation through gene expression involving peroxisomal proliferator-activated receptor (11,12). However, little is known about the origin of ac...