Peroxisomal fatty acid oxidation is a substantial source of the acetyl moiety of malonyl-CoA in rat heart.

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 malonyl-CoA 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 malonyl-CoA 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 beta-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 beta-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)(2)(3)(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 acetyl-CoA used for malonyl-CoA synthesis. Acetyl-CoA is produced predominantly in the mitochondria. The concentration of acetyl-CoA available to cytosolic ACC␤ appears to be in the low M range (13), i.e. 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). 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). 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).
The present study was undertaken to characterize the contributions of glucose, lactate/pyruvate, and fatty acids of different chain length to the acetyl moiety of malonyl-CoA in rat hearts perfused under conditions that mimic the in vivo milieu, in terms of substrate supply to the heart. We used mass isotopomer 2 analysis (23) to compare the labeling patterns of malonyl-CoA and of the acetyl moiety of citrate labeled from various 13 C-labeled substrates. Our strategy was to use combi-* This work was supported by the National Institutes of Health (Research Grants RO1DK35543 and PO1AG15585 and Training Grant DK07319) and by a grant from the Cleveland Mt. Sinai Health Care Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ To whom correspondence should be addressed:  (28) from the livers of rats that had been starved for 2 days and then re-fed for 3 days with a high glucose diet. The enzyme, isolated from the Bio-Gel column (28), was precipitated with 50% ammonium sulfate, and aliquots of the suspension (2 units/0.1 ml) were kept frozen at Ϫ80°C. Before use, the enzyme was dissolved in 1 ml of 250 mM Tris-HCl, pH 8.7, containing 5 mM dithiothreitol.
Organ Perfusion Experiments-Sprague-Dawley rats were fed ad libitum for 10 -12 days with standard laboratory chow. Hearts from overnight-fasted rats (180 -220 g) were perfused in the Langendorf mode (12 ml/min) with non-recirculating bicarbonate buffer containing dialyzed bovine serum albumin (3%, fatty acid-free, Intergen), 50 M carnitine, 8 nM insulin, and physiological concentrations of carbohydrates (glucose, lactate, and pyruvate) and a fatty acid (oleate, palmitate, or docosanoate) to mimic the in vivo conditions (29) Combinations of unlabeled and labeled substrates (glucose, lactate, pyruvate, oleate, palmitate, and/or docosanoate) were chosen to induce a wide range of 13 C enrichment of malonyl-CoA and of the acetyl moiety of citrate (Table I). All experiments started by a 15-min equilibration with all substrates being unlabeled. Then, depending on the protocol, 1-3 unlabeled substrates were replaced by the labeled substrate(s) shown in Table I. Mass isotopomer analysis allows us to use pairs of labeled substrates in the same experiments, which generate M1 and M2 mass isotopomers of both malonyl-CoA and the acetyl moiety of citrate. Interconvertible lactate and pyruvate were used both unlabeled and identically labeled. The hearts were quick-frozen after 5-40 min of perfusion with the labeled substrates.
Analytical Procedures-The concentration and 13 C labeling of malonyl-CoA were assayed as described previously (21). We developed a technique for measuring the mass isotopomer distribution of the acetyl moiety of citrate. 200 -500 mg of powdered frozen heart was extracted with 3.5 volumes of 6% perchloric acid. The acid extract was titrated with 10 M KOH to pH 12, and the alkaline solution was kept 2 h at room temperature and then kept overnight at 4°C to allow hydrolysis of extant acetyl-CoA. After centrifuging the KClO 4 , the solution was supplemented with 50 mM Tris, pH 8.7, and then with 10 mM MgCl 2 , 10 mM ATP, 0.2 mM NADH, 0.6 mM CoA, and 10 units/ml malate dehydrogenase. The reaction was started with 0.2 units of ATP-citrate lyase. The progression of the reaction was followed by the decrease in absorbance at 340 nm. When the reaction was complete (30 -40 min at room temperature), the solution was run through an Oasis SPE cartridge (Waters) preconditioned with 1 ml of methanol followed by 1 ml of water. After loading the sample, the cartridge was washed with 1 ml of 5% methanol in water, and acetyl-CoA was released with 1 ml of methanol. After evaporation in a Savant vacuum centrifuge, the residue was dissolved in 0.15 ml of 0.1 mM HCl and analyzed by ion trap liquid chromatography-mass spectrometry. The masses monitored were 808.1-810.1. The mass isotopomer distribution of calibration standards of [U-13 C 6 ]citric acid and [1,5-13 C 2 ]citric acid, precursors of M2 and M1 acetyl-CoA standards, was assayed by gas chromatography-mass spectrometry of trimethyl citrate formed by incubating the citric acid standards with methanol/HCl.
Data Presentation and Statistical Analyses-Data are expressed as molar percent enrichment (MPE) as defined previously (18,29). Briefly, mass isotopomers of metabolites containing 1 to n 13 C atoms are identified as Mi with i ϭ 1, 2, . . . n, and the absolute MPE of individual 13 C-labeled mass isotopomers (Mi) of a given metabolite was calculated as follows, where A M and A Mi represent the peak areas from ion chromatograms corrected for natural abundance, corresponding to unlabeled (M) and 13 C-labeled (Mi) mass isotopomers, respectively. We present data from about 50 heart perfusion experiments. For each of the experimental conditions chosen (Table I), we ran 6 -8 perfusions in the presence of selected 13 C-labeled substrate(s) with the time parameter being allowed to vary. Data shown in figures are the M2 and M1 13 C enrichments of tissue malonyl-CoA or acetyl moiety of citrate measured in a given heart perfusion. They represent means of duplicate gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry injections, respectively, which differed by Ͻ2%. The statistical significance of differences between the 13 C enrichment of malonyl-CoA and of the acetyl moiety of citrate obtained for the perfusions conducted for various times under a given experimental condition was tested using a paired t test (Graph Pad Prism Software version 4). The 13 C enrichment ratios (malonyl-CoA)/(acetyl moiety of citrate) obtained with a 13 C-labeled substrate in a given experimental condition are reported as means Ϯ S.E. of n perfusion experiments.

RESULTS
Experiments with Oleate-In the first two series of experiments, hearts were perfused with buffer containing 5 mM glucose, 1.0 mM lactate, 0.2 mM pyruvate, and 0.3 mM oleate (  In all groups, hearts from overnight-fasted rats were perfused with non-recirculating buffer containing 3% bovine serum albumin, 50 M carnitine, 100 microunits/ml insulin and unlabeled or labeled 4 or 5 mM glucose, 0.5 or 1 mM lactate, 0.1 or 0.2 mM pyruvate, and 0.1 to 0.4 mM fatty acid, as indicated. The last column shows the average labeling ratios (malonyl-CoA)/(acetyl moiety of citrate) for the M1 and M2 isotopomers. The ratios are presented as mean Ϯ S.E. (n ϭ 6 or 7). *, p Ͻ 0.05. **, p Ͻ 0.01. ***, p Ͻ 0.001. NS, non significant using paired paired t test.  (Fig. 1A). This was unexpected because, in the presence of [1-13 C]oleate, the maximal M1 enrichment of mitochondrial acetyl-CoA is 100/9 ϭ 11%, assuming zero contribution from glucose and lactate/pyruvate to acetyl-CoA. Therefore, the 19% M1-labeled acetyl moiety of malonyl-CoA could not be derived solely from mitochondrial acetyl-CoA. Indeed, the M1 enrichment of the acetyl moiety of citrate plateaued at 9% (Fig. 1B). Thus [1-13 C]oleate contributed 9 ϫ 9 ϭ 81% to mitochondrial acetyl-CoA. However, the M1 enrichment of the acetyl moiety of citrate (9%) was about one-half that of malonyl-CoA (19%). In contrast, the M2 enrichment of the acetyl moiety of citrate derived from [U-13 C 3 ](lactate ϩ pyruvate) or [U- 13 C 6 ]glucose plateaued at a value that was ϳ 2-fold greater than that of malonyl-CoA (5 and 18%, respectively). The combined data of Groups 1 and 2 show that the contributions of exogenous oleate, lactate plus pyruvate, and glucose to mitochondrial acetyl-CoA, 81, 4, and 18%, respectively, add up to close to 100%. These results, which concur with previous ones (16,19), indicate that there is little if any contribution of endogenous substrates to mitochondrial acetyl-CoA. Taken together, the data of Fig. 1, A and B, demonstrate that some of the malonyl-CoA label derived from [1-13 C]oleate does not arise from the mitochondrial metabolism of this substrate. To explain this finding, we hypothesized that the high M1 enrichment of malonyl-CoA was derived, at least in part, from the partial peroxisomal ␤-oxidation of [1-13 C]oleate, forming highly enriched [1-13 C]acetyl-CoA in the vicinity of ACC.
Experiments with Docosanoate-To provide support for a contribution of peroxisomal ␤-oxidation of fatty acids to malonyl-CoA formation, we used [1,2,3,4-13 C 4 ]docosanoate (Table I,  Group 3). Docosanoate is a very long-chain C 22 fatty acid that is partly oxidized in peroxisomes (25). Because of its low solubility, even in the presence of 3% bovine serum albumin, we perfused rat hearts with only 0.1 mM [1,2,3,4-13 C 4 ]docosanoate. Fig. 2 shows the high M2 labeling of malonyl-CoA, which peaked at about 43%. In contrast, the M2 enrichment of the acetyl moiety of citrate plateaued at only about 4%. Since only 2 of the 11 acetyl units of [1,2,3,4-13 C 4 ]docosanoate were labeled, complete mitochondrial oxidation of the substrate would have resulted in a maximal M2 enrichment of the acetyl moiety of citrate of 18%. In the presence of [1,2,3,4-13 C 4 ]docosanoate, the 10-fold difference in the M2 enrichments of malonyl-CoA and acetyl moiety of citrate strongly suggests that most of the acetyl moiety of malonyl-CoA is derived from peroxisomal ␤-oxidation.
Experiments with Palmitate-In the experiments reported in the above sections, the fatty acids oleate and docosanoate were 13 C-labeled on the first acetyl. These tracers do not allow probing specifically the fate of the omega acetyl, which, presumably, forms acetyl-CoA only in mitochondria. The availability of differentially 13

FIG. 1. Mass isotopomer distributions of (A) malonyl-CoA and (B) the acetyl moiety of citrate in hearts perfused with [1-13 C]oleate ؉ [U-13 C 3 ](lactate ؉pyruvate) or with [U-13 C 6 ]glucose (Groups 1 and 2).
Note the different scales for the y-axes of the two graphs. For this and subsequent figures, the mean 13 C enrichment ratios (malonyl-CoA/acetyl moiety of citrate) for the M1 and M2 isotopomers are shown in Table I (last  column) were perfused with buffer containing 4 mM glucose, 0.5 mM lactate, 0.1 mM pyruvate, and 0.4 mM palmitate (Table I, Groups 4 -6).
Lastly, one group of perfusions was conducted under the same conditions with a mixture of [16-13 C]palmitate and [1,2-13 C 2 ]palmitate (Group 6). These two 13 C substrates allow probing in the same experiment the fates of the last and the first acetyl groups of palmitate, as M1 and M2 isotopomers, respectively. Hearts were perfused with 0.2 mM [16-13 C]palmitate ϩ 0.2 mM [1,2-13 C 2 ]palmitate, each tracer being thus 50% enriched (Table I, Group 6). As shown in Fig. 4, the M1 enrichment of malonyl-CoA was ϳ 10-fold lower than its M2 enrichment. In contrast, the M1 enrichment of the acetyl moiety of citrate was 2-fold lower than its M2 enrichment. These data provide clear evidence for a differential metabolism of the ␣ and acetyl groups of palmitate. DISCUSSION According to current concepts, the acetyl-CoA used by heart cytosolic acetyl-CoA carboxylase to form malonyl-CoA (i) is of mitochondrial origin and (ii) is transferred from the mitochondria via acetylcarnitine (14) or via citrate and ATP-citrate lyase (15,16). The major finding of this study is the demonstration that a substantial proportion of malonyl-CoA is synthesized from acetyl-CoA molecules derived from extramitochondrial long-chain chain fatty acid ␤-oxidation. This was evidenced by comparing the 13 C labeling of malonyl-CoA with that of the acetyl moiety of citrate, which reflects mitochondrial acetyl-CoA. In the presence of fatty acids labeled in the first acetyl moiety, the labeling ratio (malonyl-CoA)/(acetyl of citrate) was greater than 1.0. In contrast, in the presence of labeled glucose or lactate/pyruvate, the labeling ratio (malonyl-CoA)/(acetyl of citrate) was smaller than 1.0.
In hearts perfused with [U-13 C 3 ](lactate ϩ pyruvate) and [1-13 C]oleate, the M2 enrichment of malonyl-CoA (from [U-13 C 3 ](lactate ϩ pyruvate)) plateaued at about one-half that of mitochondrial acetyl-CoA (Fig. 1B). This implies that the labeling of M2 acetyl-CoA transferred from mitochondria was diluted by unlabeled acetyl-CoA formed in the extramitochondrial space. The only known source of unlabeled extramitochondrial acetyl-CoA (in the absence of exogenous acetate) is the partial peroxisomal oxidation of fatty acids. Although [1-13 C]oleate was M1-labeled in its first acetyl, its oxidation did not generate any M2 acetyl-CoA. Therefore, all acetyl-CoA molecules derived from the partial peroxisomal oxidation of [1-13 C]oleate are "M2 unlabeled" and dilute the M2 labeling of acetyl-CoA derived from [U-13 C 3 ](lactate ϩ pyruvate). In contrast, the M1 enrichment of malonyl-CoA labeled from [1-13 C]oleate was twice that of mitochondrial acetyl-CoA. The latter finding can only be explained by the formation of M1 acetyl-CoA molecules through extramitochondrial oxidation of [1-13 C]oleate, most likely in peroxisomes.
In fact, it appears that, under the conditions of these experiments, about one-half of the extramitochondrial acetyl-CoA used to form malonyl-CoA was derived from the mitochondria, and one-half was derived from extramitochondrial ␤-oxidation of fatty acids. Since the M2 enrichment of mitochondrial acetyl-CoA derived from [U-13 C 3 ](lactate ϩ pyruvate) was diluted 2-fold after its transfer to the extramitochondrial space, we can assume that a similar 2-fold dilution occurred to the M1 enrichment of mitochondrial acetyl-CoA derived from [1-13 C]oleate (9% to 4.5%). Then, we can calculate the M1 enrichment of extramitochondrial acetyl-CoA derived from the peroxisomal ␤-oxidation of [1-13 C]oleate. Out of 100 molecules of malonyl-CoA, 50 were derived from acetyl-CoA enriched at 4.5%, and 50 were derived from acetyl-CoA enriched at X%.

FIG. 3. Mass isotopomer distributions of (A) malonyl-CoA and (B) the acetyl moiety of citrate in hearts perfused with 50% enriched [U-13 C 16 ]palmitate or with [U-13 C 6 ]glucose ؉ [3-13 C](lactate ؉ pyruvate) (Groups 4 and 5).
Solving a simple algebraic equation yields a 33.5% enrichment of acetyl-CoA derived from the peroxisomal ␤-oxidation of [1-13 C]oleate. Since one in three acetyls derived from peroxisomal ␤-oxidation was labeled, it appears that [1-13 C]oleate underwent three cycles of peroxisomal oxidation, on average. This assumes that the exogenous [1-13 C]oleate was the sole fuel of peroxisomal ␤-oxidation in these hearts. Our data are compatible with current concepts on partial peroxisomal ␤-oxidation of (very) long-chain acyl-CoAs (26). Presumably, [1-13 C]oleate was degraded in peroxisomes to an unlabeled C 12 CoA ester, which either was transferred to the mitochondria for further oxidation or alternatively could be involved in the acylation of heart proteins (30).
In a third set of perfusion experiments (Group 6), an equimolar mixture of palmitate tracers labeled in the first ([1,2-13 C 2 ]palmitate) or last ([16-13 C]palmitate) acetyl units provided more insight into the mechanisms involved in the extramitochondrial formation of acetyl-CoA. The M1 enrichment of malonyl-CoA was lower than its M2 enrichment. This was expected since (i) [16-13 C]palmitate would label malonyl-CoA only via labeling of mitochondrial acetyl-CoA, whereas (ii) [1,2-13 C 2 ]palmitate would label malonyl-CoA via mitochondrial acetyl-CoA and peroxisomal ␤-oxidation. Comparing the mass isotopomer distributions of the acetyl moiety of citrate and of malonyl-CoA in these hearts reveals that the M2 enrichment of malonyl-CoA, derived from [1,2-13 C 2 ]palmitate, is somewhat higher than the M2 enrichment of the acetyl moiety of citrate. Therefore, some of the M2 labeling of malonyl-CoA was probably derived from partial peroxisomal ␤-oxidation of [1,2-13 C 2 ]palmitate. Also, the M1 enrichment of malonyl-CoA, derived from [16-13 C]palmitate, is lower than the M1 enrichment of the acetyl moiety of citrate. This probably results from the production of unlabeled acetyl-CoA by partial peroxisomal ␤-oxidation of [16-13 C]palmitate. The degree of dilution of (M1 enrichment of acetyl moiety of citrate)/(M1 malonyl-CoA) is imprecise because of the low M1 enrichment of malonyl-CoA. If one estimates the dilution factor at about 2-3, using the rationale described for the experiments with [1-13 C]oleate, one would conclude that the labeled substrate went through one or two cycles of peroxisomal ␤-oxidation. This is compatible with the estimate of Veerkamp and Van Moerkerk's (33), derived from incubations of rat heart homogenates with [1-14 C]palmitate in the presence of antimycin and rotenone.
The interpretation of the difference in the M1 and M2 labelings of malonyl-CoA is somewhat complicated by the fact that the M2 labeling of the acetyl moiety of citrate from [1,2-13 C 2 ]palmitate was about twice that of its M1 labeling from [16-13 C]palmitate (Fig. 3). One could have expected that the two tracers would equally label the acetyl moiety of citrate. This difference can be explained by the following considerations. The transfer of label from [16-13 C]palmitate to mitochondrial acetyl-CoA is only partial because of the combined effects of (i) the incomplete isotopic equilibrium of the mitochondrial acetoacetyl-CoA thiolase reaction (34,35) and (ii) the reversibility of the mitochondrial 3-oxoacid-CoA transferase reaction that converts acetoacetyl-CoA to acetoacetate (19,35). In the reversible acetoacetyl-CoA thiolase reaction, the exchange of the C 3 ϩ 4 acetyl of acetoacetyl-CoA with the free acetyl-CoA pool is much slower than the exchange of the C 1 ϩ 2 acetyl (34). This was also observed in liver for the mitochondrial (34,36,37) and cytosolic acetoacetyl-CoA thiolase (38). Therefore, acetoacetyl-CoA derived from the last four carbons of [16-13 C]palmitate is more labeled in its C 3 ϩ 4 acetyl compared with its C 1 ϩ 2 acetyl. Through the reversible 3-oxoacid-CoA transferase reaction, the asymmetrically labeled acetoacetyl-CoA is partly converted to asymmetrically labeled acetoacetate, which is released in the perfusate (35). As a result, the mitochondrial acetyl-CoA pool is more labeled when palmitate is labeled in its first versus last acetyl moiety.
There are no precise estimates of the contribution of peroxisomal oxidation to the total rate of long-chain fatty acid oxidation in the heart (25). However, using data from this study, one can estimate the range of the rate of peroxisomal oxidation needed to sustain the production of malonyl-CoA. The turnover of rat heart malonyl-CoA is about 5 nmol⅐min Ϫ1 ⅐g of wet weight Ϫ1 (21). Since peroxisomal oxidation contributes at least one-half of the acetyl-CoA used for malonyl-CoA synthesis, the latter could be supported by a rate of peroxisomal oxidation of about 2.5 (nmol acetyl)⅐min Ϫ1 ⅐g of wet weight Ϫ1 . This rate is 100 times lower than the rate of acetyl-CoA produced from mitochondrial ␤-oxidation of long-chain fatty acids in hearts perfused with a mixture of substrates including 0.4 mM oleate (19). Therefore, a very low rate of peroxisomal fatty acid oxidation is sufficient to sustain the turnover of heart malonyl-CoA. However, acetyl-CoA formed by peroxisomal fatty acid oxidation can also be used in acetylation reactions. Also, the C 12 and C 14 acyl-CoAs derived from peroxisomal ␤-oxidation can be used in protein acylation, which is increasingly recognized as an important regulatory mechanism for many biochemical processes (30,40). Since the heart is not a lipogenic organ, the C 12 and C 14 acyl-CoAs could be derived from the partial peroxisomal oxidation of common long-chain fatty acids.
In conclusion, data from this study conducted in hearts perfused with a substrate mixture mimicking the in situ milieu show that a substantial fraction of the fatty acid carbon incorporated into malonyl-CoA is derived from peroxisomal ␤-oxidation and that this fraction increases with the fatty acid chain length. Also, using the labeling pattern of malonyl-CoA as a probe of peroxisomal ␤-oxidation, it appears that palmitate, oleate, and docosanoate are degraded in peroxisomes to C 12 -C 14 intermediates. The latter could be further oxidized in mitochondria and/or could participate in protein acylation (30). Since the concentration of cytosolic acetyl-CoA is much lower than the K m of acetyl-CoA carboxylase for acetyl-CoA, the supply of cytosolic acetyl-CoA must be a component of the regulation of malonyl-CoA metabolism. Therefore, peroxisomal ␤-oxidation, which supplies acetyl-CoA to the cytosolic site of acetyl-CoA carboxylase, may contribute to the regulation of malonyl-CoA metabolism and of mitochondrial fatty acid oxidation. Future investigations will (i) assess the impact of fatty acid concentrations on the contribution of peroxisomal ␤-oxidation to the acetyl moiety of malonyl-CoA and (ii) address the pathophysiological significance of our findings, especially under conditions where signaling through peroxisomal proliferator-activated receptor ␣ is increased, such as diabetes (39).