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

Abstract

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...

Probing peroxisomal β-oxidation and the labelling of acetyl-CoA proxies with [1-13C]octanoate and [3-13C]octanoate in the perfused rat liver

“…Thus, under our conditions, the assay of acetyl-CoA enrichment is not affected by free acetate, an ubiquitous contaminant. We described previously an assay of the enrichment of acetyl-CoA by LC-MS of the whole molecule [4]. The acetylthiophenol technique allows assaying the enrichment of acetyl-CoA by GC-MS which is more generally available than LC-MS. Also the basal enrichment of acetylthiophenol is 10.3 % compared with that of intact acetyl-CoA (26.6 %).…”

“…The 13 C-labelling of the acetyl moiety of liver citrate (a probe of mitochondrial acetyl-CoA) was assayed as outlined in [4] by cleaving citrate with ATP citrate-lyase isolated from rat liver [14] and reacting the acetyl-CoA formed with thiophenol, as above. The 13 C-labelling of the C1 + 2 fragment of BHB (another probe of mitochondrial acetyl-CoA [7,8]) was calculated as the difference between the labelling of the whole molecule and that of the C3 + 4 fragment [15].…”

“…We showed recently that, in the heart, acetyl-CoA derived from the partial peroxisomal β-oxidation of long-chain and very-longchain fatty acids is used for malonyl-CoA synthesis [4], a key regulator of the mitochondrial oxidation of long-chain fatty acids [5,6]. This conclusion was based on the comparison of the 13 Clabelling of malonyl-CoA and the acetyl moiety of citrate, a proxy for mitochondrial acetyl-CoA [4].…”

“…This conclusion was based on the comparison of the 13 Clabelling of malonyl-CoA and the acetyl moiety of citrate, a proxy for mitochondrial acetyl-CoA [4]. The labelling ratio (malonylCoA)/(acetyl moiety of citrate) was < 1.0 in the presence of substrates which yield labelled acetyl-CoA in mitochondria .…”

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

“…Since the K m 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 (8 -10). Future studies will investigate the respective roles of the various sources of cytosolic acetyl-CoA, which include not only mitochondria but also peroxisomes (18).…”