The movement of lipids across mitochondrial membranes represents a rate-limiting step in fatty acid oxidation within the heart. A key regulatory point in this process is flux through carnitine palmitoyltransferase-I (CPT-I), an enzyme located on the outer mitochondrial membrane. Malonyl-CoA (M-CoA) is a naturally occurring inhibitor of CPT-I; therefore, the abundance of M-CoA has long been considered a major regulator of fatty acid oxidation. A recent paper published in the Biochemical Journal by Altamimi et al. (Biochem. J. (2018) 475, 959–976) provides evidence for a novel mechanism to produce M-CoA. Specifically, these authors identified carnitine acetyltransferase within the cytosol and further show that flux in the reverse direction forms acetyl-CoA, which is the necessary substrate for the subsequent synthesis of M-CoA. The elegant study design and intriguing data presented by Altamimi et al. provide further insights into the reciprocal regulation of substrate selection within the heart, with implications for fuel utilization and the development of cardiac diseases.
The oxidation of fat and carbohydrates represents the major energy-producing processes within the heart, and while the utilization of these substrates is intimately regulated, the underlying mechanisms remain incompletely understood. Nevertheless, a key control point implicated in reciprocal substrate utilization is the capacity for fatty acid transport into mitochondria, a process requiring flux through carnitine palmitoyltransferase-I (CPT-I). While it has long been established that malonyl-CoA (M-CoA) impairs CPT-I flux in a variety of tissues and experimental conditions, the source of cytosolic M-CoA implicated in this process has remained speculative. Seminal work conducted in perfused heart preparations by Randle et al.  nearly 60 years prior proposed that the mitochondrial tricarboxylic acid cycle (TCA) intermediates acetyl-CoA and citrate were key substrates in this process. Specifically, during times of accelerated carbohydrate and TCA flux, excess citrate was capable of traversing the mitochondrial membrane and accumulating within the cytosol. The flux of cytosolic citrate through ATP citrate lyase (ACL) results in the synthesis of acetyl-CoA, for the subsequent production of M-CoA. Coined the glucose-fatty acid cycle, Randle et al.  proposed that high rates of carbohydrate oxidation were capable of attenuating mitochondrial fatty acid utilization in a reciprocal manner, mediated by the accumulation of citrate and acetyl-CoA.
In a recent study by Altamimi et al. published in the Biochemical Journal , an alternative pathway to produce M-CoA has been supported. This is based on a similar logic to Randle's seminal work, namely that in conditions of excessive energy provision to the mitochondria, a compound ‘leaves’ the mitochondrial matrix to inhibit fatty acid uptake. Specifically, the authors have focused on the near-equilibrium carnitine acetyltransferase (CrAT) as a potential source of cytosolic acetyl-CoA. This enzyme is known to ‘buffer’ excess mitochondrial acetyl-CoA while producing acetylcarnitine in situations of energy surplus, a process primarily thought to prevent back inhibition of pyruvate dehydrogenase and maintain carbohydrate flux. It is well documented that carnitine-acylcarnitine translocase mediates the subsequent movement of mitochondrial acetylcarnitine into the cytosolic space . While the metabolism of cytosolic acetylcarnitine has remained unknown, similar to the mitochondrial and cytosolic enzymatic pools of CPT, it has been speculated that CrAT additionally exists in multiple subcellular compartments, one of which may be localized within the cytosol [4,5]. Despite the fact that the theory of cytosolic CrAT localization was proposed in the late 1960s, this has remained a contentious issue [4,5]. In 1998, Abbas et al.  performed experiments in isolated and permeabilized rat cardiomyocytes to provide further insights into the potential activity of cardiac mitochondrial and cytosolic CrAT. These authors determined that the relative activity of a cytosolic CrAT pool represented only 5% of total cellular CrAT activity , concluding the near absence, and thus negligible functional relevance, of cytosolic localized CrAT. However, in a recent review, Alrob and Lopaschuk  estimated that the predicted cytosolic CrAT activity considered insignificant by this previous work  was capable of replenishing the cytosolic M-CoA pool 4–5 times per minute, provided that the cytosolic CrAT enzyme could catalyze the reverse reaction to form acetyl-CoA. This relationship and historical perspective had yet to be directly tested until Lopaschuk and colleagues performed the work published recently in the Biochemical Journal .
In an elegant study design, the authors investigated the existence, and potential reverse activity, of cytosolic CrAT using a variety of approaches . They provide compelling evidence that reverse CrAT (rCrAT), and in particular cytosolic rCrAT, exists in the cardiac tissue where it is capable of reforming cytosolic acetyl-CoA. As a substrate for acetyl-CoA carboxylase (ACC), this cytosolic acetyl-CoA is therefore suggested to lead to the production of M-CoA, a potent allosteric inhibitor of CPT-I. Altogether, this pathway is proposed as a mechanism in which CrAT can further promote carbohydrate flux and glucose utilization at the expense of fatty acid oxidation, ultimately enhancing cardiac efficiency. This effect may be of functional relevance specifically within the heart, as the maximal homogenate activity of cardiac CrAT and rCrAT were 9- and 6-fold greater than that determined within the liver of the same animals. In support of this data implying the importance of rCrAT within cardiomyocytes, the activity of ACL was 2.5-fold lower in the heart compared with the liver, further indicating cytosolic rCrAT as an alternative source of acetyl-CoA in states of attenuated synthesis from the traditional citrate pathway. Moreover, while the cytosolic rCrAT activity could only be calculated and not directly measured due to mitochondrial breakage during fractionation, the estimated rCrAT activity (∼1.5 nmol/min/mg protein) in cardiac tissue represented 4.6% of total activity within the heart, remarkably similar to previous estimates of cytosolic CrAT . Altogether, these data suggest that rCrAT could provide a relevant source of cardiac cytosolic acetyl-CoA, and ultimately M-CoA, implicated in the inhibition of CPT-I.
To examine the biological relevance of submaximal CrAT flux, forward and reverse CrAT kinetic assays were also performed, establishing an apparent Km of 66 µM CoA for cardiac rCrAT and an apparent Km of 54 µM carnitine for cardiac CrAT. Furthermore, to address the concern of mitochondrial contamination in cytosolic fractions, Altamimi et al. performed experiments examining CrAT and rCrAT activity using mild subcellular fractionation and digitonin lysis procedures on mouse hearts, rat hearts, and H9C2 myoblasts. This yielded comparable results with varying lesser degrees of mitochondrial contamination, suggesting that the calculated rCrAT was not an artifact from matrix-derived CrAT. Moreover, while these data suggest a role of cytosolic CrAT in cardiac metabolism, the authors  further examined the regulation of CrAT in various models of metabolic perturbations to solidify this relationship. In support of the supposition that CrAT is an important regulator of M-CoA concentrations, in a model of attenuated M-CoA synthesis (ACC2−/− mice), CrAT levels and activity were lower, while conversely CrAT was increased following the consumption of a high-fat diet, a situation associated with a decreased acetyl-CoA/CoA ratio. Altogether, Altamimi et al.  provide a compelling and intriguing argument to suggest that cytosolic CrAT activity is important for substrate selection during various metabolic perturbations.
Altamimi et al.  present insightful data that cytosolic rCrAT may, in theory, be sufficient to produce cytosolic acetyl-CoA; however, several limitations, which the authors acknowledged, hinder our understanding of biological flux through this pathway. As a near-equilibrium reaction, the direction of flux is expected to be predominantly driven by substrate–product concentrations. A major technical limitation when trying to extrapolate in vitro kinetic analysis to ‘biological relevance’ is determining the concentrations of substrates and products within the matrix and cytosolic compartments. Unfortunately, this limitation extends to all enzymatic analysis, and while precise quantification of metabolites localized within each cellular compartment (matrix vs. cytosol) is a current methodological challenge, it is believed that cytosolic carnitine content remains substantially greater than that of acetylcarnitine [8,9]. Thus, given biological substrate–product inhibition, the rate of cytosolic rCrAT flux in vivo may be lower than the reported maximal 4.6%. Moreover, the apparent Km for CrAT (54 µM carnitine) is well below estimated intramuscular carnitine content, and therefore, carnitine concentrations may be saturating the forward CrAT reaction. In contrast, the apparent Km for rCrAT (66 µM CoA) appears greater than estimates of cytosolic CoA content [7,10]. It is, therefore, possible that the forward cytosolic CrAT reaction may occur as the predominant direction of enzymatic flux, which would challenge the notion that rCrAT occurs biologically. Therefore, in the future, it is imperative to establish the flux rates of cytosolic-specific rCrAT in the presence of biological substrate and product concentrations; however, before this can be accomplished, accurate estimates of cytosolic metabolite concentrations need to be determined. Despite these limitations, Altamimi et al. have provided compelling evidence for the presence of cytosolic rCrAT.
The role of CrAT in regulating cardiac metabolism is an intricate relationship to which knowledge is limited, and thus, Altamimi et al.  should be commended for their thought process and detailed experimental work. If physiologically possible, the cytosolic reverse CrAT flux to produce acetyl-CoA, a substrate for M-CoA production and thus CPT-I inhibition, could have implications in regulating cardiac substrate selection and augmenting cardiac function. However, at this time, it remains unclear the extent to which cytosolic reverse flux can occur, and until current knowledge gaps can be bridged, the translation of in vitro activity to in vivo flux remains a challenge.
The Authors declare that there are no competing interests associated with the manuscript.