Branched-chain keto acids (BCKA) metabolism involves several well-regulated steps within mitochondria, requires cofactors, and is modulated according to the metabolic status of the cells. This regulation has made it challenging to utilize in vitro approaches to determine the contribution of branched-chain amino acid oxidation to energy production. These methodological issues were elegantly addressed in a recent publication within the Biochemical Journal. In this issue, Goldberg et al. [Biochem. J. (2019) 476, 1521–1537] demonstrated in a well-designed system the dependence of ATP and bicarbonate for BCKA full oxidation. In addition, the utilized system allowed the authors to characterize specific biochemical routes within mitochondria for each BCKA. Among them, a quantitative analysis of the participation of BCKA on mitochondrial flux was estimated between tissues. These findings are milestones with meaningful impact in several fields of metabolism.
Branched-chain amino acids (BCAA: l-valine, l-leucine, and l-isoleucine) are a group of essential amino acids which have in common branched-chain aliphatic structure. The denomination of ‘essential' is because of the dependence of diet ingestion since mammalian metabolism, including humans, is incapable of synthesizing these amino acids. BCAAs are necessary to stimulate the synthesis of new proteins and also participate in signaling processes within diverse cells. In an alternative and poorly characterized process, BCAAs are metabolized within mitochondria, which in turn eventually become available to the tricarboxylic acid (TCA) cycle . Briefly, the first reaction is the transamination of BCAAs structures by branched-chain amino acid transaminase (BCAAT), which requires α-ketoglutarate to receive nitrogen and produces glutamate. This first reaction is reversible and produces three metabolites collectively called branched-chain keto acids (BCKA), such as ketoisocaproate (KIC), ketomethylvalerate (KMV), and ketoisovalerate (KIV) from leucine, isoleucine, and valine, respectively. The next reaction, catalyzed by BCKA dehydrogenase complex (BCKDH), is irreversible and yields isovaleryl-CoA, α-methylbutyryl-CoA, and isobutyryl-CoA from KIC, KMV, and KIV, respectively . Although the complete sequence of reactions is well-described, the integration between BCKA catabolism with mitochondrial function has been poorly demonstrated. This lack of detail arises from some unique properties of BCKA metabolism, such as the dependence of ATP and bicarbonate in the intermediate reactions, which are not necessary for fatty acids and glucose-pyruvate metabolism. In the last few decades, amino acid metabolism has been proposed to be involved in the progression of diseases such as diabetes, cancer, and heart failure. In addition, patients with nitrogen retention regimes (i.e., chronic kidney disease), may be benefited from the use of BCKA treatment. Therefore, the aforementioned scenarios highlight the importance of fully understanding of BKCA metabolism in integrated systems as well as the integration with other substrates (i.e. glucose, fatty acids).
In a recent study published by Goldberg et al.  in Biochemical Journal, the dependence of ATP and bicarbonate for BCKA oxidation was addressed in an integrated system. The authors have focused on physiological ATP/ADP ratios, by using a recycling clamp with creatine/phosphocreatine, and the participation of bicarbonate to highlight the dependence of BCKA metabolism of these molecules. Their work is based on the theoretical underestimation of the mitochondrial utilization in the previous studies because of experimental limitations. In addition, using several other mitochondrial substrates (pyruvate, malate, glutamate, succinate, and octanoyl-carnitine), it was possible to estimate the participation of BKCA to total mitochondrial flux capacity and reactive oxygen species (ROS) production.
In this work, several interesting findings were observed, most notably the importance of ATP free energy (−ΔGATP) and bicarbonate in the mitochondrial matrix to maintain BCKA flux. This finding has important implications, for example, in some extreme metabolic states, such as starvation, exercise or cancer, when protein catabolism is stimulated. As BCAAT is a reversible enzyme working to keep relative equal concentrations of BCAA and BCKA, it is possible that BCAA catabolism may represent a viable process for mitochondria to maintain aerobic energy production in a resting cell where ATP levels are not limiting. However, according to the recent data published by Goldberg et al. , a reduction in BCKA oxidation occurs within the liver once ATP/ADP ratios and PCr/Cr increase. Since skeletal muscle and heart did not present the same dependence of ATP free energy to oxidase BCKA than liver, the use of BCKA during exercise/stressed situations, where ATP/ADP and PCr/Cr ratios presumably decrease, and NADH concentrations likely increase inhibiting key enzymes of BCKA metabolism, remains debatable. This view may also help explain the observed minimal contribution of BCKA metabolism within overall heart mitochondrial bioenergetics, as this tissue is never truly ‘resting’. The possible decreased reliance on BCKA metabolism in these situations could also represent a means to maintain α-ketoglutarate concentrations, preventing depletion of this TCA cycle intermediate and avoiding unnecessary anaplerotic reactions to replenish TCA cycle, especially in an active muscle. In addition, while the present study, and previous work , has demonstrated that BCKA inhibits maximal respiration, this likely represents a finding of limited biological relevance, as BCKA metabolism is only stimulated in resting conditions when electron transport chain flux is limited. Nevertheless, clearly, BCKA metabolism is highly regulated and integrated within overall mitochondrial metabolism.
Interestingly, BCKA oxidation in liver mitochondria presented a unique response to −ΔGATP. From the lowest −ΔGATP value (12.9 kcal/mol) to an intermediate value (14.08 kcal/mol), BCKA-stimulated oxygen consumption was virtually the same. In contrast, higher −ΔGATP decreased mitochondrial O2 consumption, while in the heart and muscle BCKA-supported respiration was stimulated in this environment. The unique −ΔGATP dose-dependence on the liver and peripheral mitochondria is difficult to place into biological context. As fasting would be expected to decreased PCr/Cr ratios, which based on the data by Goldberg et al. , would promote liver utilization of BCKA and prevent peripheral tissue metabolism, when the opposite metabolic situation would appear favorable (i.e. liver production of BCKA and peripheral utilization). Nevertheless, the difficulties of extrapolating in vitro data to in vivo situations are numerous, therefore, although the tissue-dependent responses are interesting and unique, these findings remain to be placed into a broader relevance.
The first reaction of BCKA metabolism is catalyzed by BCKDH, which also produces the anion radical superoxide. Several necessary reactions to fully oxidized BCKA result in the production of ROS, an issue that was also addressed in the current study. Goldberg et al. , found that even with relatively lower oxygen consumption, BCKA oxidation produced more ROS per O2 consumed (% leak fraction) than multi substrates oxidation. In this respect, once mitochondria rely on BCKA metabolism for the energetic demand of cells, it could be hypothesized that ROS emission would increase, which could activate diverse (mal)adaptive processes. Additionally, higher relative ROS production under BCKA oxidation could be a negative feedback to BCAA oxidation, since BCAAT is sensitive to the redox environment . However, the integration between mitochondrial redox control of BCAA catabolism still needs to be elucidated. The authors further characterized the effects of BCKA on mitochondrial bioenergetics by examining individual keto acids. BCKA share the same enzymatic complex in the first reaction, however, after isovaleryl-CoA, α-methylbutyryl-CoA, and isobutyryl-CoA production by BCKDH, the enzymes involved in the following reactions to produce the end metabolites to enter in the TCA cycle are different. In this scenario, when analyzed separately, BCKA showed some interesting unique properties. Specifically, since the KIC oxidation only produces acetyl-CoA, the intermediate reactions are under acetyl-CoA allosteric regulation, which could decrease KIC catabolism and underestimate the participation of this keto acid in mitochondrial metabolism. In this work, Goldberg et al.  insightfully resolved this issue by adding malate, which can be condensed with acetyl-CoA to produces citrate by citrate synthase enzyme; or adding carnitine, which by the action of carnitine acetyltransferase (CrAT) decreases the mitochondrial acetyl-CoA concentration. Therefore, the allosteric regulation of KIC metabolism by acetyl-CoA suggest that under energy overload (i.e. high-fat diets or obesity), KIC catabolism, and possibly leucine, would be decreased by inhibition of the key enzymes in the process. This regulation could explain, at least partially, the observation of higher blood circulating BCAA observed in obese insulin-resistant people [6,7].
The work performed by Goldberg et al.  addresses important issues surrounding BCKA oxidation within mitochondria, however, some important questions remain open. Exogenous bicarbonate was used in the present study to stimulate BCKA metabolism, however, there is controversy surrounding the possibility of bicarbonate transport across mitochondrial membranes, or alternatively it has been suggested that bicarbonate transport is electrogenic and influenced by mitochondrial membrane potential, which may help explain why BCKA respiration was minimal in the presence of a smaller −ΔGATP [8,9]. Additionally, it has been demonstrated in some experiments that bicarbonate influences post-translational regulation of ETS proteins , and therefore it is still unknown if the bicarbonate effects observed in the present study are only related to BCKA oxidation or some side effects on ETS regulation and function. Importantly, in the present study bicarbonate did not affect mitochondrial respiration driven by PMGSO, which could suggest the absence of the bicarbonate effects on ETS activity. However, the use of multi substrates produces high mitochondrial respiration (∼10 000 JO2 pmol/sec/mg) which may make it difficult to detect subtle changes in mitochondrial function related to bicarbonate (∼200–400 JO2 pmol/sec/mg). Nevertheless, the present paper by Goldberg et al.  highlights the complexity of BCKA metabolism, and although some questions remain, several important issues were addressed. In the future, studies should take advantage of the experimental designs published by Goldberg et al.  to determine BCKA metabolism in certain extreme metabolic disturbs, such as diabetes, muscle waste, cancer, and nitrogen retention therapies.
The Authors declare that there are no competing interests associated with the manuscript.