Ketone bodies as an energy source: regular‐grade, premium, or super‐fuel to power the mitochondrial engine?

Ketone bodies (KB) are lipid-derived molecules that are naturally produced by the liver during starvation, low carbohydrate (CHO) intake and in response to prolonged exercise. Under these conditions, KBs serve as an alternative fuel source for peripheral tissues such as the brain, heart and skeletal muscle. The three KBs are acetone, acetoacetate (AcAc) and beta-hydroxybutyrate (βHB), with the latter two being the most physiologically relevant. Nutritional ketosis (blood KBs >0.5 mm) can be achieved through adherence (over days to weeks) to a very low CHO ketogenic diet (KD) (Pinckaers et al. 2017). As a response to low CHO intake, the body transitions to burning fat as a primary fuel source, with the subsequent production of KBs. Alternatively, the use of exogenous ketone supplements (e.g. ketone salts and ketone esters) can induce an acute state of ketosis within minutes without the requirement of CHO or energy restriction. Currently, there is substantial and revamped interest in the application of KDs and the use of exogenous ketone supplements within the athletic community as a means to alter fuel metabolism and improve performance. Traditional fuelling strategies to support exercise performance have focused on optimizing CHO availability, as stores of this precious fuel substrate are finite. However, alternative fuelling strategies based on a high fat KD have been suggested to enhance the body's reliance on fat as fuel, spare finite CHO stores, and increase the availability of KBs as an alternative and additional fuel source for working muscles. Therefore, adherence to a KD and increased KB availability is thought to have the potential to shift the preference and hierarchy of fuel substrates for mitochondrial energy provision away from CHO metabolism and towards FFA and KB oxidation. Alternatively, as the rise in endogenously produced KBs with a KD is dependent upon the depletion of muscle and liver CHO stores, there may be a reduction in the capacity to utilize CHO as fuel, thereby compromising moderate- to high-intensity exercise performance. Orally ingested ketone supplements can rapidly increase circulating KB concentration in the presence of fully loaded muscle and liver CHO stores, thereby introducing an additional fuel source not normally available. However, although the KD and exogenous ketone supplements both increase circulating KB availability, they lead to different metabolic states and appear to have distinct effects on substrate availability and metabolism. Therefore, more research is required in order to better understand what order KBs fall within the mitochondrial fuel substrate ladder and how they influence metabolic pathways. Although the hierarchy of energy substrate metabolism and utilization in the body is relatively well understood, the role of KBs as a driver of mitochondrial respiration (MR) remains unclear, and even more so when seeking to understand their contribution as a fuel source in the presence of CHO, lipids and their derivatives. In a recent issue of The Journal of Physiology, Petrick et al. (2020) set out to determine the ability of βHB and AcAc to drive MR in isolated mitochondria and permeabilized fibres (PmFbs). Through a number of elaborate in vitro experiments, they investigated the extent to which KBs contribute to mitochondrial energy production in both rodent and human skeletal muscle. Under physiological conditions, KBs will never be the only fuel substrate available for driving MR. Therefore, they tested the ability of KBs to drive MR alone, as well as in the presence of CHO-derived pyruvate. In the animal experiments, the group surgically removed red gastrocnemius tissue (RG), and left ventricle tissue (LV) from rats either at rest or following an acute bout of exercise. In a small human experiment, muscle biopsies were obtained from the vastus lateralis from four highly trained, healthy, active young adults following a 4-h fast, with the samples subsequently prepared for permeabilization of fibres and mitochondrial isolation. The researchers exposed these PmFbs and isolated mitochondria to various derivatives of CHO and lipid metabolism, notably CHO-derived pyruvate, ADP, malate and palmitoylcarnitine. Ultimately, they determined that the ability of KBs to drive MR in isolated mitochondria is minimal compared with CHO-derived substrates. In both the presence and absence of ADP, pyruvate was shown to be the main driver of MR compared to βHB and AcAc in both LV and RG isolated mitochondria. This result was found regardless of the order of addition of the substrates. Therefore, even under situations where isolated mitochondria from the LV and RG were initially primarily fuelled by KB, the subsequent addition of pyruvate resulted in a ∼3- to 4-fold increase in MR. This increase was ∼10-fold in human skeletal muscle under the same conditions. The consensus to be made from these isolated mitochondria experiments is that the ability of KBs to drive MR is minimal and virtually undetectable in the presence of pyruvate, following a single dose of KBs. Even though there was no direct assessment of the individual contribution of each substrate to MR, ketone bodies cannot stimulate MR above pyruvate under these conditions. PmFbs were also analysed, as they better depict intracellular structure compared to isolated mitochondria. In this set of experiments, KB and pyruvate titrations were conducted in order to determine the influence of their concentrations on their respective utilization during MR. In the LV tissue, KBs were only able to maximally increase MR by ∼30% of the level achieved by pyruvate. At saturating concentrations of KBs (10 mm), the addition of a submaximal concentration of pyruvate (100 μm) had no impact on their ability to drive MR. Notably, these experiments were done with supraphysiological concentrations of KBs, orders of magnitude greater than the concentration of KBs that would exist under normal physiological conditions. When the concentration of KBs were brought down to physiologically relevant concentrations (100 μm), submaximal concentrations of pyruvate were able to blunt the ability of βHB to drive MR. However, this effect was not observed with AcAc. Therefore, the submaximal presence of pyruvate can preferentially drive MR to the detriment of βHB but does not outcompete AcAc. KB contribution to energy production incrementally declined as increasing amounts of pyruvate were titrated into the intramuscular environment. With saturating concentrations of pyruvate, KBs were unable to further stimulate MR beyond its pyruvate-supported activity. The experiments on PmFbs demonstrate that pyruvate is clearly the prioritized substrate in the context of the in vitro mitochondrial environment, which may be either outcompeting or causing product inhibition of KBs. Similar to these experiments using tissues from rested rats, there was no increase in the ability of KBs to drive MR in the LV and RG of exercised rats. Every which way the researchers manipulated the protocol, KBs made little to no contribution towards mitochondrial ATP production in PmFbs and isolated mitochondria. These results offer a novel perspective into the mechanistic role of KB metabolism and utilization in the presence of CHO-derived substrates like pyruvate. Petrick et al. (2020) suggested that when pyruvate and KBs are both present in the mitochondria, pyruvate metabolism supersedes and inhibits ketolysis via product inhibition by which by-products of pyruvate oxidation, such as acetyl-CoA and NADH, inhibit ketolytic enzymes. However, the in vivo condition represents a more complex environment for interaction and competition between substrates, particularly during exercise. Further, there is also the added complication of differences that might arise depending on whether the KBs are sourced exogenously through supplementation or endogenously through KD. This in vitro work by Petrick et al. (2020) examining the extent to which KBs drive MR at the mitochondrial level is in line with a recent in vivo investigation by Dearlove et al. (2020) in which a stable isotope-labelled ketone monoester supplement was applied at various doses to determine whole-body exogenous βHB oxidation rates during exercise. Irrespective of exercise intensity, the contribution of exogenous βHB oxidation rates to energy expenditure was very low (<5%). Despite an initial seminal study showing that co-ingestion of ketone monoester + CHO in endurance athletes led to improved time trial performance compared to CHO alone (Cox et al. 2016), results from recent studies suggest that raising KB availability via ketone supplements produces equivocal effects on exercise performance (Margolis & O'Fallon, 2020). While, there is certainly more work to do, e.g. exploring the intricacies of different types of ketone supplements, the effects of long-term exogenous ketone supplementation, and more prolonged ketogenic diet interventions on KB uptake and utilization as a fuel source, another interesting avenue of research is the role of KBs in enhancing exercise recovery and/or adaptation. For example, KBs appear to regulate whole-body and muscle protein turnover, inflammatory processes, oxidative stress, and muscle regeneration via enhancing satellite cell activation and differentiation. They may therefore influence post-exercise recovery processes and/or adaptive tissue remodelling in response to prolonged exercise training. This is an exciting avenue for future research. Overall, while KBs are a critical fuel source under certain circumstances, the work of Petrick et al. (2020) suggests that in vitro, they are more of a regular-grade fuel for the mitochondria that can be overtaken by CHO- and lipid-derived substrates. These findings may shift the focus away from KBs as an energy source to fuel exercise and reorient the spotlight back on CHO as the true exercise superfuel. No competing interests declared. Both authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. None. The authors would like to thank Drs Arthur Cheng and Tyler Churchward-Venne for editing the article.

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