Acyl-CoA thioesterase-2 facilitates β-oxidation in glycolytic skeletal muscle in a lipid supply dependent manner

Reviewer #1 (Public Review):

This study examined whether mitochondrial acyl-CoA thioesterase-2 (ACOT2) regulates mitochondrial matrix acyl-CoA levels. Acot2 deletion in murine skeletal muscle (SM) resulted in acyl-CoA build-up. When energy demand and pyruvate availability were elevated, a lack of ACOT2 activity promoted glucose oxidation. This preference for glucose over fatty acid oxidation was recapitulated in C2C12 myotubes with acute depletion of Acot2. In mice fed a high-fat diet, ACOT2 enabled the accretion of acyl-CoAs and ceramide derivatives in glycolytic SM, and this was associated with worse glucose homeostasis compared to when ACOT2 was absent. The authors suggest that ACOT2 supports CoASH availability to facilitate β-oxidation in glycolytic SM when lipid supply is modest. However, when lipid supply is high, ACOT2 enables acyl-CoA and lipid accumulation, CoASH sequestration, and poor glucose homeostasis. Thus, ACOT2 regulates matrix acyl-CoA concentration in glycolytic muscle, and its impact depends on lipid supply.

Based on the data provided in this study, the authors propose that ACOT2 regulates mitochondrial matrix acyl-CoA levels in white skeletal muscle to facilitate fatty acid oxidation β-oxidation. However, I do not believe the data supports this concept, since ACOT2 deletion actually increased fatty acid oxidation in the mitochondrial JO2 studies. In addition, there are some problems with the experimental data that the authors need to address. This includes the experimental conditions used to assess JO2 in the mitochondria, and not using Cre control mice.

Reviewer #2 (Public Review):

Summary:

The manuscript from Bekeova et al. entitled "Acyl-CoA thioesterase-2 facilitates P-oxidation in glycolytic skeletal muscle in a lipid supply dependent manner" examines whether loss of acyl-CoA thioesterase-2 (ACOT2) in the mitochondrial matrix of skeletal muscle alters mitochondrial fatty acid metabolism. The authors generate data demonstrating that under normal chow conditions, loss of ACOT2 increases mitochondrial respiration of long-chain fatty acid, but also increases susceptibility to the build-up of metabolic intermediates. However, during short-term high-fat feeding (7 days), mice with knockout of skeletal muscle ACOT2 had better glucose and insulin tolerance. Interestingly, skeletal muscle ACOT2 knockout mice on chow and high-fat diet utilized more glucose during the active (dark cycle) portion of the day. These data suggest that ACOT2 may be a potential therapeutic target to improve glucose homeostasis.

Strengths:

The use of creatine kinase cre recombinase to specifically target striated muscle localizes the genetic manipulation, thus increasing the rigor of these experiments by limiting potential off-site changes in ACOT2 expression. Also, the assessment of mitochondrial respiration and response to changes in energy change via the creatine kinase clamp technique is a strength. These data provide a measurement of isolated mitochondrial respiration at physiologically relevant concentrations of ATP and ADP, while also allowing for assessment of how these mitochondria respond to changes in free energy (Fisher-Wellman et al. 2018). The indirect calorimetry data provides systemic physiological context to the striated muscle-specific genetic manipulation, while also allowing for the examination of how this change in skeletal muscle ACOT2 impacts systemic responses to different energy challenges. Finally, the extensive metabolomics, transcriptomics, and lipidomics analysis, not only provides a wealth of data but is used to further the authors' investigation of skeletal muscle ACOT2 activity in mitochondrial fatty acid oxidation and glucose homeostasis.

Weaknesses:

Several general confounding factors exist in the experimental design that could potentially impact the interpretation of the observed outcomes. First, all mice were housed at housing temperatures (22C) below the thermoneutral zone, which has been well described by many investigators to result in dramatically increased energy expenditure. Changes in total and resting energy expenditure could alter the skeletal muscle and systemic utilization of lipids, response to high-fat diet, and glucose homeostasis. Second, no dietary control was observed in these experiments. While this did not impact outcomes when the diets were not compared, once the authors began to compare normal chow to high-fat diet, numerous differences in the composition of these diets could impact the outcomes. Third, the extended food withdrawal before the glucose- and insulin tolerance tests puts the mouse in a state of extreme energy stress more akin to starvation than fasting, which can negatively impact outcomes (Ayala et al. 2010, Virtue & Vidal-Puig 2021). Fourth, the use of the Seahorse platform for the assessment of respiration of isolated mitochondria is highly debatable (Schmidt et al. 2021), particularly when the investigators also used high-resolution respirometry specifically designed for the purpose of measuring isolated mitochondrial oxygen consumption. Importantly, the use of the Seahorse platform to assess cellular respiration in this investigation is quite appropriate. Finally, while the authors present data demonstrating that ACOT2 expression is highest in Type I fibers compared to the various Type II fiber types, a large number of the experiments are performed in a muscle that is primarily composed of Type II fibers. The authors briefly acknowledge this limitation. But, is important for the reader to keep this in mind when trying to consider how these findings would translate to humans.

Impact:

The authors have generated data that implicates skeletal muscle mitochondrial coenzyme A handling as a therapeutic target in the improvement of glucose homeostasis. While the exact role of increased tissue lipid burden on insulin action, glucose uptake, and substrate metabolism is still debated, the association between increased tissue lipid and impaired tissue- and systemic glucose handling is very strong. The data herein suggest that ACOT2 represents a pharmaceutical target to improve systemic glucose homeostasis in the population with obesity.

Reviewer #3 (Public Review):

Cells can oxidize diverse substrates in the mitochondria to sustain cellular energy metabolism. However, all of these substrates require covalent thioester linkage to coenzyme A (CoA). Thus, multiple energy metabolism substrates could potentially compete for a limited pool of mitochondrial CoA. Cells encode a set of mitochondrial acyl-CoA thioesterases (ACOTs) that free CoA up by removing attached substrates. The authors hypothesized that ACOT2, a mitochondrial ACOT with a preference for long-chain acyl-CoA substrates that arise during the oxidation of lipids as a fuel source, could regulate the balance of substrates used in the mitochondria by reducing the oxidation of lipids by removing them from CoA and freeing the mitochondrial pool of CoA for use by other substrates.

To test this hypothesis, the authors generated mice with loss of ACOT2 in the skeletal muscle, where this is most expressed, and assayed the CoA composition of muscle and their glucose/fatty acid catabolism in mice that were challenged with different diets, fasting or exercise to expose the muscle to different substrates conditions. These experiments were complemented with biochemical analysis of mitochondria isolated from the muscle of control and ACOT2 animals exposed to a variety of substrates and challenged with different simulated energy demands.

On the basis of these convincing experiments, the authors argue that loss of ACOT2 both in vivo and in vitro interestingly increases glucose oxidation, while not increasing oxidation of lipids. This is particularly surprising as the CoA competition model would predict that ACOT2 loss would increase lipid oxidation while hindering glucose oxidation. The authors argue that ACOT2 facilitates lipid oxidation due to ACOT2 reversal of lipid ligation to CoA preventing feedback inhibition of the lipid oxidation pathway that occurs when lipid supply outstrips the ability of the lipid oxidation pathway to metabolize the lipids. These findings will be valuable for the field of metabolism providing insight into how ACOTs regulate substrate catabolism in cells and tissues.