Hepatic conversion of acetyl-CoA to acetate plays crucial roles in energy stresses

Reviewer #1 (Public Review):

The authors investigate the roles of ACOT12/8 in the production of acetate by the liver. They observe that acetate concentration parallels ketone concentrations during fasting and T1DM. They show that acetate is produced from fatty acids in hepatocytes. They also provide data from human subjects who were classified as either "healthy" or "diabetic," but there is no other characterization or description of these people, making it difficult to ascertain the context by which they were studied. Nevertheless, these findings could be gleaned from the literature, and yet there remains surprising uncertainty regarding the mechanism of acetate production by the liver. The authors use ShACOT12/8 and liver-specific ACOT12/8 knockout mice to demonstrate that these acetyl-CoA hydrolases are largely necessary for acetate production. There is data on this role for ACOTs in the literature, but they have yet to be widely studied. Using a 3H-palmitate assay, the authors then find that loss of these ACOTs inhibit fatty acid oxidation and propose that the mechanism involves scavenging CoA, analogous to the canonical role of ketogenesis. The idea is plausible but only partially proven. A related finding is that loss of these ACOTs inhibit ketogenesis, which the authors attribute to the loss of function of HMGC2S, partially through acetylation. These mechanisms suffer some limitations based on the cytosolic and mitochondrial compartmentation of the two processes, but the observations appear sound. Finally, the authors try to demonstrate that hepatic ACOT-mediated acetate production is necessary for normal motor function. The tracer data used to support the importance of acetate metabolism do not include loss of function models and generally need to be reported more transparently. Conceptually, one may be skeptical of the rather dramatic loss of motor function in the context of a relatively minor circulating nutrient. This may be a significant finding but requires more supporting evidence. Overall, the authors convincingly show that ACOT12/8 are critical for hepatic acetate production in mice, which will be helpful for the field, but the ramifications will require further investigation.

Reviewer #2 (Public Review):

Catabolic conditions lead to increased formation of ketone bodies in the liver, which under these conditions play an important role in supplying energy to metabolically active organs. In this manuscript, the authors explore the concept of whether and to what extent hepatic formation of acetate might contribute to energy supply under metabolic stress conditions. The authors show that patients with diabetes have increased acetate levels, which is explained as a consequence of the increased fatty acid flux from adipose tissue to the liver. This is confirmed in a preclinical model for type 1 diabetes, where acetate concentrations are in a similar range to ketone bodies. Acetate concentrations also increase under physiological conditions of fasting. Using stable isotopes, the authors show that palmitate is used as the primary source for acetate production in primary hepatocytes. Using cell culture studies and adenoviral-mediated knockdown in mice, it can be shown that the conversion of acetyl-CoA to acetate is catalyzed in peroxisomes by acyl-CoA thioesterase8 (ACOT8) and after transport of citrate from mitochondria and subsequent conversion to acetyl-CoA in the cytosol by ACOT12. Remarkably, ACOT8/12 not only regulate the formation of acetate but play a crucial role in the maintenance of cellular CoA concentration. Accordingly, depletion of ACOT8/12 activity leads to a reduction of other CoA derivatives such as HMG-CoA, which resulted in the inhibition of ketone body synthesis. In diabetic mice, ACOT 8 or ACOT12 knockdown appears to lead to some limitations in strength and behavior.

In summary, the authors clearly demonstrate that hepatic release-mediated by ACOT8 and ACOT12-determines the plasma concentration of acetate. This is a very remarkable observation, since most studies assume that short-chain fatty acids in plasma are primarily generated by fermentation of dietary fiber by intestinal bacteria. The authors demonstrate in very well performed studies the metabolic changes that result from impaired thiolysis. On the other hand, the ACOT12 phenotype has been demonstrated in a recently published study (PMID: 34285335). In this study, ACOT12 deficiency caused NAFLD, thus it would be worth to determine whether deficiency of ACOT12 and/or ACOT8 promotes de novo lipogenesis under the conditions of the present study. As a further limitation, it should be noted that the relevance of acetate production for the energy supply of peripheral organs including the central nervous system could not be clearly demonstrated. For instance, impaired ketone body production due to impaired CoA availability could affect the metabolic activity of various organs. Moreover, the human cohort is not very well described, e.g. it is unclear whether the patients have type 1 or type 2 diabetes.

Reviewer #3 (Public Review):

Wang et al. investigated the role of acetate production, a byproduct of fatty acid oxidation, in the context of metabolic stressors, including diabetes mellitus and prolonged fasting. Mechanistically, they show the importance of the liver enzymes ACOT8 (peroxisome) and ACOT12 (cytoplasm) in converting FFA-derived acetyl-CA into acetate and CoA. The regeneration of CoA allows for subsequent fatty acid oxidation. Inhibiting the generation of acetate has negative motor and behavioral consequences in streptozocin-treated mice, which are mitigated with acetate injection.

This paper's strengths include using multiple mouse models, metabolic stressors (db/db-/-, streptozocin, and prolonged starvation), numerous cell lines, precise knockout and rescue experiments, and complimentary use of mass spectrometry and nuclear magnetic resonance analytical platforms. The presented data support the conclusions of this paper, but some aspects need to be clarified.

For example, for all animal studies, please list the age and sex of the animals at the time of the experiments. Sex and age are important biological variables that can affect metabolism, and such characteristics are needed when comparing results from different research groups.

In clinical medicine, common ketones that are measured are acetoacetate, beta-hydroxybutyrate, and acetone. However, the data presented here suggest the importance of measuring acetate when patients present with ketoacidosis in uncontrolled diabetes or starvation.