1. Reviewer #3 (Public Review):

    Volatile anesthetics (VA) are thought to cause developmental defects in newborns and the authors previously studied the metabolic consequences of VA on newborn mice. Surprisingly, they found VA exposure rapidly and dramatically dropped circulating levels of the ketone beta-hydoxybutyrate (BHB). Newborn mice use ketones as energetic substrates (compared to glucose in weaned animals) so perturbing ketone metabolism could underpin some of the detrimental side-effects of VA. Therefore, the authors sought to determine why VA cause this drop in ketone availability in newborns.

    The authors first found that multiple VAs rapidly (half maximal effect occurs in ~10min) and at subanesthetic doses decrease BHB levels from ~2mM to <1mM in newborn but not in older (older than P19) mice. Extended VA exposure (>60min) also caused a decrease in circulating glucose. BHB levels could be rescued by IP injection prior to anesthesia. Why do VAs cause this effect? Ketones are known to be produced by fatty acid oxidation in the liver. The authors therefore indirectly assessed fatty acid oxidation by measuring levels of acylcarnitines (an intermediate metabolite in fatty acid oxidation) in newborn livers after VA treatment and found lower levels of acylcarnitines consistent with lower levels of fatty acid oxidation in the liver upon VA treatment. Pharmacologically inhibiting fatty acid oxidation could also drop BHB levels in newborn plasma as well. Thus, the authors provide compelling evidence that VA exposure blocks fatty acid oxidation and ketogenesis in the liver of newborns and this underlies the drop in BHB in the circulation.

    The authors next asked why VAs decreased fatty acid oxidation. VAs are thought to inhibit the electron transport chain (ETC) which would cause redox imbalances (particularly in the NAD/NADH ratio) that could lead to altered TCA cycle metabolic activity that could potentially impact fatty acid oxidation. The authors therefore indirectly tested this hypothesis by measuring TCA cycle intermediates and did by VA exposure altered newborn liver levels of several TCA cycle metabolites including citrate. Citrate is metabolized by the enzyme ACLY to generate cytosolic Ac-CoA which is used by the enzyme ACC to produce malonyl-CoA, an intermediate in lipid synthesis. Malonyl-CoA is also known to inhibit the production of acylcarnitines and fatty acid oxidation. Therefore, the higher levels of citrate in VA exposed livers prompted the authors to determine if VA exposure specifically in neonates increased malonyl-CoA and if this blocked fatty acid oxidation and ketogenesis. The authors measured malonyl-CoA in newborn livers and observed an increased upon VA exposure. ACC inhibitions have been developed and the authors found that ACC inhibition (which presumably would prevent malonyl-CoA formation) could partially rescue the drop in BHB brought on by VA exposure in newborns. Thus, this study delineates how altered fatty acid oxidation and ketogenesis in the liver underlies the drop in BHB elicited upon VA exposure and opens the door to future studies determining if the drop in BHB contributes to newborn sensitivity to VAs and future studies elucidating exactly how VA exposure alters the TCA cycle and citrate metabolism to block fatty acid oxidation.

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  2. Reviewer #2 (Public Review):

    Here the author reported that Volatile anesthetics VA induce a rapid depletion of circulating ß-HB and the induction of hypoglycemia by VA in neonates, but not in adults. The phenomenon is very interesting and robust, however it has already been described. Whats new here is that through a metabolomics analysis they demonstrate a role of ACC and CPT1 in this phenomenon. Intermediates of the TCA cycle are reduced as would be expected and this is interesting, but chiefly descriptive, and not mechanistic. The key question what causes these derangements in TCA cycle and for sure it's altered enzymatic activity but again what accounts for these and that questions answered would get at the mechanism, but this study here remains descriptive. Is this a cell autonomous effect? For example could you replicate this in a dish with isolated hepatocyte or myotubes from neonates versus adults?

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  3. Reviewer #1 (Public Review):

    Stokes, et al. describe the effects of isoflurane on metabolism in post-natal day 7 mice, and older mice. They demonstrate that blood levels of glucose and ß-hydroxybutyrate fall quickly in response to isoflurane, and that the magnitude of the decrease increases with the length of the exposure. Mice 30 days post-natal do not exhibit these changes in response to isoflurane. The authors document the much higher circulating levels of ß-hydroxybutyrate in the post-natal day 7 mice, highlighting the importance of this substrate for supporting the energetics of the developing brain. Important control experiments, administering 100% oxygen without anesthetic to post-natal day 7 mice, as well as administering anesthetics to 30 day old mice on a ketogenic diet, did not result in significant decreases in glucose and ß-hydroxybutyrate blood levels. Remarkably, they observed significant decreases in response to very small, subanesthetic doses of isoflurane, halothane and sevoflurane in post-natal day 7 mice. Administration of bolus glucose corrects the glucose level for these mice under anesthesia, but not the level of ß-hydroxybutyrate, while administration of bolus ß-hydroxybutyrate corrects both levels.

    The authors then proceed to a series of measurements in an attempt to determine a direct target of volatile anesthetics on metabolism, focusing on hepatic metabolism. This is something of a Procrustean bed, given that the there is ample evidence that volatile anesthetics affect a large number of different membrane bound processes. Nonetheless, these experiments provided valuable data demonstrating anesthetic induced decreases in fatty acid oxidation. This reviewer finds the arguments regarding impairment of the citric acid cycle a bit unconvincing: 7 and 30 day old mice exhibit the same increase in citrate and isocitrate levels, yet only the 7 day old mice show elevated lactate levels. Rather than exhibiting increased metabolic flexibility, as the authors suggest, this finding seems to argue that 7 day old mice have less metabolic flexibility. The authors demonstrate that several perturbations of fatty acid metabolism can result in depression of ß-hydroxybutyrate, leading them to focus on carnitine palmitoyl transferase-1. They demonstrate that inhibition of this enzyme produces a decrease in ß-hydroxybutyrate; however, they also find that mice with a knockout of this enzyme do not have decreased ß-hydroxybutyrate levels.

    The authors are circumspect in their conclusions regarding the targets responsible for the metabolic changes observed in neonatal mice in response to anesthetics. They do correctly highlight the potential importance of these metabolic effects. It will be crucial for future research to determine whether these effects can be directly correlated to measures of cerebral function during anesthesia, e.g., EEG or evoked potentials, and to measures of neuropathological change. Of great interest to clinicians will be demonstration of whether co-administration of glucose or ß-hydroxybutyrate together with anesthetics can abrogate such changes.

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  4. Evaluation Summary:

    This manuscript reports that Volatile anesthetics VA induce a rapid depletion of circulating ß-HB and the induction of hypoglycemia by VA in neonates, but not in adults. The phenomenon is very interesting and robust, however it has already been described. Whats new here is that through a metabolomics analysis they demonstrate a role of ACC and CPT1 in this phenomenon.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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