All animal use was in accordance with the National Institutes of Health (NIH) Guide for the care and use of laboratory animal and conformed to the principles specified in protocols approved by the University of Kansas Medical Center and University of Minnesota Institutional Animal Care and Use Committees (IACUC).

Wild-type C57BL/6J male mice (8 – 20 weeks of age) were fed ad libitum with a normal chow diet and housed on a 12:12 dark: light cycle. Neuron-specific SCOT knockout mice were developed by crossing the Synapsin 1-Cre driver to the floxed Oxct1 allele on a C57BL/6NJ background (14). Wild-type C57BL/6J mice were used initially to establish the KB supported respiration protocol, with follow up studies focused on SCOT KO in C57BL/SNJ occurring separately. No between strain comparisons were performed. Mice were anesthetized and euthanized by phenobarbital injection and exsanguination followed by removal of the brain for isolation of mitochondria. The entire left hemisphere was transferred to pre-cooled glass Potter homogenizer with 8 mL of brain isolation buffer A (320 mM sucrose, 10 mM Tris-Cl, 1 mM EDTA, 2.5 g/L BSA). The tissue was homogenized with 4–6 strokes at 200 rpms. The resulting homogenate was centrifuged for 10 min at 1,000 x g at 4°C. Then, the supernatant was transferred to a new tube and centrifuged for 10 min at 10,000 x g at 4°C. After centrifugation, the supernatant was discarded, the mitochondrial pellet was resuspended in 6 mL isolation buffer B (320 mM sucrose, 10 mM Tris-Cl, 1 mM EDTA) and recentrifuged for 10 min at 9,000 x g at 4 °C. Afterwards, the pellet was collected and resuspended in 1600 μL of isolation buffer B. Isolated brain mitochondria were stored on ice until use.

Respiration of isolated brain mitochondria was determined using Oxygraph-2k (O2k, OROBOROS Instruments, Innsbruck, Austria). Experiments using isolated brain mitochondria from wild type C57BL/6J male mice were performed in modified MiR05 (110 mM sucrose; 60 mM K-MES; 0.5 mM EGTA; 3 mM MgCl₂; 10 mM KH₂PO₄; 20 mM HEPES; 0.1% fatty acid-free BSA; and 20 mM glucose; adjusted to pH 7.1 with KOH). All experiments were performed at 37◦C. 75–100 μg of mitochondria in buffer (25–30 μL) were added to each 2-ml respiration chamber. Substrate concentrations for respirometry experiment are shown as following: 2 mM Malate; 0.01 mM CoA; 2.5 mM Carnitine; 10 mM ADP; 1 mM αKG; 1 mM AcAc; 1 mM D-βOHB. Since KBs putatively provide reducing agents to both complex I and II, we investigated the entry path of electrons from KBs to the ETC, leveraging malonic acid (5 mM Mna, complex II inhibitor) and rotenone (0.002 mM Rot, complex I inhibitor). Following all respirometry experiments, data was normalized to the mitochondrial protein via BCA assay. Acetoacetate was synthesized as previously described (15). Briefly, ethyl acetoacetate underwent base-catalyzed hydrolysis using an equimolar amount of sodium hydroxide at 60°C, followed by neutralization with HCl, sterile filtration and storage at −80°C. Chemical purity was assessed via ¹H-NMR and concentration was quantified via UHPLC-MS/MS.

30–32 weeks old C57BL/6NJ SCOT-Control and neuron-specific SCOT-KO male mice were placed on a reverse light cycle (dark 10:00 −22:00), with ad libitum assess to water and normal chow diet. We used 30–32-week-old middle-aged mice as metabolism changes are often noticed of the middle-aged brain (16), to explore the ability of neuron cells to oxidize ketone bodies and determine the effects of ketone oxidation on acetyl CoA and energy charge in isolated brain mitochondria. Brain mitochondria were isolated as above, protein concentration determined by BCA, and 150 ug of mitochondrial protein was used. Respiration experiments were performed in the same modified MiR05 buffer as above at 37◦C and substrate concentrations were the same as above. Briefly, following additions of different fuel substrates, brain mitochondria were added, and a baseline respiration value was reached. The respiratory flux was then assessed following sequential adding substrates. After each respiration experiment, contents of the reaction wells were transferred to microcentrifuge tubes and centrifuged at 8,000 x g at 4°C for 15 minutes. The supernatant was discarded, and the pellet was collected and frozen in liquid nitrogen and stored at −80 °C until processing.

Mitochondria were homogenized using pestle with cold 0.4 M perchloric acid, 0.5 mM EGTA extraction solution containing [ ¹³C₁₀, ¹⁵N₅] ATP sodium salt (100 mM), [¹³C₁₀, ¹⁵N₅] AMP sodium salt (100 mM), [1,2-¹³C₂] acetyl-CoA lithium salt (5 mM), and [1,2,3-¹³C₃] malonyl-CoA lithium salt (5 mM) purchased from Sigma. After incubation on ice for 10 min, samples were centrifuged at 15,000xg for 15 min at 4⁰C. The resulting supernatants were neutralized with freshly prepared 0.5 M K₂CO₃, vortexed, and centrifuged at 15,000xg for 30 min at 4⁰C. Final extracts were then analyzed by LC-MS/MS as previously described, with modifications (17). Briefly, analysis of energy acetyl CoA and energy charge was performed using a Vanquish LC system. Separation was achieved on a reverse-phase C18 column (Waters Xbridge, 150 × 2.1 mm, 3.5 μm) using the following mobile phases: A) 95% water/5% methanol with 4 mM dibutylammonium acetate (DBAA, Millipore Sigma) and B) 25% water/75% acetonitrile with 4 mM DBAA. The ion-pairing mobile phase additive DBAA provided adequate chromatographic separation. Samples were separated using the following binary gradients: 0–80% B for 8 min, 80–100% B for 5 min, 100% B for 3 min, and 100–0% B for 5 min. The flow rate was 100 μL/min, with a diverter valve used for the first 2 min to minimize matrix components entering the MS. The column was maintained at 30⁰ C and the injection volume was 2 μL. The LC system was hyphenated to a Thermo Q Exactive Plus MS equipped with heated electrospray ionization. The MS was operated in positive ionization mode with PRM mode used for quantitatively targeted analysis. The isolation window was set to m/z 1.0; the resolution was 17,500, and the collision energy was 35 (arbitrary units). Retention times and m/z transitions for acetyl CoA are detailed in Supplemental_Table_1. (DOI: 10.6084/m9.figshare.28653461). Energy charge was calculated from the concentration of quantified nucleotides using the following formula: Energy Charge = ([ATP] + 0.5[ADP]) / ([ATP] + [ADP] + [AMP]). (18)

Fig. 1, Fig. 2 and Fig. 3A&B were analyzed using one-way ANOVA, followed by a Fisher’s LSD post hoc analysis. Fig. 3C was analyzed using unpaired t test to directly compare AcAc vs D-βOHB supported induction of respiration. In Fig. 4E, Fig. 5, and Fig. 6, unpaired t test was used to examine SCOT control and SCOT KO under different substrate conditions. For Fig. 4B, 4D and 4F, the effect of genotype, substrates and their interaction were analyzed by a two-way ANOVA analysis, followed by a Fisher’s LSD post hoc analysis. All data are presented as means ± SEM and outliers are removed using Grubbs’ test. Statistical significance was defined as P < 0.05, and all statistics were run using GraphPad Prism 10.0 (GraphPad Software Inc., La Jolla, CA).

We determined maximal ADP-dependent respiration of isolated brain mitochondria from Wild-type C57BL/6J male mice in response to increasing concentrations of AcAc (0.1 –10 mM, Fig. 1A). Respiration of AcAc only increased above basal by 15% at a concentration ranging from 0.1 to 0.5 mM, decreasing after a concentration of 1 mM, and continued to decrease to 10 mM (~20– 35% less than basal at 8 mM: Fig. 1B). In contrast to other metabolic substrates, KB metabolism provides a surplus flux of acetyl-CoA (19). Other studies have shown AcAc alone inhibited α-ketoglutarate dehydrogenase activity due to sequestration of free CoASH which inhibits KB utilization (20, 21). L-carnitine is another essential molecule involved in fatty acid metabolism that transports fatty acid chains into the mitochondrial matrix for utilization (22). Thus, we tested if low KB-supported respiration rate in brain mitochondria was due to the low availability of CoASH and carnitine. Since D-βOHB is upstream of AcAc oxidation and generates reducing equivalents as NADH, we added D-βOHB (1 mM) to examine if the extra step in breakdown is impacted by various treatments (Fig. 1C). Again, KB supported respiration rates in the isolated brain mitochondria were low: 5–10% increase for AcAc and 35%−40% increase for D-βOHB (Fig. 1D). This increase with D-βOHB may contribute to the production of NADH via βOHB dehydrogenase. Overall, our findings indicate that KB respiration in isolated mitochondria is relatively low when considering respiration rates of other mitochondrial substrates (23).

Given the modest KB supported respiration rate after addition of CoASH and carnitine, we hypothesized that other substrates were needed for maximal mitochondrial KB respiration. Ketone body oxidation requires activation of AcAc by succinyl-CoA to generate AcAc-CoA through the activity of SCOT. Thiolytic cleavage of AcAc-CoA generates acetyl-CoA that enters the TCA cycle for terminal oxidation. Succinyl-CoA is generated from αKG in the presence of free CoASH by α-ketoglutarate dehydrogenase. However, the common addition of malate to mitochondrial respiration experiments depletes endogenous αKG by antiport with malate via the malate-aspartate shuttle, potentially limiting mitochondrial succinyl-CoA availability for AcAc activation via the SCOT reaction. Therefore, we posited that KB respiration in isolated mitochondrial requires supplementation of succinyl-CoA. To test this, we added αKG to our respiration protocol (Fig. 2A), expecting that it would increase succinyl-CoA and enhance KB respiration (Fig. 1E). Compared to adding AcAc alone (respiration 5–10% increase to basal: Fig. 1D), adding AcAc following the addition αKG increases AcAc respiration 75% higher than basal (Fig. 2B). To determine the KB concentration for maximal mitochondrial respiration in the presence of αKG, we again performed a titration of AcAc (Fig. 2C). Dose-dependent increase of AcAc respiration was observed up to 1 mM AcAc, with decreases observed at higher concentrations (Fig. 2D). Increasing αKG concentrations failed to augment AcAc respiration further (Fig. 2E–F). To determine the extent to which succinyl-CoA synthetase-derived succinate drove the effect of αKG on respiration, we used malonic acid, a complex II (succinate dehydrogenase) inhibitor, which diminished αKG-dependent respiration only by 15% suggesting that αKG sourcing of succinyl-CoA synthetase-derived succinate was a minor contributor (Fig. 2G– H). This suggests the αKG effect is primarily complex I-dependent, via αKG dehydrogenase-dependent NADH and succinyl-CoA. In summary, our experiments show that αKG at 1 mM drives maximal respiration of AcAc. Finally, the addition of αKG likely resulted in significant complex I-mediated respiration.

Since KBs putatively provide reducing agents to both complex I and II (24), we next investigated the entry path of electrons from KBs to the ETC using the protocol shown in Fig. 3A, leveraging malonic acid (Mna, complex II inhibitor) and rotenone (Rot, complex I inhibitor). Again, we observed increased respiration after the addition of AcAc and D-βOHB compared to αKG alone (Fig. 3B&3C). We then calculated this increased respiration using the respiration of αKG alone and after the addition of KBs (Respiration _{(αKG +AcAc or D-βOHB)} - Respiration _(αKG)) (Fig. 3D). We found no significant difference in respiration rates fueled by AcAc or D-βOHB (Fig. 3D). Fig. 3D directly compares AcAc vs. D-βOHB supported induction of respiration (Fig. 3D). A complex I inhibitor, rotenone (Rot), was added and elicited a bigger decrease than Mna for both substrates (Fig. 3B&3C), suggesting that αKG +KB respiration are complex I driven via αKG dehydrogenase and D-βOHB dehydrogenase (Fig. 3D). To our surprise, Mna resulted in a bigger decrease in D-βOHB supported respiration compared to respiration with AcAc in isolated brain mitochondria, suggesting that D-βOHB was more complex II driven than AcAc via succinate (Fig. 3D). In summary, our findings suggest that αKG is necessary for maximizing KB respiration in isolated brain mitochondria and KBs contribute to ETC mainly through complex I, however, D-βOHB contributes more to complex II than AcAc.

As previously stated, ketone utilization is regulated by SCOT mediated covalent activation of AcAc by succinyl-CoA to form AcAc-CoA, which is converted to acetyl-CoA for entry into the TCA cycle (14). Since AcAc is downstream of D-βOHB and focusing on the regulating step: the exchange between succinyl-CoA and AcAc via SCOT, we compared how control mice vs. SCOT-KO would respire with AcAc as a substrate and if loss of SCOT would differently impact metabolite pools within brain mitochondria. Since neuronal cells display high energy demand (25), we suspected that the lack of SCOT in neurons would impair AcAc respiration rate. Importantly however, mitochondria isolated from brain includes neuronal and non-neuronal sources including astrocytes and oligodendrocytes that still express the SCOT enzyme (26–29). First, we tested if αKG was essential for AcAc respiration in middle-aged control and SCOT KO C57BL/6NJ mice (30–32 weeks) (Fig. 4) and again showed that without αKG there was a modest minimal AcAc respiration (Fig. 5A&4B). No significant differences were noticed between the control and KO when comparing mitochondrial alone respiration and AcAc respiration. We then tested if AcAc would increase αKG respiration (Fig. 5C). Both control and KO demonstrated significant increase in respiration _{(αKG +AcAc)} compared to the respiration with αKG alone (2-fold increase (WT) and 68% increase (KO) to basal) (Fig. 5D). No significant difference was observed in respiration _(αKG) between the control and KO but there was a significant decrease in SCOT KO respiration _{(αKG +AcAc)} (Respiration 14.7% lower in SCOT KO to control). Since mitochondria were isolated from the whole left hemisphere of the brain and SCOT was eliminated only in neurons, we suspected residual AcAc respiration from other cell types but that a small decrease in AcAc respiration was attributable to lacking SCOT in neurons. As expected, controls showed a significantly higher AcAc respiration (Respiration _{(αKG +AcAc)} - Respiration _(αKG)) than SCOT-KO (Fig. 5E). We then used Rot and Mna in separate respiration experiments to distinguish how AcAc contributes to Complex I and Complex II, respectively. Like what we observed before, adding Rot had a larger inhibition of respiration than Mna, indicating respiration _{(αKG +AcAc)} was predominantly supported by complex I (Fig. 5F). However, no significant difference was notable between the control and KO in AcAc contribution to complex I or complex II (Fig. 5F). Again, maintenance of AcAc respiration in neuronal SCOT KO mitochondria is likely due to maintenance of SCOT expression in mitochondria isolated from other cell types in the brain.

Previously, KB metabolism has been shown to affect metabolite pools within various tissues that use KB as their fuel source (7). With neurons being the biggest energy consumer, we expected that lacking ketolysis in neurons would influence brain mitochondria metabolite profile. Providing brain mitochondria from the control and SCOT-KO with AcAc, we quantified the metabolite contents such as acetyl-CoA and calculated energy charge (EC) from ADP, AMP and ATP (18) with mass spectrometry. Energy charge is defined as (ATP+0.5 ADP)/(ATP+ADP+AMP), which is a measure of the relative concentrations of substrates and products of ATP-generating and ATP-utilizing reactions (18). Since αKG is the co-substrate for maximizing AcAc respiration, we first evaluated the metabolite contents in the control and SCOT-KO with 1 mM of αKG. A larger quantity of brain mitochondria (150 μg) was used for a more accurate measurement of acetyl CoA and energy charge (Fig. 4). We again used Mna and Rot to examine the impacts of αKG on our experimental conditions (Fig. 6A). At the end of each respiration experiment, we measured respiration rate, and it showed no significant difference between the control and KO under following conditions: αKG, αKG+Mna, αKG+Rot (Fig. 6B). Comparing respiration rate among three conditions, inhibiting complex I reduced the respiration rate (αKG+Rot) greater than complex II (αKG+Mna) (Fig. 6B). It again suggested that αKG was primarily complex I driven via αKG dehydrogenase which produces NADH. We then measured metabolite substrates: acetyl CoA and calculated energy charge using AMP, ADP, and ATP (18). Again, the control and KO showed no significant difference (Fig. 6D&6E).

To determine the role of neuronal SCOT in AcAc-dependent changes in acetyl-CoA and energy charge, we used AcAc and αKG+AcAc to test any differences between the control and KO (Fig. 7A). Complex I and II inhibitors were added to examine the flow of electrons from αKG+AcAc to the ETC within mitochondria. The mitochondrial respiration status showed no difference between the control and KO despite trends for lower respiration in KO without inhibitors and with Rot (Fig. 7B). As expected for αKG+AcAc, acetyl-CoA levels were significantly decreased in the SCOT-KO across the three conditions, suggesting that diminished procession from AcAc to acetyl-CoA on brain mitochondria in which SCOT had been eliminated from neurons (Fig. 7C). This diminished acetyl-CoA pool phenotype are dependent on both αKG and AcAc and not seen in either αKG alone (Fig. 6C) or AcAc alone (Fig. 7C). Again, it suggests αKG as a source of succinyl-CoA for the procession from AcAc to acetyl-CoA. Mitochondrial energy charge was not different between the control and KO (Fig. 7D).