Fig 1.
Sodium butyrate rapidly increase oxygen consumption in a dosage dependent manner.
(A) Quantification of oxygen consumption levels upon addition of SB. Time zero refers to the last measure prior to the addition of vehicle/SB. (B+C) Area under the curve for each condition plotted in a scatter plot for the 2–7 (7.5–45 minutes) measurements (B) after the injection and 7–12 (45–82.5 minutes) measurements (C) after the injection. Control (orange, n = 28), 3mM SB (light green, n = 16), 15mM SB (dark green, n = 22), 30mM SB (brown, n = 15). Error bars indicate standard error of mean in all graphs. One-way ANOVA, Tukey´s post-hoc test, ns p>0.05; * p≤0.05; ** p≤0.01; *** p≤0.001; **** p≤0.0001. Detailed statistical information is presented in S1 and S2 Tables in S1 File. Each measurement was normalized to the first measurement (time point ‘0’), which is the measurement prior to the introduction of the vehicle/SB/.
Fig 2.
Sodium butyrate does not impact the levels of proteins and acetylation of proteins after 20 minutes.
(A) Heatmap showing comparison of 2827 detected proteins between control (orange, n = 4) and 15mM SB (green, n = 4) treated fly heads after 20 min. (B) Heatmap comparing 557 detected acetylation sites of 267 proteins between control (orange, n = 4) and 15mM SB (green, n = 4) treated fly heads after 20 min. FC (fold change). Levels of key mitochondrial proteins related to TCA cycle were not significantly altered after 20 min between control (orange, n = 4) and 15mM SB (green, n = 4) group. Genes encoding proteins are as follows: (C) CG3861 (Citrate synthase), (D) CG9244 (Aconitate hydratase), (E) CG7176 (Isocitrate dehydrogenase), (F) CG1544 (2-oxoglutarate dehydrogenase), (G) CG5214 (Dihydrolipoyllysine-residue succinyltransferase), (H) CG7430 (Dihydrolipoyl dehydrogenase), (I) CG11963 (Succinyl-CoA ligase), (J) CG4094 (Fumarate hydratase), (K) CG5362 (Malate dehydrogenase). Error bars indicate ±S.E.M in all graphs. Student’s t-test was performed and detailed statistical information is presented in S3 Table in S1 File. Levels of key mitochondrial proteins were not significantly altered after 20 min between control (orange, n = 4) and 15mM SB (green, n = 4) group. Genes encoding proteins are as follows: (L) Complex I: CG6343 (ND-42), CG2286 (ND-75), (M) Complex II: CG17246 (SdhA), CG6666 (SdhC), (N) Complex III: CG8764 (Ox), CG7361 (Rieske), (O) Complex IV: CG14724 (COX5A), CG9603 (COX7A) and (P) Complex V. Error bars indicate ±S.E.M in all graphs. Student’s t-test was performed and detailed statistical information is presented in S3 Table in S1 File.
Fig 3.
Pyruvate and proline rapidly and transiently increase oxygen consumption in fly heads.
(A) Representative tracing for oxygen level changes in control and 25mM pyruvate and proline (PP)-treated fly heads. Each measurement lasted 2 min and consisted of 10 sub-measurements (ticks). The last measurement before (3) and the measurements after injection (4–18) are shown. The arrowhead indicates the addition of PP or control. (B) Quantification of OCR based on (A). (C+D) Area under the curve for each condition plotted in a scatter plot for the (C) 2–7 (7.5–45 minutes) measurements or (D) 7–12 (45–82.5 minutes) measurements after the injection. Control (orange, n = 11), 5mM PP (blue, n = 15), 25mM PP (purple, n = 7). One-way ANOVA, Tukey´s post hoc test, ns p>0.05; * p≤0.05; ** p≤0.01. Error bars indicate ±S.E.M in all graphs. Detailed statistical information is presented in S4 and S5 Tables in S1 File. Each measurement was normalized to the first measurement (time point ‘0’), which is the measurement prior to the introduction of the vehicle/pyruvate+proline.
Fig 4.
Butyrate is rapidly incorporated into butyric derivatives.
(A) Schematic overview of key metabolic pathways (red) involving the measured metabolites. (B-D) Changes in pool size in response to 13C4-labeled SB treatment in fly heads over time (red, 0 min; blue, 20 min; green, 60 min). n = 6 for all metabolites. Error bars indicate ±S.E.M in all graphs. One-way ANOVA, Tukey´s post-hoc test, ns p>0.05; * p≤0.05; ** p ≤ 0.01; *** p≤0.001; **** p≤0.0001. Detailed statistical information is presented in S6 Table in S1 File.
Fig 5.
Butyrate is rapidly incorporated into TCA cycle metabolites.
(A) Analysis of Mass Isotopomer Distribution (MID) of acetyl-CoA. 13C4-sodium butyrate treatment to determine its incorporation at different time points by mass spectrometry. (B-H) Analysis of Mass Isotopomer Distribution (MID) in TCA cycle metabolites after 13C4-SB treatment to determine its incorporation over time by mass spectrometry. n = 6 for all metabolites and time points. M0 unlabeled mass of isotope, M+n native metabolite mass + number of isotopically labeled carbons. Error bars indicate ±S.E.M in all graphs. Detailed statistical analysis was performed using one-way ANOVA, Tukey´s post-hoc test, results are presented in S7 Table in S1 File.