Figure 7. The effects of 3OHB on mitochondrial respiratory efficiency depend on BDH1 flux.

(A–C) Freshly isolated mitochondria from heart ventricles of BDH1^fl/fl (controls), csBDH1^–/–, or WT C57BL/6N mice were used to assess the impact of 3OHB on the relationship between (A) oxygen consumption rate (JO₂), (B) membrane potential (ΔΨ ) in millivolts (mV), and (C) NAD(P)H/NAD(P)⁺ redox state versus the estimated Gibbs energy of ATP hydrolysis (ΔG_ATP). Mitochondria were fueled with pyruvate + malate (110 μM each) and 50 μM L-octanoylcarnitine in the absence (purple) or presence of 2 mM of the (R, red) or (S, green) enantiomer of 3OHB, or acetoacetate (AcAc, gray). (D) Mitochondrial respiratory efficiency was evaluated by plotting JO₂ against ΔΨ. Dotted lines separate the submaximal and maximal portions of JO₂ vs. ΔG_ATP. Triangle denotes the changing concentrations of ATP relative to ADP (ATP:ADP), resulting in a reciprocal change in energy demand. Data represent mean ± SEM (n = 4–6). Comparisons between BDH1^fl/fl versus csBDH1^–/– mitochondria were analyzed by 3-way ANOVA followed by Tukey HSD (^#ketone/genotype interaction; ^§ketone effect; P< 0.05 and family-wise error rate [FWER] < 0.05) using measurements (A–C) made at submaximal JO₂. Energy fluxes representing maximal JO₂ (ΔG_ATP = –12.95) were analyzed by 2-way ANOVA and Tukey HSD (*FWER < 0.05, relative to vehicle control). Comparison of R and S enantiomers were analyzed by 2-way ANOVA (submaximal JO₂) and 1-way ANOVA (maximal JO₂) each followed by Tukey HSD (^§effect of R at submaximal JO₂; ^¤effect of S at maximal JO₂; FWER < 0.05). Effects of AcAc were analyzed by 2-way ANOVA at submaximal JO₂ (^†main effect of ketone; ^‡ketone/ΔG_ATP interaction, P < 0.05) and t test at maximal JO₂ (*P < 0.05).