Ketone flux through BDH1 supports metabolic remodeling of skeletal and cardiac muscles in response to intermittent time-restricted feeding

Abstract

Time-restricted feeding (TRF) has gained attention as a dietary regimen that promotes metabolic health. This study questioned if the health benefits of an intermittent TRF (iTRF) schedule require ketone flux specifically in skeletal and cardiac muscles. Notably, we found that the ketolytic enzyme beta-hydroxybutyrate dehydrogenase 1 (BDH1) is uniquely enriched in isolated mitochondria derived from heart and red/oxidative skeletal muscles, which also have high capacity for fatty acid oxidation (FAO). Using mice with BDH1 deficiency in striated muscles, we discover that this enzyme optimizes FAO efficiency and exercise tolerance during acute fasting. Additionally, iTRF leads to robust molecular remodeling of muscle tissues, and muscle BDH1 flux does indeed play an essential role in conferring the full adaptive benefits of this regimen, including increased lean mass, mitochondrial hormesis, and metabolic rerouting of pyruvate. In sum, ketone flux enhances mitochondrial bioenergetics and supports iTRF-induced remodeling of skeletal muscle and heart.

Keywords: acylcarnitines; beta-oxidation; fiber type; intermittent fasting; ketones; metabolic flux; mitochondria; proteomics; striated muscles; time-restricted feeding.

Figure 1.. Intermittent TRF promotes metabolic resilience, preserves lean mass, and remodels skeletal muscle.

(A) TRF experimental design. (B) Daily food intake during TRF weeks 4–10. (C) Body mass (BM) pre-special diet (e.g., standard chow diet, pre-diet), post-special diet (post-diet) and at TRF weeks 1–9 at ZT3 or ZT21 (ZT = zeitgeber time). (D) Body mass (BM) gained represented as a sum of fat mass and lean mass. (E) Blood glucose (BG, top) and blood 3OHB (bottom) post-special diet and at TRF weeks 2–9 at ZT3 or ZT21 (ZT = zeitgeber time). (F) Acute fasting challenge (AFC) change (Δ) in body mass (BM), fat mass (FM) and lean mass (LM) from fed to fasted (fasting) and fasted to refed (refed). (G) AFC BM change from baseline. (H) AFC blood glucose, NEFAs and 3OHB. (I) Volcano plot of skeletal muscle (SkM, quadriceps) proteomics. (J) Proteomics pathway analysis. (K) Volcano plot of SkM mitochondrial (mito) proteins. (L) Number of SkM mito proteins up or down regulated by pathway. (M) Heatmap of 12h fasted quadriceps muscle (skm) metabolites represented as the mean z-score. (B-H) Data are mean ± SEM. (B) N=2–6 per group. Mice were group housed; thus data represent an estimate of food intake per day per mouse. (C-H) N=10 per group. Data were analyzed by two-tailed Student’s t-test. (*) significant difference between Ad Lib and TRF mice. (I-L) N=4–5 per group. *P_adjusted≤0.05. N represents biological replicates. See also Figure S1.

Figure 2.. Ketone flux in skeletal muscle depends on fiber type and fuel supply.

(A) Ketone oxidation pathways in muscles. (B) [U-¹³C] 3OHB tracing experimental design. (C) Soleus (SOL) and EDL [U-¹³C] 3OHB labeling strategy. M4 3OHB generates M2 acetyl-CoA and M2 citrate. M2 Ac-CoA derived from 3OHB can be converted to M2 3OHB. (D) Buffer and tissue 3OHB average ¹³C-labeling (%) from fasted SOL and EDL muscles incubated in fasted buffer. (E) Average ¹³C-labeling (%) of TCAC intermediates from fasted SOL and EDL muscles incubated in fasted buffer. Citrate (Cit) enrichment normalized to tissue 3OHB labeling from (F) fasted SOL and EDL muscles incubated in fasted buffer, (G) SOL and (H) EDL muscles incubated in fed or fasted (fst) buffers and (I) SOL muscles isolated from fed or fasted mice incubated in fasted buffer. (J) Ketolytic enzyme abundance in mitochondria isolated from heart (H), red muscle (R) and white muscle (W). (K) Correlation plot of BDH1 enzyme activity vs. BDH1 protein abundance. (D-I) Data are means ± SEM. (D-I) N=5–6 per group. (J) Representative image with n=2/tissue type. (K) N=5 per group. (D-I) Data were analyzed by two-tailed Student’s t-test. (*) significant differences between soleus vs. EDL or fed vs. fasted buffers. (K) Data were analyzed by linear regression. *P≤0.05. N represents biological replicates. See also Figure S2.

Figure 3.. mBDH1 deficiency compromises exercise tolerance and exacerbates metabolic bottlenecking during acute fasting.

(A) Mouse model of skeletal muscle (Skm) and heart (Hrt) BDH1 deficiency (mBDH1 KO mice, left) and western blot validation (right). (B) BDH1 activity in mitochondria from mixed skm (gastroc and quad), heart, and liver from 18h fasted mice. (C) Blood 3OHB in fed and 18h fasted states. (D) Time and distance to exhaustion during a treadmill test after an 18h fast. (E) Blood glucose before and after the exercise test. (F) Acylcarnitine profiles of quadriceps (Quad), red gastrocnemius (RG), heart and plasma from mice in the 12h fasted state represented as mean of the z-score. (G) ¹³C parallel tracing experimental design and (H) ¹³C parallel tracing strategy in fasted soleus (SOL) and EDL muscles from mice incubated in fasted buffer with ¹³C 3OHB (dark blue), ¹³C AcAc (light blue) or ¹³C palmitate (PA, purple). (I) Ketone and (J) palmitate ¹³C-labeling (%) in fasted SOL. (K) C4OHAC average ¹³C-labeling (%) in fasted SOL. (L) C4OHAC labeling from ketones (¹³C 3OHB+AcAc) as MIDs. (M) Short chain (SC) carbon circuit in SkM. (N) Semi-quantitative estimate for metabolite pool sizes of AcAc, C4OH AC and C2AC in fasted SOL. Metabolite peak areas were normalized to internal standard peak areas (norvaline, AcAc; d₉-carnitine, C4OH AC and C2AC) and tissue mass. (B-E, I-L, and N) Data are means ± SEM. (A) Representative image with N=1 per group. (B) N=4 per group. (CF, I-L and K) N=6–8 per group. Data were analyzed by two-tailed Student’s t-test. (*) significant differences between FC and mKO mice. *P≤0.05. N represents biological replicates. See also Figure S3.

Figure 4.. Cardiac BDH1 deficiency leads to metabolic bottlenecking upon fasting and lipid exposure.

(A) ¹³C-tracing experimental design and (B) ¹³C-palmitate (PA) tracing strategy in fasted, Langendorff-perfused hearts. (C) TCAC intermediate ¹³C-labeling (%) in fasted hearts. (D) M+2 abundance (%) in TCAC intermediates. (E) Semi-quantitative estimate for pool sizes of acyl carnitines in fasted hearts. Acyl carnitine peak areas were normalized to d₉-carnitine standard and tissue mass. Acyl carnitine pool sizes are segregated by the tracer-derived contribution (¹³C, dark purple) and the unlabeled fraction (¹²C, light purple). (F) Short-chain acyl CoA labeling (%) in fasted hearts. (G) Semi-quantitative estimate for pool sizes of acyl CoA’s and (H) free CoA in fasted hearts. Acyl CoA and free CoA peak areas were normalized to d₉-pentanoyl-CoA standard and tissue mass. (C-H) Data are represented as means ± SEM. N=7–9 per group and data were analyzed by two-tailed Student’s t-test. (*) represents significant differences between FC and mKO mice. *P≤0.05. (E) (*) and (#) represent genotype differences (P≤0.05) between ¹³C-labeled fraction and unlabeled fraction, respectively.

Figure 5.. Muscle BDH1 flux is required for iTRF-induced building of lean mass.

(A) iTRF experimental design in FC and mKO mice. Blood (B) glucose and (C) 3OHB during an acute fasting challenge (AFC). Longitudinal measures of (D) body mass, (E) fat mass and (F) lean mass. (G) Soleus and EDL muscle weights. (H) Experimental design and (I) tracing scheme for ¹³C glucose studies in soleus and EDL muscles from 5h fasted Ad Lib and TRF FC and mKO mice under fed buffer conditions. Light and dark red circles represent carbons from PDH flux or anaplerotic fluxes (malic enzyme (ME) and pyruvate carboxylase (PC)), respectively. Average ¹³C-labeling (%) of (J) pyruvate, (K) citrate, (L) glutamine (gln), (M) C2AC, and (N) C4OHAC. (O) Citrate and (P) glutamine mass isotopomer distributions (MIDs). (B-G and J-P) Data are represented as mean ± SEM. (B-D) N=12–16 per group. (E-F) N=10–16 per group. (G) N=9–16 per group. (J-P) N=5–8 per group. (B-G) Data were analyzed by two-tailed Student’s t-test. (J-P) Data were analyzed by two-tailed student’s t-test and two-way ANOVA. (*) significant differences between FC Ad Lib and FC TRF or mKO Ad Lib and mKO TRF mice. (#) main effect of genotype. *P≤0.05. N represents biological replicates. See also Figure S4.

Figure 6.. Deficiency of BDH1 in heart compromises iTRF-induced reprogramming of mitochondrial efficiency.

(A) Function of freshly isolated heart mitochondria was examined using the Oroboros-O2K respirometry system paired with the creatine kinase (CK) energetic clamp technique. Parallel measurements of membrane potential ($\text{ΔΨ}$), redox potential (NAD(P)H/NAD(P)+), and JH₂O₂ emissions were obtained via spectrofluorometric assays using a QuantaMaster Spectrofluorometer. (B) Example of respiratory conductance (JO₂ vs. Gibb’s Free Energy of ATP hydrolysis or $\text{Δ}{\text{G}}_{\text{ATP}}$). A steeper slope represents higher conductance (closed circles) whereas a lower slope indicates lower conductance (open circles). (C) Example of respiratory efficiency (JO₂ vs. $\text{ΔΨ}$). The rightward shift shows that mitochondria are maintaining a more polarized $\text{ΔΨ}$ for any given rate of oxygen consumption (JO₂), indicative of increased respiratory efficiency or a higher P:O ratio. (D) JO₂ vs. $\text{Δ}{\text{G}}_{\text{ATP}}$, (E) mitochondrial respiratory efficiency represented as JO₂ plotted against $\text{ΔΨ}$ and (F) electron leak, expressed as a percentage of oxygen flux (JH₂O₂/JO₂*100 = % Electron Leak) measured in heart mitochondria fueled by octanoyl-carnitine+malate (OcM), pyruvate+palmitoyl-carnitine+malate (PyrMPc) or pyruvate+malate (PyrM). (D-F) Data are means ± SEM. (D-F) N=5–7 per group. (F) Data were analyzed by two-tailed student’s t-test. (E) The black (FC) and red (mKO) arrows indicate the direction of the respiratory efficiency shift. (*) significant differences between FC Ad Lib and FC TRF or mKO Ad Lib and mKO TRF mice. *P≤0.05. N represents biological replicates. See also Figure S5.

Figure 7.. mBDH1 deficiency compromises iTRF-induced remodeling of the cardiac mitochondrial proteome.

(A) Proteomics workflow using both TMT and label free methods. Volcano plot of the mitochondrial proteome derived from hearts of 5h fasted (B) Ad Lib FC vs. Ad Lib mKO mice, (C) Ad Lib vs. TRF FC mice and (D) Ad Lib vs. TRF mKO mice. Red and blue circles represent proteins upregulated and downregulated, respectively (P_adjusted<0.05). (Data are means of the z-score. (B-D) N=5/group. N represents biological replicates (E) Mechanisms by which BDH1 impacts FAO and overall mitochondrial efficiency: (1) redox transfer to Complex I optimizes electron partitioning; (2) a mild redox-induced brake fine tunes beta-oxidation and prevents severe FAO bottlenecking; (3) provision of acetyl CoA supports TCAC flux as well as (4) reverse MKT flux, which defends against CoA trapping and bioenergetic instability.