Nutritional ketosis improves exercise metabolism in patients with very long-chain acyl-CoA dehydrogenase deficiency

Jeannette C Bleeker, Gepke Visser, Kieran Clarke, Sacha Ferdinandusse, Ferdinand H de Haan, Riekelt H Houtkooper, Lodewijk IJlst, Irene L Kok, Mirjam Langeveld, W Ludo van der Pol, Monique G M de Sain-van der Velden, Anita Sibeijn-Kuiper, Tim Takken, Ronald J A Wanders, Michel van Weeghel, Frits A Wijburg, Luc H van der Woude, Rob C I Wüst, Pete J Cox, Jeroen A L Jeneson, Jeannette C Bleeker, Gepke Visser, Kieran Clarke, Sacha Ferdinandusse, Ferdinand H de Haan, Riekelt H Houtkooper, Lodewijk IJlst, Irene L Kok, Mirjam Langeveld, W Ludo van der Pol, Monique G M de Sain-van der Velden, Anita Sibeijn-Kuiper, Tim Takken, Ronald J A Wanders, Michel van Weeghel, Frits A Wijburg, Luc H van der Woude, Rob C I Wüst, Pete J Cox, Jeroen A L Jeneson

Abstract

A maladaptive shift from fat to carbohydrate (CHO) oxidation during exercise is thought to underlie myopathy and exercise-induced rhabdomyolysis in patients with fatty acid oxidation (FAO) disorders. We hypothesised that ingestion of a ketone ester (KE) drink prior to exercise could serve as an alternative oxidative substrate supply to boost muscular ATP homeostasis. To establish a rational basis for therapeutic use of KE supplementation in FAO, we tested this hypothesis in patients deficient in Very Long-Chain acyl-CoA Dehydrogenase (VLCAD). Five patients (range 17-45 y; 4 M/1F) patients were included in an investigator-initiated, randomised, blinded, placebo-controlled, 2-way cross-over study. Patients drank either a KE + CHO mix or an isocaloric CHO equivalent and performed 35 minutes upright cycling followed by 10 minutes supine cycling inside a Magnetic Resonance scanner at individual maximal FAO work rate (fatmax; approximately 40% VO2 max). The protocol was repeated after a 1-week interval with the alternate drink. Primary outcome measures were quadriceps phosphocreatine (PCr), Pi and pH dynamics during exercise and recovery assayed by in vivo 31 P-MR spectroscopy. Secondary outcomes included plasma and muscle metabolites and respiratory gas exchange recordings. Ingestion of KE rapidly induced mild ketosis and increased muscle BHB content. During exercise at FATMAX, VLCADD-specific plasma acylcarnitine levels, quadriceps glycolytic intermediate levels and in vivo Pi/PCr ratio were all lower in KE + CHO than CHO. These results provide a rational basis for future clinical trials of synthetic ketone ester supplementation therapy in patients with FAO disorders. Trial registration: ClinicalTrials.gov. Protocol ID: NCT03531554; METC2014.492; ABR51222.042.14.

Keywords: VLCADD; fatty acid oxidation; in vivo 31P MRS; ketone ester; mitochondrial energy transduction; muscle; nutritional ketosis; very long-chain acyl-CoA dehydrogenase.

Conflict of interest statement

The intellectual property and patents covering the uses of ketone bodies and esters are owned by BTG Ltd, The University of Oxford, the NIH, and TΔS Ltd. Should royalties ever accrue from these patents, K.C. and P.J.C. as named inventors may receive a share of royalties as determined by the terms of the respective institutions. K.C. is director of TΔS Ltd, a University of Oxford company with the aim of developing and commercialising products based on the ketone ester. P.J.C. is a former employee of TdeltaS. J.C.B., G.V., S.F., F.H.d.H., R.H.H., L.I., I.L.K., M.L., W.L.v.d.P., M.G.M.d.S.‐v.d.V., A.S.‐K., T.T., R.J.A.W., M.v.W., F.A.W., L.H.v.d.W., R.C.I.W., and J.A.L.J. declare that they have no conflict of interest.

© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.

Figures

Figure 1
Figure 1
Study protocol and effects of dietary substrates during exercise on tolerability and cardiopulmonary exercise testing. A, Studyprotocol; B, Maximum scores of complaints during study protocol reported by patients; C, Concentration of creatine kinase in plasma after CHO (blue) or KE + CHO (red) ingestion; D, Subjective exertion score reported by patients after CHO (blue) or KE + CHO (red) ingestion; E and F, Heart rate (E) and respiratory exchange ratio measured during upright bicycling bout of protocol after CHO (blue) or KE + CHO (red) ingestion. N = 5, data are presented as mean ± SEM. Differences between groups were analysed with two‐way ANOVA for repeated measures with Bonferroni post‐hoc analysis
Figure 2
Figure 2
Effects of dietary substrates during exercise on plasma metabolites in VLCADD patients. A‐D, Concentration of beta‐hydroxybutyrate (A) glucose (B) in whole blood, insulin (C) and lactate (D) in plasma after CHO or KE + CHO ingestion. E‐H, Fold change from baseline concentration of C14:1‐carnitine (C), sum of C14 + C16 + C18‐carnitine (D), free fatty acids (E) and C2‐carnitine (F) in plasma. In A, C‐F n = 5 for CHO and KE + CHO. In B n = 5 for KE + CHO, n = 5 for CHO in t = 0 and 3 hours after exercise, n = 4 for CHO in t = 30‐85. Data are presented as mean ± SEM. Differences between groups were analysed with two‐way ANOVA for repeated measures with Bonferroni post‐hoc analysis. The red bar in graph A represents the differences between t = 75 and t = 85 for KE + CHO analysed with paired –t test. The blue bar in graph B represents the difference between t = 75 and t = 85 for CHO analysed with paired –t test. **** indicates P value <.001, *** indicates P value <.001, ** indicates P value <.01, and * indicates P value <.05
Figure 3
Figure 3
Effects of dietary substrates and exercise on muscle glucose and fat metabolism in VLCADD patients before and after exercise. A, Intramuscular concentrations of beta‐hydroxybutyrate before and after exercise after ingestion of CHO or KE + CHO. B, Sum of intramuscular concentrations of glycolytic intermediates (Hexose‐P, Fructose‐1,6‐diphosphate, Glyceraldehyde‐3P, 1,3‐Diphosphoglyceric acid, 2‐/3‐Phosphoglyceric acid and Phosphoenolpyruvate) before and after exercise after ingestion of CHO or KE + CHO. C, Intramuscular concentrations of fructose 1,6‐diphosphate before and after exercise after ingestion of CHO or KE + CHO. D, Sum of intramuscular concentrations of tricyclic acid cycle intermediates (citrate/isocitrate, α‐ketoglutarate, succinate, fumarate, malate) before and after exercise after ingestion of carbohydrates or ketone ester. E‐P, Intramuscular concentrations of acylcarnitine species before and after exercise after ingestion of CHO or KE + CHO. Values on the Y‐axis are the ratio of peak area over internal standard (PA), corrected for the total adenosine nucleotides (ATP + ADP + AMP) (TAN), per sample. Error bars are mean ± SD. N = 4 for all conditions. ** = P < .01 with 2 way ANOVA
Figure 4
Figure 4
Effects of dietary substrates on in vivo muscle energetics during and after cycling in VLCADD patients. A, Transversal T1‐weighted MR images of the right upper leg of patients #4 (A.1) and #1 (A.2). Subjects were positioned feet‐first. The slightly flattened left side of the thigh image indicates the position of the 31P surface coil overlying the m. vastus lateralis. Note the large diameter of the subcutaneous fat layer surrounding the thigh muscles in female patient #1. B, in vivo 31P Magnetic Resonance spectra of the vastus lateralis muscle of patient ID4 recorded during stationary exercise at individual FATMAX workload after either CHO (top trace) or KE + CHO ingestion (bottom trace), respectively. ATP, adenosine triphosphate; Pi, inorganic phosphate; PCr, phosphocreatine. C and D, Mean in vivo concentration ratio of inorganic phosphate (Pi) and phosphocreatine (PCr) during stationary exercise (C) and mean recovery time constant (D) of the vastus lateralis muscle of 4 VLCADD patients after CHO vs KE + CHO ingestion, respectively. * indicates P value <.05; two tailed paired t test. Data are presented as mean ± SEM

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