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Observational Study
. 2020 Mar 6;12(3):e11589.
doi: 10.15252/emmm.201911589. Epub 2020 Feb 28.

Metabolic effects of bezafibrate in mitochondrial disease

Hannah Steele #  1 , Aurora Gomez-Duran #  2   3 , Angela Pyle  1   4 , Sila Hopton  4   5 , Jane Newman  4   6 , Renae J Stefanetti  6 , Sarah J Charman  7 , Jehill D Parikh  7 , Langping He  4   5 , Carlo Viscomi  3 , Djordje G Jakovljevic  7 , Kieren G Hollingsworth  7 , Alan J Robinson  3 , Robert W Taylor  4   5   6 , Leonardo Bottolo #  8   9   10 , Rita Horvath #  2 , Patrick F Chinnery #  2   3
Affiliations
Observational Study

Metabolic effects of bezafibrate in mitochondrial disease

Hannah Steele et al. EMBO Mol Med. .

Abstract

Mitochondrial disorders affect 1/5,000 and have no cure. Inducing mitochondrial biogenesis with bezafibrate improves mitochondrial function in animal models, but there are no comparable human studies. We performed an open-label observational experimental medicine study of six patients with mitochondrial myopathy caused by the m.3243A>G MTTL1 mutation. Our primary aim was to determine the effects of bezafibrate on mitochondrial metabolism, whilst providing preliminary evidence of safety and efficacy using biomarkers. The participants received 600-1,200 mg bezafibrate daily for 12 weeks. There were no clinically significant adverse events, and liver function was not affected. We detected a reduction in the number of complex IV-immunodeficient muscle fibres and improved cardiac function. However, this was accompanied by an increase in serum biomarkers of mitochondrial disease, including fibroblast growth factor 21 (FGF-21), growth and differentiation factor 15 (GDF-15), plus dysregulation of fatty acid and amino acid metabolism. Thus, although potentially beneficial in short term, inducing mitochondrial biogenesis with bezafibrate altered the metabolomic signature of mitochondrial disease, raising concerns about long-term sequelae.

Keywords: bezafibrate; metabolomics; mitochondrial DNA; mitochondrial disorder; mitochondrial encephalomyopathy.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Summary of the study design.
Figure EV1
Figure EV1. Additional clinical characteristics of the six patients with the m.3243A>G MTTL1
Renal function of the participants: urea, creatine and estimated glomerular filtration rate (GFR). Dotted lines represent the mean ± SD.
Figure 2
Figure 2. Metabolic effects of bezafibrate in patients with the m.3243A>G MTTL1 mutation
W0, W6 and W12 refer to the week of study.

  1. Serum FGF‐21 and GDF‐15 levels before, during and after treatment (two‐sided Wilcoxon signed‐rank test, P‐value = 0.031 in all significant cases) (see Fig EV2 for details where P1–P6 refer to the individual patients).

  2. Volcano plot showing differences in metabolite levels between 0 and 6 and 12 weeks of treatment. Metabolites in histidine, alanine, aspartate and glutamine metabolism, and the TCA cycle pathways are annotated at 10% false discovery rate (see Dataset EV1 for the whole list of differentially regulated metabolites. Although some other metabolites showed a more pronounced difference, these did not fit into a recognised pathway). Metabolite labels are colour‐coded according to the identified KEGG pathways (dark red = “Alanine, aspartate and glutamate metabolism”, dark green = “Histidine metabolism” and dark blue = “Citrate cycle (TCA cycle)”, with transition colour code if the metabolite belongs to more than one KEGG pathways).

  3. Scatterplot depicting P‐values from the metabolites Pathway Enrichment Analysis (y‐axis) and impact values from Pathway Topology Analysis (x‐axis) (see Dataset EV2 for details). KEGG pathways at 10% adjusted Holm–Bonferroni P‐value are highlighted.

  4. Volcano plot of acyl‐carnitine metabolites between 0 and 6 and 12 weeks of treatment with highlighted differential metabolites at 10% FDR.

  5. Respiratory chain complex and citrate synthase activity in skeletal muscle before and after 12 weeks of treatment. No significant differences were detected before and after 12 weeks of treatment by using the two‐sided Wilcoxon signed‐rank test with an empirical level of significance.

Data Information: In (A) and (E), horizontal dotted lines denote the mean values in healthy age‐matched controls. In (E), solid horizontal lines and bars represent the mean ± SD of the technical replicates at each time point. Horizontal lines on (A) and (E) refer to statistical comparisons of the mean values, where ns = non‐significant, *P‐value ≤ 0.05.
Figure EV2
Figure EV2. Serum FGF‐21 and GDF‐15 levels before, during and after treatment
Colour code and patterns represent different samples at different time points. Plain bars specify patients before treatment, whereas striped and dotted bars represent the same patients at 6 and 12 weeks after treatment, respectively. Horizontal dotted lines denote the mean values in healthy age‐matched controls. Bars and error bars represent the mean ± SD of the technical replicates at each time point. Statistical testing was performed by using the Kruskal–Wallis test followed by Tukey honest significant differences procedure on the ranked data (Serum FGF‐21, 6 vs. 0 weeks, P1–P6: P‐value = 0.0045; 12 vs. 6 weeks, P1, P3, P4, P5 and P6: P‐value = 0.0045; 12 vs. 0 weeks, P1–P6: P‐value = 3 × 10−5; GDF‐15, 6 vs. 0 weeks, P3–P6: P‐value = 0.0067, 0.0045 and 0.0177; 12 vs. 6 weeks, P1, P2, P4 and P5: P‐value = 0.0041, 0.0045, 0.0045 and 0.0045; 12 vs. 0 weeks, P1–P6, P‐value: 2 × 10−5, 3 × 10−5, 0.0172, 3 × 10−5, 3 × 10−5, 0.0053) (***P‐value ≤ 0.001, **P‐value ≤ 0.01, *P‐value ≤ 0.05).
Figure EV3
Figure EV3. Additional clinical characteristics of the six patients with the m.3243A>G MTTL1
Bodyweight and basal surface area (BSA) before and after 12 weeks of treatment. Colour codes represent different samples. No significant differences were detected before and after treatment using the two‐sided Wilcoxon signed‐rank test with an empirical level of significance.
Figure EV4
Figure EV4. Metabolic response to Bezafibrate
  1. A

    Unsupervised k‐means cluster analysis of serum metabolites identified three groups: untreated (group 1 in red) and two groups for the treated patients (groups 2 and 3 in blue and green, respectively).

  2. B–D

    Volcano plot of the differentially regulated metabolites (FDR < 0.1) with (B) no differences observed between 6 weeks and 12 weeks of treatment, (C) after 6 weeks and (D) after 12 weeks. Metabolites in “Alanine, aspartate and glutamate metabolism”, “Histidine metabolism” and “Citrate cycle (TCA cycle)” are highlighted in dark red, dark green and dark blues, respectively, with transition colour code if the metabolite belongs to more than one KEGG pathways.

  3. E, F

    Volcano plot with differentially regulated metabolites (FDR < 0.1) of the acyl‐carnitine metabolites (E) after 6 weeks and (F) 12 weeks.

Figure EV5
Figure EV5. Mitochondrial enzyme activity in individual patients before and after bezafibrate treatment
  1. A, B

    (A) Mitochondrial respiratory chain enzyme activities (expressed relative to citrate synthase activity) and (B) citrate synthase activity (expressed relative to total protein) in individual patients before (plain bars) and after 12 weeks (dotted bars) of treatment. Horizontal dotted lines indicate the mean values in healthy age‐matched controls. Bars and error bars represent the mean ± SD of the technical replicates at each time point. Statistical testing was performed by using the two‐sided Mann–Whitney test with an empirical level of significance (CII/CS, P1, P2, P3, P5, P6, empirical P‐value = 0.0318, 0.0360, 0.0260, 0.0354 and 0.0018; CIII/CS, P3–P6, empirical P‐value = 0.0376, 0.0360, 0.0324 and 0.0323; CIV/CS, P3, P5 and P6, empirical P‐value = 0.0340, 0.0487, 0.0310; citrate synthase, P1, P3, P4, P5 and P6, empirical P‐value: 0.0318, 0.0360, 0.0261, 0.0348 and 0.0354) (**P‐value ≤ 0.01, *P‐value ≤ 0.05).

Figure 3
Figure 3. Mitochondrial biogenesis and clinical effects of bezafibrate in patients with the m.3243A>G MTTL1 mutation
P1–P6 refer to individual patients.

  1. Representative examples of the quadruple immunofluorescence quantification of skeletal muscle fibres before and after treatment. The box in the top right‐hand corner indicates the normal range, using two antibodies to NDUFB8 and COX I.

  2. Quantitative quadruple immunofluorescence of skeletal muscle fibres in the six patients showing a decrease (two‐sided Wilcoxon signed‐rank test with an empirical level of significance) in the proportion of complex IV‐deficient skeletal muscle fibres before and after 12 weeks of treatment (empirical P‐value = 0.048).

  3. Hierarchical cluster analysis of skeletal muscle RNA‐seq transcripts before and after treatment showing no clear separation between untreated and treated patients.

  4. Volcano plot depicting differentially expressed RNA‐seq genes at 10% FDR after 12 weeks of treatment. Significantly different mammalian mitochondrial genes (MitoCarta 2.0) are annotated.

  5. Reactome Pathway Analysis of the differentially expressed genes at 10% FDR after 12 weeks of treatment (see Expanded View document for details).

  6. Percentage level m.3243A>G in blood, urinary sediment and skeletal muscle. No differences were detected by using the two‐sided Wilcoxon signed‐rank test with an empirical level of significance. W = week of the study.

  7. Cardiac parameters before and after treatment (two‐sided Wilcoxon signed‐rank test with an empirical level of significance). ES = end‐systolic volume (ml) (empirical P‐value = 0.047) and index (ml/m2) (empirical P‐value = 0.046), measured by MRI. Horizontal lines on (B), (F) and (G) refer to statistical comparisons of the mean values, where ns = non‐significant, *P‐value ≤ 0.05. For normal reference ranges, see Hollingsworth et al (2012).

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