Heptanoic and medium branched-chain fatty acids as anaplerotic treatment for medium chain acyl-CoA dehydrogenase deficiency

Abstract

Triheptanoin (triheptanoylglycerol) has shown value as anaplerotic therapy for patients with long chain fatty acid oxidation disorders but is contraindicated in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In search for anaplerotic therapy for patients with MCAD deficiency, fibroblasts from three patients homozygous for the most common mutation, ACADM^G985A/G985A, were treated with fatty acids hypothesized not to require MCAD for their metabolism, including heptanoic (C7; the active component of triheptanoin), 2,6-dimethylheptanoic (dMC7), 6-amino-2,4-dimethylheptanoic (AdMC7), or 4,8-dimethylnonanoic (dMC9) acids. Their effectiveness as anaplerotic fatty acids was assessed in live cells by monitoring changes in cellular oxygen consumption rate (OCR) and mitochondrial protein lysine succinylation, which reflects cellular succinyl-CoA levels, using immunofluorescence (IF) staining. Krebs cycle intermediates were also quantitated in these cells using targeted metabolomics. The four fatty acids induced positive changes in OCR parameters, consistent with their oxidative catalysis and utilization. Increases in cellular IF staining of succinylated lysines were observed, indicating that the fatty acids were effective sources of succinyl-CoA in the absence of media glucose, pyruvate, and lipids. The ability of MCAD deficient cells to metabolize C7 was confirmed by the ability of extracts to enzymatically utilize C7-CoA as substrate but not C8-CoA. To evaluate C7 therapeutic potential in vivo, Acadm^-/- mice were treated with triheptanoin for seven days. Dose dependent increase in plasma levels of heptanoyl-, valeryl-, and propionylcarnitine indicated efficient metabolism of the medication. The pattern of the acylcarnitine profile paralleled resolution of liver pathology including reversing hepatic steatosis, increasing hepatic glycogen content, and increasing hepatocyte protein succinylation, all indicating improved energy homeostasis in the treated mice. These results provide the impetus to evaluate triheptanoin and the medium branched chain fatty acids as potential therapeutic agents for patients with MCAD deficiency.

Keywords: ACADs, acyl-CoA dehydrogenases; Anaplerosis; Dojolvi(TM); Fatty acid oxidation disorders; Heptanoic acid; Lysine succinylation; MCAD deficiency; Medium branched-chain fatty acids; Triheptanoin.

Figure 1.

Bioenergetic parameters determined from oxygen consumption rate (OCR) measurements in control cells (Fb826) after sequential addition of the electron transport chain inhibitors/stress compounds oligomycin, FCCP, and rotenone/antimycin A. Panels from top heptanoic acid (C7), dimethylheptanoic acid (dMC7), 6-amino-2,4-dimethylheptanoic acid (AdMC7), and 4,8-dimethylheptanoic acid (dMC9). Bar graphs are comparison of C7 and MBCFAs treatment groups are relative, normalized in percentage terms to untreated (0 μM FA), to untreated cells in the absence of glucose/pyruvate cells. Statistical analyses were carried out on original raw data provided in Supplement Data section Figure 1s, where Tukey multiple range test was used. Data are means ± SD of n=6–8 wells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, statistical comparison of C7 and MBCFAs treatment groups are relative to untreated cells (0 μM FA) in the absence of glucose/pyruvate. Percent changes in red to draw attention to the significantly different Y-axis scale.

Figure 2.

Comparative, normalized in percentage terms to untreated, real time oxygen consumption rate (OCR) measurements in three MCAD deficient patient fibroblast cell lines Fb831, Fb786, and Fb787 following treatment of medium chain fatty acids (FA). Panels from top to bottom: (A) C7, (B) dMC7, (C) AdMC7, and (D) dMC9. Graphs from left to right: Basal respiration, ATP-linked respiration, maximal respiration, and spare capacity. Statistical significance was transferred from the original data provided in Supplement Data section Figures 2s, 3s, and 4s, that were means ± SD of n=6–8 wells; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, statistical comparison of C7 and MBCFAs treatment groups relative to no treatment (0 μM FA). Statistical analysis used Tukey multiple range test.

Figure 2.

Comparative, normalized in percentage terms to untreated, real time oxygen consumption rate (OCR) measurements in three MCAD deficient patient fibroblast cell lines Fb831, Fb786, and Fb787 following treatment of medium chain fatty acids (FA). Panels from top to bottom: (A) C7, (B) dMC7, (C) AdMC7, and (D) dMC9. Graphs from left to right: Basal respiration, ATP-linked respiration, maximal respiration, and spare capacity. Statistical significance was transferred from the original data provided in Supplement Data section Figures 2s, 3s, and 4s, that were means ± SD of n=6–8 wells; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, statistical comparison of C7 and MBCFAs treatment groups relative to no treatment (0 μM FA). Statistical analysis used Tukey multiple range test.

Figure 3.

Comparative, normalized in percentage terms to untreated, real time basal extracellular acidification rate (ECAR) measurements in control fibroblast (Fb826) and three MCAD deficient patient fibroblast cell lines Fb831, Fb786, and Fb787 in response to incubation for 72 hours with C7, dMC7, AdMC7, and dMC9. Percent change in basal ECAR calculated relative to the measurement at 0 μM FA (no treatment). Data are means ± SD of n=6–8 wells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, statistical comparison of C7 and MBCFAs treatment groups relative to no treatment (0 μM FA). Statistical analysis used Tukey multiple range test.

Figure 4.

Immunofluorescence of Fb826 control (A) and Fb831, Fb786, Fb787 patient fibroblast cell lines (B-D) stained with antisuccinyllysine antibody (Ksu; Green) and anti-MTCO1 (Red) antibodies and DAPI (Blue) counterstain for nuclei after 72h treatment with 90 μM C7, dMC7, AdMC7 and dMC9. Scale bar = 20 μM.

Figure 4.

Immunofluorescence of Fb826 control (A) and Fb831, Fb786, Fb787 patient fibroblast cell lines (B-D) stained with antisuccinyllysine antibody (Ksu; Green) and anti-MTCO1 (Red) antibodies and DAPI (Blue) counterstain for nuclei after 72h treatment with 90 μM C7, dMC7, AdMC7 and dMC9. Scale bar = 20 μM.

Figure 5.

Measurements of selected Krebs cycle intermediates in control (Fb826) and in MCAD deficient (Fb831) fibroblast cells cultured in T175 flasks. All media had no additional glutamine or pyruvate, and lipid stripped FBS was included instead of regular FBS in all treatments. The number of biological replicates (n) submitted for analysis was six. Data are means ± SD of n=4–6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, statistical comparison of C7 and MBCFAs treatment groups relative to no treatment (0 μM FA); #P < 0.1, ##P < 0.01, ####P < 0.0001 statistical comparison of untreated cells in the absence of glucose/pyruvate relative to untreated cells in the presence of glucose/pyruvate. Statistical analysis used Tukey multiple range test.

Figure 6.

Measurements of aspartate and glutamate in control (Fb826) and MCAD deficient (Fb831) fibroblast cells. All media had no additional glutamine or pyruvate, and lipid stripped FBS was included instead of regular FBS in all treatments. The number of biological replicates (n) was six. Error bars reflect at least four biological replicates used in the data analysis. Data are means ± SD of n=6–8. **P < 0.01 and ****P < 0.0001, statistical comparison of C7 and MBCFAs treatment groups relative to no treatment (0 μM FA); ####P < 0.0001 statistical comparison of untreated cells in the absence of glucose/pyruvate relative to untreated cells in the presence of glucose/pyruvate. Statistical analysis used Tukey multiple range test.

Figure 7.

Biochemical and histological evidence of efficacy of triheptanoin in Acadm^−/− mice. Mice were treated twice daily with triheptanoin [0%, 5%, 7.5%, and 12.5% of diet (wt:wt)] using oral gavage for seven days before histological examination. Livers from the various treatments were sectioned at different regions. (A) Oil Red O staining of mouse liver tissue section showing dose-dependent improvement of macrovesicular and microvesicular steatosis (B) H&E staining showing oil droplets disappearing; and (C) PAS staining showing glycogen liver content increasing (more purple) with increasing amounts of triheptanoin. Yellow (or White) bar in bottom right corner of images represents 110 μm. Inset bar graph is the heptanoylcarnitine (C7-carnitine), valerylcarnitine (C5-carnitine), and propionylcarnitine (C3-carnitine) plasma levels after four days of dosing triheptanoin at 0%, 5%, 7.5% and 12.5% of diet (wt:wt) to Acadm^−/− mice (n=4 per treatment group).

Figure 8.

Immunofluorescence confocal microscopy images of mice liver sections stained with antisuccinyllysine (Ksu; Green) and anti-MTCO1 (Red) antibodies and DAPI (Blue) counterstain. Wildtype untreated (A) and Acadm^−/− untreated and triheptanoin treatment [0%, 5%, 7.5%, and 12.5% of diet (wt:wt)] using oral gavage for seven days before histological examination. Images were captured using Zeiss LSM 710 confocal microscope with 20x objective Zoom 1.5x. Scale bar = 20 μM.