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. 2011 Nov;15(11):2317-25.
doi: 10.1111/j.1582-4934.2010.01223.x.

A metabolic shift induced by a PPAR panagonist markedly reduces the effects of pathogenic mitochondrial tRNA mutations

Tina Wenz  1 , Xiao Wang, Matteo Marini, Carlos T Moraes
Affiliations

A metabolic shift induced by a PPAR panagonist markedly reduces the effects of pathogenic mitochondrial tRNA mutations

Tina Wenz et al. J Cell Mol Med. 2011 Nov.

Abstract

Mutations in mitochondrial DNA-encoded tRNA genes are associated with many human diseases. Activation of peroxisome proliferator-activated receptors (PPARs) by synthetic agonists stimulates oxidative metabolism, induces an increase in mitochondrial mass and partially compensates for oxidative phosphorylation system (OXPHOS) defects caused by single OXPHOS enzyme deficiencies in vitro and in vivo. Here, we analysed whether treatment with the PPAR panagonist bezafibrate in cybrids homoplasmic for different mitochondrial tRNA mutations could ameliorate the OXPHOS defect. We found that bezafibrate treatment increased mitochondrial mass, mitochondrial tRNA steady state levels and enhanced mitochondrial protein synthesis. This improvement resulted in increased OXPHOS activity and finally in enhanced mitochondrial ATP generating capacity. PPAR panagonists are known to increase the expression of PPAR gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. Accordingly, we found that clones of a line harbouring a mutated mitochondrial tRNA gene mutation selected for the ability to grow in a medium selective for OXPHOS function had a 3-fold increase in PGC-1α expression, an increase that was similar to the one observed after bezafibrate treatment. These findings show that increasing mitochondrial mass and thereby boosting residual OXPHOS capacity can be beneficial to an important class of mitochondrial defects reinforcing the potential therapeutic use of approaches stimulating mitochondrial proliferation for mitochondrial disorders.

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Figures

Fig 1
Fig 1
Bezafibrate induces a metabolic shift in cultured cells. (A) Relative expression of PGC-1α and TFAM in mutant and control cell lines grown in bezafibrate-supplemented media. Cells grown in media with vehicle (DMSO) were used as a reference (n= 3 and bars represent S.D.). P < 0.001 for all samples. (B) Lactate concentration of bezafibrate and DMSO-treated mutant and control cell lines in media after 24 hrs growth (n= 3 and bars represent S.D.). (C) Glucose consumption of mutant and control cell lines after 24 hrs growth (n= 3 and bars represent S.D.). (D) Growth curves of MELAS and MERRF mutant cells treated with bezafibrate and cultured in glucose medium. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2
Fig 2
Bezafibrate-treated cells have enhanced aerobic ATP synthesis and increased ATP levels. Rate of oligomycin-sensitive ATP-synthesis via complex I (A) and complex II (B) in digitonin-extracts from mutant and control cells grown in presence of bezafibrate and DMSO (n= 3 and bars represent S.D.). Activities of control cells (nmol/min/mg): CI: wt (MELAS): 33.56, wt (MERRF): 31.5, SUB52: 30.5, W20: 36.9; CII: wt (MELAS): 23.8, wt (MERRF): 22.0, SUB52: 21.0, W20: 25.8. (C) ATP levels in cells treated with DMSO or bezafibrate. Values were normalized by total protein content and by ATP content of DMSO-treated wild-type cells (n= 3 and bars represent S.D.). ATP levels of controls cells (nmol/mg protein): wt (MELAS): 21.2, wt (MERRF): 19.7, SUB52: 18.9, W20: 20.3. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3
Fig 3
Bezafibrate increases overall OXPHOS enzyme activity and steady state levels by increasing mitochondrial mass. (A) and (B) COX and combined CI + III activity in bezafibrate and DMSO-treated mutant and control cell lines (n= 3 and bars represent S.D.). (C) CS activity in bezafibrate and DMSO-treated mutant and control cell lines (n= 3 and bars represent S.D.). (D)–(E) Western blot analysis of bezafibrate and DMSO-treated mutant and control lines. Antibodies against ND39 (complex I), SDH (flavoprotein of complex II), COXI and COXIV (complex IV), ATPase subunit β (complex V) and VDAC1 (outer membrane protein) were used. An antibody against tubulin was used as loading control. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4
Fig 4
Bezafibrate increases affected tRNA levels and enhances mitochondrial protein synthesis. (A) Levels of affected tRNA in bezafibrate and DMSO-treated mutant and control cell lines (n= 3 and bars represent S.D.). The right panel shows the slot blots used to quantitate the respective affected tRNA levels. The graph on the left shows the relative levels of these tRNAs. (B) Western blot analysis of bezafibrate and DMSO-treated mutant and control lines. An antibody against the small mitoribosomal subunit DAP3 was used. (C) In vivo labelling of mitochondrial translation products in bezafibrate and DMSO-treated mutant cell lines. W20 and MELAS cell lines were used as controls. Western blot against tubulin was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5
Fig 5
PGC-1α expression is increased in suppressor cell lines of mtDNA G5703A mutation. Relative expression of PGC-1α and TFAM in suppressor lines WG4 and WL4 compared to parental W72 treated with bezafibrate. W72 cells grown in media with vehicle (DMSO) were used as a reference (n= 3 and bars represent S.D.). P < 0.001 for all samples.
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