Bezafibrate induces hepatomegaly and fatty acid oxidation in Mut and WT mice.

<p>(A) Liver weight of 10 month-old mice (n = 4–6/group). (B) H&E staining of the liver of 10 month-old mice showing hepatocytes and central vein (n = 4/group). (C) The level of liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood of 10 month-old mice (n = 6/group). (D) Gene expression of <i>PGC-1</i> coactivators and <i>PPARs</i> in the liver of 10 month-old mice normalized to actin (n = 4/group). (E) The mRNA level of markers of fatty acid oxidation in the liver of 10 month-old mice normalized to actin (n = 4/group). Acyl-coenzymeA oxidase 1 (<i>ACOX</i>), cluster of differentiation 36 (<i>CD36</i>), carnitine palmitoyl transferase (<i>CPT1</i>) and short-chain-acyl-coenzymeA dehydrogenase (<i>SCAD</i>). *, <i>P</i><0.05; **, <i>P</i><0.01, ***<i>P</i><0.001, Student’s <i>t</i>-test. Error bars represent the SEM.</p

Bezafibrate improved spleen size and structure of Mut mice.

<p>(A) Spleen weight of 10 month-old mice and (B) picture of spleen from Mut mice (n = 4–6/group). (C) Quantification of cleaved caspase-3 immunostaining in paraffin sections from the spleen of 10 month-old mice (n = 3–4/group). *, <i>P</i><0.05, one-way analysis of variance followed by Bonferroni’s multiple comparison test. (D) H&E staining of the spleen of 10 month-old mice showing the organization of white pulp (purple) and red pulp (pink) (n = 3/group). (E) Results from complete blood cell count in 10 month-old mice showing RBC (red blood cells, x10⁶µl ) (n = 5–6/group) (F) <i>PGC-1α</i> and <i>PPARγ</i> mRNA levels in the spleen of 10 month-old mice normalized to actin. (G) Quantification of western blot showing mitochondrial protein levels in total homogenate from the spleen of 10 month-old mice normalized to actin. NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8; subunit of complex I), succinate dehydrogenase subunit B (SDHB; subunits of complex II), ubiquinol-cytochrome <i>c</i> reductase core protein 2 (UQCRC2; subunit of complex III), and ATP synthase subunit 5α (ATP5A; subunit of complex V). *, <i>P</i><0.05; **, <i>P</i><0.01, Student’s <i>t</i>-test. Error bars represent the SEM.</p

Systemic effects of bezafibrate on Mut and WT mice.

<p>(A) Body weight of mice from 2 to 14 month-old (n = 11–26/group for 2 to 11 months; 2–6/group for 12 to 14 months). The difference in body weight between WTSD and MutSD mice is statistically significant from 3 to 14 months, between WTSD and WTBD mice is significant at every time point and the difference between MutSD and MutBD is significant from 3 to 14 months. Measurement of total (B) bone mineral density (BMD), (C) bone mineral content (BMC) (D), body area (cm²), lean mass (g), total body fat (g) and percent (%) fat of 10 month-old mice (n = 5–7/group). (E) Percent survival of mice (n = 11–15/group). *, <i>P</i><0.05; **, <i>P</i><0.01, Student’s <i>t</i>-test. Error bars represent the SEM.</p

Bezafibrate delayed hair loss and restored the skin structure of Mut mice.

<p>(A) Pictures of mice at 7 month-old and 10 month-old showing their coat phenotype (n = 6/group). Squares highlight area of coat being described. (B) Dorsal skin sections from 10 month-old mice showing hematoxylin and eosin (H&E) staining to depict structural changes (black arrow indicates break in the epidermis layer of MutSD mice), Verhoeff’s Van Geison (EVG) staining for elastic fibers shown in black/dark brown (yellow arrow) and Masson’s trichrome staining showing collagen in blue (n = 2/group). (C) Western blot showing total Smad3 and glyceralgehyde 3-phosphate (GAPDH) protein levels in total skin homogenate from 10 month-old mice and quantification of total Smad3 band intensity normalized to GAPDH (n = 4/group). Error bars represent the SEM.</p

The effect of bezafibrate on the skeletal muscle of Mut and WT mice.

<p>(A) Western blot of PGC-1α and loading control actin in total quadricep homogenate from 10 month-old mice and quantification of PGC-1α band intensity normalized to actin (n = 4/group). (B) Gene expression of <i>PGC-1β</i> and <i>PPARs</i> in the quadricep of 10 month-old mice normalized to actin (n = 4/group). (C) Citrate synthase activity in the total quadricep homogenate from 10 month-old mice (n = 4/group). (D) Quantification of western blot of mitochondrial proteins in the total homogenate from the quadricep of 10 month-old mice (n = 4/group). NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8; subunit of complex I), succinate dehydrogenase subunit B (SDHB; subunits of complex II), ubiquinol-cytochrome <i>c</i> reductase core protein 2 (UQCRC2; subunit of complex III), mitochondrial cytochrome c oxidase subunit 1 (MTCO1; subunit of complex IV) and ATP synthase subunit 5α (ATP5A; subunit of complex V) (E) Quantification of mtDNA levels in the quadriceps of 10 month-old mice based on ND1 (subunit of complex I) levels normalized to glyceraldehyde 3-phosphate (GAPDH) (n = 4/group). (F) Gene expression of markers of fatty acid oxidation, acyl-coenzymeA oxidase 1 (ACOX), cluster of differentiation 36 (CD36), carnitine palmitoyl transferase (CPT1) and short-chain-acyl-coenzymeA dehydrogenase (SCAD) in the quadriceps of 10 month-old mice normalized to actin (n = 4/group) (G) Skeletal muscle weight of 10 month-old mice (n = 4–6/group). (H) The number of falls of mice when put to run on a treadmill for 3 minutes at 9 meters/minute (n = 5–10/group). *, <i>P</i><0.05; **, <i>P</i><0.01, ***<i>P</i><0.001, Student’s <i>t</i>-test. Error bars represent the SEM.</p

The effect of bezafibrate treatment in mouse models of disease.

<p>The effect of bezafibrate treatment in mouse models of disease.</p

Kaplan-Meier survival curves of D257A/D257A males and females.

<p>Survival curves of control diet (solid black line) and calorie restricted (red dashed line) D257A/D257A males (left) and females (right). n = 23–29. D257A/D257A = <i>Polg</i>^D257A/D257A.</p

Survival parameters of PolgD257A/D257A mice on control and calorie-restricted diets.

<p>Survival parameters of PolgD257A/D257A mice on control and calorie-restricted diets.</p

Echocardiography of 13–16 months old wild-type and <i>Polg</i>^D257A/D257A mice on control and calorie-restricted diets.

<p>Echocardiography of 13–16 months old wild-type and <i>Polg</i>^D257A/D257A mice on control and calorie-restricted diets.</p

Body weight, skeletal muscle mass, and testis weight of mitochondrial mutator mice.

<p>Body weights (A), vastus lateralis (VL) and gastrocnemius (GN) muscle mass (B) of 13–16 months old +/+, +/D257A, and D257A/D257A males (left) and females (right) under control diet or calorie restricted conditions. (C) Testes weights of 10–18 months old +/+, +/D257A, and D257A/D257A males under control diet or calorie restricted conditions. ‡P < 0.05 control diet vs CR diet within genotype. n = 8–16. *P < 0.05 +/+ vs D257A/D257A within diet. #P < 0.05 +/+ vs +/D257A within diet. §P < 0.05 +/D257A vs D257A/D257A within diet. +/+ = <i>wild-type</i>, +/D257A = <i>Polg</i>^+/D257A, and D257A/D257A = <i>Polg</i>^D257A/D257A.</p