Long-term bezafibrate treatment improves skin and spleen phenotypes of the mtDNA mutator mouse

Abstract

Pharmacological agents, such as bezafibrate, that activate peroxisome proliferator-activated receptors (PPARs) and PPAR γ coactivator-1α (PGC-1α) pathways have been shown to improve mitochondrial function and energy metabolism. The mitochondrial DNA (mtDNA) mutator mouse is a mouse model of aging that harbors a proofreading-deficient mtDNA polymerase γ. These mice develop many features of premature aging including hair loss, anemia, osteoporosis, sarcopenia and decreased lifespan. They also have increased mtDNA mutations and marked mitochondrial dysfunction. We found that mutator mice treated with bezafibrate for 8-months had delayed hair loss and improved skin and spleen aging-like phenotypes. Although we observed an increase in markers of fatty acid oxidation in these tissues, we did not detect a generalized increase in mitochondrial markers. On the other hand, there were no improvements in muscle function or lifespan of the mutator mouse, which we attributed to the rodent-specific hepatomegaly associated with fibrate treatment. These results showed that despite its secondary effects in rodent's liver, bezafibrate was able to improve some of the aging phenotypes in the mutator mouse. Because the associated hepatomegaly is not observed in primates, long-term bezafibrate treatment in humans could have beneficial effects on tissues undergoing chronic bioenergetic-related degeneration.

Figure 1. Systemic effects of bezafibrate on Mut and WT mice.

(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). *, P<0.05; **, P<0.01, Student’s t-test. Error bars represent the SEM.

Figure 2. Bezafibrate delayed hair loss and restored the skin structure of Mut mice.

(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.

Figure 3. Bezafibrate improved spleen size and structure of Mut mice.

(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). *, P<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) PGC-1α and PPARγ 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 c reductase core protein 2 (UQCRC2; subunit of complex III), and ATP synthase subunit 5α (ATP5A; subunit of complex V). *, P<0.05; **, P<0.01, Student’s t-test. Error bars represent the SEM.

Figure 4. The effect of bezafibrate on the skeletal muscle of Mut and WT mice.

(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 PGC-1β and PPARs 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 c 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). *, P<0.05; **, P<0.01, ***P<0.001, Student’s t-test. Error bars represent the SEM.

Figure 5. Bezafibrate induces hepatomegaly and fatty acid oxidation in Mut and WT mice.

(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 PGC-1 coactivators and PPARs 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 (ACOX), cluster of differentiation 36 (CD36), carnitine palmitoyl transferase (CPT1) and short-chain-acyl-coenzymeA dehydrogenase (SCAD). *, P<0.05; **, P<0.01, ***P<0.001, Student’s t-test. Error bars represent the SEM.