The histone deacetylase inhibitor butyrate improves metabolism and reduces muscle atrophy during aging

Abstract

Sarcopenia, the loss of skeletal muscle mass and function during aging, is a major contributor to disability and frailty in the elderly. Previous studies found a protective effect of reduced histone deacetylase activity in models of neurogenic muscle atrophy. Because loss of muscle mass during aging is associated with loss of motor neuron innervation, we investigated the potential for the histone deacetylase (HDAC) inhibitor butyrate to modulate age-related muscle loss. Consistent with previous studies, we found significant loss of hindlimb muscle mass in 26-month-old C57Bl/6 female mice fed a control diet. Butyrate treatment starting at 16 months of age wholly or partially protected against muscle atrophy in hindlimb muscles. Butyrate increased muscle fiber cross-sectional area and prevented intramuscular fat accumulation in the old mice. In addition to the protective effect on muscle mass, butyrate reduced fat mass and improved glucose metabolism in 26-month-old mice as determined by a glucose tolerance test. Furthermore, butyrate increased markers of mitochondrial biogenesis in skeletal muscle and whole-body oxygen consumption without affecting activity. The increase in mass in butyrate-treated mice was not due to reduced ubiquitin-mediated proteasomal degradation. However, butyrate reduced markers of oxidative stress and apoptosis and altered antioxidant enzyme activity. Our data is the first to show a beneficial effect of butyrate on muscle mass during aging and suggests HDACs contribute to age-related muscle atrophy and may be effective targets for intervention in sarcopenia and age-related metabolic disease.

Keywords: aging; butyrate; histone deacetylase; metabolism; sarcopenia; skeletal muscle.

Figure 1

Butyrate reduces fat mass and improves glucose metabolism in C57Bl/6 mice during aging. Butyrate increases histone acetylation in (A) brain and (B) liver (*P < 0.05 vs. control). (C) Body weight changes over the 10‐month study. (D) Percent fat mass and (E) percent lean mass measured by quantitative magnetic resonance imaging in old mice (n = 14–22). Intraperitoneal (F) glucose, (G) insulin, and (H) pyruvate tolerance tests in old mice (n = 14–15 for GTT and PTT; n = 7 for ITT; control aged – white; butyrate aged – gray. *P < 0.05 analyzed by ANOVA with repeated measures).

Figure 2

Butyrate prevents muscle atrophy during aging. (A) Hindlimb muscle mass of 12 and 26‐month‐old C57Bl/6 female mice relative to body weight (n = 14–17). (B) Cross‐sectional area and (C) minimum Feret's diameter (n = 3) from muscle sections stained with hematoxylin and treosin. (D) Four‐paw grip strength (n = 5–8). (E) Muscle fiber type determined by MHC isoform composition (n = 3–4). (F) Representative images used to quantify CSA and MFD (*P < 0.05 vs. diet‐matched young; ^# P < 0.05 vs. age‐matched control; control young – white, control aged – black, butyrate young – light gray, butyrate aged – dark gray). (G) Oil Red O staining of lipid droplets in gastrocnemius muscle (Sections from three animals were examined per group; 4× and 10× images are from two different animals per group). All data are displayed as means with standard error.

Figure 3

Butyrate increases mitochondrial biogenesis in skeletal muscle. Protein levels of (A) porin and (B) TFAM during aging and butyrate treatment were determined by Western blot. mRNA levels of (C) PGC‐1α and (D) TFAM in young and old animals treated with butyrate were determined by qPCR. (E) Western blot quantified in (A) and (B) (^& P < 0.05 significant effect of diet; control young – white, control aged – black, butyrate young – light gray, butyrate aged – dark gray). (F) Oxygen consumption over 24 h; dark cycle only is statistically significant (n = 8). (G) 40‐h spontaneous activity in old mice (n = 3–4) (control aged – white; butyrate aged – gray). (H) Top – Succinate dehydrogenase activity in gastrocnemius muscle from young control, old control, and old butyrate groups. Soleus muscle in the bottom left corner of each image. Bottom – Cytochrome C activity in gastrocnemius muscle with soleus muscle in the bottom of each image (Sections from three animals were examined per group).

Figure 4

Effect of butyrate and aging on HDAC4/myogenin signaling, oxidative stress, and apoptotic markers. (A) HDAC4 protein was determined by (B) Western blot (n = 3). (C) Expression of HDAC4, myogenin, atrogin‐1, MuRF1, and Musa1 was determined by qPCR (n = 8). (D) Expression of embryonic MHC (eMHC), AChRα, Runx1, and neural cell adhesion molecule (NCAM) was determined by qPCR (n = 8; *P < 0.05 vs. diet‐matched young determined by ANOVA with Tukey's post‐test; data are displayed as means with standard error). (E) Central nuclei in a muscle fiber stained with hematoxylin and treosin (arrow). (F) Frequency of central nuclei per 100 muscle fibers (chi‐squared test was used to determine significance; *P < 0.05 vs. diet‐matched young; ^# P < 0.05 vs. age‐matched control; control young – white, control aged – black, butyrate young – light gray, butyrate aged – dark gray).

Figure 5

Effect of butyrate and aging on proteasome activity in skeletal muscle. Proteasome‐specific (A) chymotrypsin‐like, (B) trypsin‐like, and (C) peptidylglutamyl peptide hydrolyzing activities were determined using fluorescent substrates (n = 5–6). (D) Protein levels of 20S α7 subunit were determined by Western blot. (E) Western blot quantified in (D, G). (F) Results of a cell death ELISA in young and old animals treated with butyrate. (G) Protein levels of XIAP were determined by Western blot. (H) mRNA levels of Gadd45 (n = 8). *P < 0.05 vs. diet‐matched young determined by ANOVA with Tukey's post‐test; control young – white, control aged – black, butyrate young – light gray, butyrate aged – dark gray. All data are displayed as means with standard error.

Figure 6

Butyrate modulates antioxidant enzyme activity in skeletal muscle during aging. (A) Protein carbonyls in young and old mice fed control butyrate diets. Catalase (B) protein and (C) activity were determined in muscle from young and old animals treated with control or butyrate diets. (D) MnSOD, (E) CuZnSOD, and (F) GPx activities were determined in young and old mice fed control and butyrate diets (*P < 0.05 vs. diet‐matched young; ^% P < 0.05 significant effect of diet; control young – white; control aged – black; butyrate young – light gray; butyrate aged – dark gray). All data are displayed as means with standard error.