The gut microbiota influences skeletal muscle mass and function in mice

Abstract

The functional interactions between the gut microbiota and the host are important for host physiology, homeostasis, and sustained health. We compared the skeletal muscle of germ-free mice that lacked a gut microbiota to the skeletal muscle of pathogen-free mice that had a gut microbiota. Compared to pathogen-free mouse skeletal muscle, germ-free mouse skeletal muscle showed atrophy, decreased expression of insulin-like growth factor 1, and reduced transcription of genes associated with skeletal muscle growth and mitochondrial function. Nuclear magnetic resonance spectrometry analysis of skeletal muscle, liver, and serum from germ-free mice revealed multiple changes in the amounts of amino acids, including glycine and alanine, compared to pathogen-free mice. Germ-free mice also showed reduced serum choline, the precursor of acetylcholine, the key neurotransmitter that signals between muscle and nerve at neuromuscular junctions. Reduced expression of genes encoding Rapsyn and Lrp4, two proteins important for neuromuscular junction assembly and function, was also observed in skeletal muscle from germ-free mice compared to pathogen-free mice. Transplanting the gut microbiota from pathogen-free mice into germ-free mice resulted in an increase in skeletal muscle mass, a reduction in muscle atrophy markers, improved oxidative metabolic capacity of the muscle, and elevated expression of the neuromuscular junction assembly genes Rapsyn and Lrp4 Treating germ-free mice with short-chain fatty acids (microbial metabolites) partly reversed skeletal muscle impairments. Our results suggest a role for the gut microbiota in regulating skeletal muscle mass and function in mice.

Fig. 1.. Skeletal muscle mass and function in GF mice.

(A) Weights of soleus, gastrocnemius, tibialis anterior (TA), quadriceps, and extensor digitorum longus (EDL) muscles from PF mice, GF mice, and C-GF mice. The number of mice used per experimental group is the following: Soleus muscle (PF, n = 13; GF, n = 14; C-GF, n = 10), gastrocnemius muscle (PF, n = 15; GF, n = 14; C-GF, n = 13), TA muscle (PF, n = 14; GF, n = 13; C-GF, n = 12), quadriceps muscle (PF, n = 15; GF, n = 14; C-GF, n = 13), and EDL muscle (PF, n = 13; GF, n = 12; C-GF, n = 13). (B) Shown are changes in expression of genes encoding myosin heavy chain (MyHC) isoforms in TA muscles of PF (n = 7), GF (n = 7), and C-GF (n = 10) mice. (C) Shown are changes in expression of Atrogin-1, Murf-1, and FoxO3 genes in TA muscles from PF (n = 7), GF (n = 7), and C-GF (n = 9) mice. (D) Shown are changes in expression of genes encoding the skeletal muscle-specific transcription factors MyoD and Myogenin in TA muscle samples from PF (n = 7), GF (n = 7), and C-GF (n = 9) mice. (E) Shown is immunoblot analysis of protein lysates from TA muscles harvested from PF, GF, and C-GF mice, indicating expression of Atrogin-1 (n = 4 mice per group), Murf-1 (n = 4 mice per group), and phosphorylated AMPK (p-AMPK; n = 5 mice per group). (F) The results in the histogram are expressed as the ratio of relative intensity of Atrogin-1 and Murf-1 protein expression normalized to tubulin as a loading control and the intensity of p-AMPK expression relative to total AMPK expression. Data are expressed as means ± SEM. Data were analyzed using ANOVA followed by Tukey’s post hoc test and were considered statistically significant at *P < 0.05, **P < 0.01, and ****P < 0.0001 between indicated groups.

Fig. 2.. Branched chain amino acid (BCAA) metabolism in skeletal muscle of GF mice.

(A) Shown are measurements of serum corticosterone concentrations in PF (n = 17), GF (n = 16), and C-GF (n = 10) mice. (B) Shown is expression of the Klf15 gene in TA muscles from PF (n = 7), GF (n = 7), and C-GF (n = 9) mice. (C) Shown are changes in expression of genes involved in BCAA catabolism (Bcat2, Bckdk, and Bckdh) in TA muscles of PF (n = 6), GF (n = 7), and C-GF (n = 9) mice. (D) Shown are changes in the expression of the genes Igf1 and Igf-binding proteins (Igfbps) in TA muscle of PF mice (n = 7), GF mice (n = 7), and C-GF (n = 9) mice. Data are expressed as means ± SEM. Data are analyzed using ANOVA followed by Tukey’s post hoc test and were considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001 between indicated groups.

Fig. 3.. Oxidative capacity of the skeletal muscle of GF mice.

(A) Representative images of TA muscle sections from PF, GF, and C-GF mice stained for the enzyme SDH. (B) Shown is expression of the Sdh gene in TA muscle from PF (n = 7), GF (n = 7), and C-GF (n = 9) mice. (C) Quantitative analysis of the ratio of mitochondrial DNA (mtDNA) to nuclear DNA in gastrocnemius muscles from PF (n = 4), GF (n = 5), and C-GF (n = 5) mice. (D and E) Shown are changes in expression of the Pgc1α and Tfam genes (D), and the CoxVa, CoxVIlb, and CytC genes (E) in TA muscles of PF (n = 6), GF (n = 7), and C-GF (n = 9) mice. (F) Shown are changes in expression of genes involved in glucose metabolism (Pfk, Pk, Ldh, and Pdh) in TA muscles of PF (n = 6), GF (n = 7), and C-GF (n = 9) mice. (G) Shown are changes in expression of genes involved in the fatty acid oxidation pathway (Lead, Mcad, and Cpt1b) in TA muscles of PF (n = 7), GF (n = 7), and C-GF (n = 9) mice. (H) Shown is the amount of glycogen in quadriceps muscles of PF (n = 4), GF (n = 3), and C-GF (n = 4) mice. Data are expressed as means ± SEM. Data were analyzed using ANOVA followed by Tukey’s post hoc test and were considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001 between indicated groups.

Fig. 4.. Metabolite analysis of the muscle, liver, and serum from GF mice.

(A to C) Shown is the average ¹H NMR spectrum of hydrophilic phase after Folch extraction for 25 metabolites. The ¹H NMR spectrum is shown for (A) gastrocnemius muscle from PF (n = 8), GF (n = 8), and C-GF (n = 10) mice; (B) liver tissue from PF (n = 7), GF (n = 8), and C-GF (n = 10) mice; (C) serum from PF (n = 8), GF (n = 8), and C-GF (n = 9) mice. 1, taurocholic acid; 2, bile acids; 3, low-density lipoprotein (LDL); 4, very-low-density lipoprotein (VLDL); 5, leucine; 6, 3-hydroxybutyrate; 7, alanine; 8, acetate; 9, glutamine; 10, glutamate; 11, pyruvate; 12, glutathione; 13, hypotaurine; 14, dimethylamine; 15, sarcosine; 16, trimethylamine; 17, dimethylglycine; 18, unknown (δ 3.11) (s); 19, choline; 20, glycerophosphorylcholine; 21, taurine; 22, betaine; 23, glycine; 24, unknown (δ 3.59) (d); and 25, unknown (δ 3.71) (s). Metabolites in red were found in higher concentrations in GF mice compared to C-GF or PF mice; metabolites in blue were found in lower concentrations in GF mice compared to C-GF or PF mice. s, singlet; d, doublet; a.u., arbitrary units.

Fig. 5.. Multicompartment metabolic reaction network.

Metabolites are connected on the basis of the shortest paths of reactions that are mediated by enzymes encoded in the Mus musculus genome or on the basis of nonenzymatic reactions from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Metabolites in orange were found in higher concentrations in GF mice compared to PF or C-GF mice. Conversely, metabolites in blue were found in lower concentrations in GF mice compared to PF and C-GF mice. Alanine was higher in the skeletal muscle (gastrocnemius) and lower in the liver. The background shading indicates the three different subnetworks for gastrocnemius muscle (purple), liver (green), and serum (pink). Overlap exemplifies similarity between affected metabolic compartments.

Fig. 6.. Differential expression of mouse neuromuscular junction proteins between GF and PF mice.

(A) Shown are changes in expression of genes encoding acetylcholine receptor subunits (Chrn) in the TA muscle of GF, PF, and C-GF mice. Genes include Chrna1 (PF, n = 7; GF, n = 7; C-GF, n = 9), Chrnb (PF, n = 7; GF, n = 7; C-GF, n = 8), Chrnd (PF, n = 7; GF, n = 7; C-GF, n = 7), and Chrne (PF, n = 7; GF, n = 7; C-GF, n = 7). (B) Shown are changes in expression in TA muscle of PF, GF, and C-GF mice of genes encoding the receptor-associated protein of the synapse (Rapsyn; PF, n = 7; GF, n = 7; C-GF, n = 9), low-density lipoprotein receptor-related protein 4 (Lrp4), and Agrin (Agrn) (PF, n = 7; GF, n = 7; C-GF, n = 9). (C) Shown are changes in expression of the gene encoding fast-twitch troponin (Tnn) in TA muscle of PF (n = 6), GF (n = 7), and C-GF (n = 9) mice. (D) Analysis of hindlimb grip strength using the weights test in PF, GF, and C-GF mice (n = 6 per group). (E to F) Shown is the spontaneous activity of GF, PF, and C-GF mice in the open-field test measured by cumulative distance traveled (E) and cumulative vertical activities (F). PF (n = 8), GF (n = 9), and C-GF (n = 8) mice were monitored over a 2-hour period. All data are expressed as means ± SEM. Data were analyzed using ANOVA followed by Tukey’s post hoc test and were considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001 between indicated groups.

Fig. 7.. Effects of bacterial metabolites on skeletal muscle of GF mice.

(A) Shown are the effects of SCFAs on dexamethasone (Dex)-induced muscle atrophy in C2C12 mouse myotubes in vitro. Differentiated myotubes were treated with a cocktail of SCFAs (10 mM) in the presence or absence of dexamethasone (1 mM) for 24 hours, and changes in expression of Atrogin-1 and Murf-1 were analyzed (n = 3 per group). For the control group, C2C12 myotubes were treated with solvent (0.1% dimethyl sulfoxide) only as vehicle. (B and C) Shown are the effects of SCFA treatment on the expression of genes encoding mitochondrial proteins in C2C12 myotubes in vitro. Differentiated myotubes were treated with a cocktail of SCFAs (10 mM) for 24 hours, and relative gene expression of (B) Pgc1α and Tfam (n = 3 per group) and (C) CoxVa, CoxVIlb, and CytC (n = 3 per group) was analyzed. (D) Shown are weights of soleus, gastrocnemius, TA, quadriceps, and EDL muscles from GF mice either untreated or treated with SCFAs (GF + SCFAs) (n = 6 mice per group). (E to G) Shown are changes in gene expression of Atrogin-1, Murf-1, and MyoD (E), Pgclα and Tfam (F), and CoxVa, CoxVIIb, and CytC (G) in TA muscles from untreated and SCFA-treated GF mice (n = 6 per group). (H) Analysis of hindlimb grip strength in untreated and SCFA-treated GF mice using the weights test (n = 6 per group). (I) Changes in expression of Rapsyn, Lrp4, and Agrn genes in TA muscle from untreated and SCFA-treated GF mice (n = 6 mice per group). All data are expressed as means ± SEM. For (A), data were analyzed using ANOVA, followed by Tukey’s post hoc test. For all other panels, data were analyzed using two-tailed Student’s t test. Data were considered statistically significant at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 between indicated groups.