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. 2024 May 15;14(1):62.
doi: 10.1186/s13578-024-01244-2.

Microbiota-derived 3-phenylpropionic acid promotes myotube hypertrophy by Foxo3/NAD+ signaling pathway

Penglin Li #  1 , Xiaohua Feng #  1 , Zewei Ma  1 , Yexian Yuan  1 , Hongfeng Jiang  1 , Guli Xu  1 , Yunlong Zhu  1 , Xue Yang  1 , Yujun Wang  1 , Canjun Zhu  1 , Songbo Wang  1 , Ping Gao  1 , Qingyan Jiang  2   3   4 , Gang Shu  5   6   7
Affiliations

Microbiota-derived 3-phenylpropionic acid promotes myotube hypertrophy by Foxo3/NAD+ signaling pathway

Penglin Li et al. Cell Biosci. .

Abstract

Background: Gut microbiota and their metabolites play a regulatory role in skeletal muscle growth and development, which be known as gut-muscle axis. 3-phenylpropionic acid (3-PPA), a metabolite produced by colonic microorganisms from phenylalanine in the gut, presents in large quantities in the blood circulation. But few study revealed its function in skeletal muscle development.

Results: Here, we demonstrated the beneficial effects of 3-PPA on muscle mass increase and myotubes hypertrophy both in vivo and vitro. Further, we discovered the 3-PPA effectively inhibited protein degradation and promoted protein acetylation in C2C12 and chick embryo primary skeletal muscle myotubes. Mechanistically, we supported that 3-PPA reduced NAD+ synthesis and subsequently suppressed tricarboxylic acid cycle and the mRNA expression of SIRT1/3, thus promoting the acetylation of total protein and Foxo3. Moreover, 3-PPA may inhibit Foxo3 activity by directly binding.

Conclusions: This study firstly revealed the effect of 3-PPA on skeletal muscle growth and development, and newly discovered the interaction between 3-PPA and Foxo3/NAD+ which mechanically promote myotubes hypertrophy. These results expand new understanding for the regulation of gut microbiota metabolites on skeletal muscle growth and development.

Keywords: 3-Phenylpropionic acid; Acetylation; Gut microbiota metabolites; Muscle hypertrophy; NAD+.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Effect of 3-PPA on muscle mass and C2C12 myotubes hypertrophy. a Body weight gain in 6 week-old male mice with7 weeks’ treatment of 0.5%3-PPA (n = 10). b Lean mass and fat mass of mice after 6 weeks’ treatment of 3-PPA by QMR (n = 10). c Gastrocnemius weight of mice after 7  weeks’ treatment (n = 10). d–e The laminin immunofluorescent staining (d) and frequency histogram of gastrocnemius muscle fiber cross-sectional area statistical analysis (e) in C57BL/6 male mice after 3 weeks 3-PPA treatment (n = 3). f Schematic representation of C2C12 treated with 3-PPA for 2 days. g–h Immunofluorescence images (g) and statistics of myotube diameter (h) of C2C12 myotubes in 3-PPA treatment for 2 days (n = 4). i–j Immunofluorescence images (i) and statistics of myotube diameter (j) of chick embryo primary skeletal muscle cells in 3-PPA treatment for 2 days (n = 3)
Fig. 2.
Fig. 2.
3-PPA attenuated protein degradation instead of protein synthesis in C2C12 myotubes. a–b Immunoblots (a) and quantification (b) of the puromycin-labeled protein expression of C2C12 myotubes with 3-PPA treatment for 2 days (n = 8). c–d Immunoblots (c) and quantification (d) of total ubiquitination protein expression of C2C12 myotubes with 3-PPA treatment for 2 days (n = 4). e–f Immunoblots (e) and quantification (f) of P-Foxo3 and Foxo3 protein expression of C2C12 myotubes with 3-PPA treatment (n = 3). g–h Immunoblots (g) and quantification (h) of the total ubiquitination protein expression of chick embryo primary skeletal muscle myotubes with 3-PPA treatment for 2 days (n = 3-4). (i–j) Immunoblots (i) and quantification (j) of P-Foxo3 and Foxo3 protein expression in chick embryo primary skeletal muscle myotubes (n = 4)
Fig. 3
Fig. 3
Protein acetylation modification mediates the effect of 3-PPA in myotube hypertrophy. a–d The glucose content (a), LD content (b), ATP content (c), ROS content (d) in C2C12 myotubes with 3-PPA treatment for 2 days (n = 3 to 4). (e) Mitochondria biogenesis and function related gene mRNA expression in C2C12 myotubes with 3-PPA treatment for 2 days (n = 4 to 8). f Mitochondria autophagy related gene mRNA expression in C2C12 myotubes with 3-PPA treatment for 2 days (n = 4). g Tricarboxylic acid cycle related gene mRNA expression in C2C12 myotubes with 3-PPA treatment for 2 days (n = 6). h Relative Acetyl-CoA content in C2C12 myotubes with 3-PPA treatment for 2 days (n = 6). i–j Immunoblots (i) and quantification (j) of Pan-acetylation protein expression of C2C12 myotubes with 3-PPA treatment for 2 days (n = 3). k–l Immunofluorescence images (k) and statistics of myotube diameter (l) of C2C12 myotubes in C646 and 3-PPA co-treatment (n = 3). m The ratio of NAD+/NADH in in C2C12 myotubes with 3-PPA treatment for 2 days (n = 3). n mRNA expression of NANAT2 and NAMPT in C2C12 myotubes with 3-PPA treatment for 2 days (n = 6). o mRNA expression of SIRT1and SIRT3 in C2C12 myotubes with 3-PPA treatment for 2 days (n = 6)
Fig. 4
Fig. 4
Foxo3/NAD+ pathway is required for 3-PPA to increase protein acetylation modification. a–b Immunoblots (a) and quantification (b) of Foxo3 protein expression in nuclei and cytoplasm of C2C12 myotubes with 3-PPA treatment for 2 days (n = 4). c–d Immunofluorescence images (c) and statistics (d) of relative nuclei Foxo3 protein in C2C12 myotubes in 3-PPA treatment for 2 days (n = 3). e–f Immunoblots (e) and quantification (f) of Foxo3 protein expression in C2C12 myotubes with different siRNA sequence treatment (n = 3 to 4). g–h Immunofluorescence images (g) and statistics (h) of myotube diameter in C2C12 myotubes with siFoxo3 and 3-PPA co-treatment (n = 3). i mRNA expression of NAMPT in C2C12 myotubes with si Foxo3 and 3-PPA co-treatment (n = 4). j–k Immunoblots (j) and quantification (k) of Foxo3 protein expression in C2C12 myotubes with P7C3 and 3-PPA co-treatment (n = 3). (l-m) Immunoprecipitation (l) and statistics (m) of Ace- Foxo3 in C2C12 myotubes with 3-PPA treatment for 2 days (n = 4). (n–o) Immunofluorescence images (n) and statistics (o) of Foxo3 of C2C12 myotubes in C646 and 3-PPA co-treatment (n = 3). (p) Binding region of 3-PPA and Foxo3 protein. (q) Calculated binding energy of Foxo3 and GPR40. r–s Immunoblots (r) and quantification (s) of relative Foxo3 protein expression in CETSA assay by 3-PPA treatment (n = 3)
Fig. 5
Fig. 5
Schematic diagram of 3-PPA’s function on myotube hypertrophy
See this image and copyright information in PMC

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