Figure 4. Short-term high-fat diet (HFD) limits fibro/adipogenic progenitor (FAP) persistence in dystrophic muscles and restores β-catenin expression enhancing their promyogenic abilities.

(A) Schematic representation of the main molecular events reverted by HFD treatment in mdx FAPs. (B) Representative confocal images of PDGFRα-positive FAPs (green) from 49-d-old wt and mdx mice fed with low-fat diet (LFD) and HFD (60× magnification; scale bar, 20 μm). Fibers (red) were stained using antibodies directed against the MyHC isoforms. Representative micrograph (20× magnification; scale bar, 100 μm) showing proliferating FAPs by coupling PDGFRα staining (green) with anti-Ki67 antibodies (red). Nuclei (blue) were revealed with Hoechst 33342. (C) Bar plot reporting the number of PDGFRα-positive FAPs per cm² of muscle section (wt LFD n = 4; wt HFD n = 4; mdx LFD n = 3; mdx HFD n = 6). (D) Bar plot reporting the fraction of Ki67-positive cells in PDGFRα-positive FAPs in TA cross-sections (wt LFD n = 4; wt HFD n = 4; mdx LFD n = 3; mdx HFD n = 6). Statistical significance was estimated by two-way ANOVA. (E) Mass spectrometry–based quantitation of β-catenin and Mest in wt and mdx FAPs from mice fed with LFD and HFD. (F) Quantitative PCR for β-catenin and Mest in mdx FAPs from mice fed with HFD and LFD mice. (G) Quantitative PCR of Follistatin in wt and mdx FAPs from mice fed with LFD and HFD (wt LFD n = 3 mice; mdx LFD n = 3 mice; mdx HFD n = 4 mice). Statistical significance was estimated by one-way ANOVA. (H) Representative scheme summarizing the experimental procedure to treat, ex vivo, FAPs with BSA-coupled palmitate/oleate (50 μM/50 μM) and 100 μM carnitine. (H, I) Quantitative PCR of Ctnnb1 and Fst transcripts in mdx FAPs treated as shown in (H). Statistical significance was estimated by t test. (J) Representative scheme summarizing the experimental procedure to treat, ex vivo, mdx FAPs with 20 nM LY2090314. (K) Quantitative PCR of Ctnnb1 and Fst transcripts in mdx FAPs treated with 20 nM LY2090314 for 48 and 72 h. Statistical significance was estimated by Two-way ANOVA (n = 3). (L) Bar plot reporting the concentrations of Follistatin in FAP-derived supernatants. Follistatin concentrations were analyzed via ELISA assay. (M) Representative immunofluorescence (20× magnification; scale bar, 100 μm) of muscle satellite cell (MuSC)–derived myotubes (red) upon incubation with the control and LFD/HFD mdx FAP-derived supernatants. Proliferating myoblasts (green) were detected using a Ki67 specific antibody. (N) Bar plot reporting the fusion index (n = 6) of differentiated MuSCs in each treatment condition. (O) Bar plot reporting the fraction of Ki67-positive MuSCs in each treatment condition. Statistical significance was estimated by One-way ANOVA. All data are represented as mean ± SEM and Statistical significance is defined as *P < 0.05; **P < 0.01; ***P < 0.001.