Figure 5. Short-term high-fat diet (HFD) ameliorates the mdx phenotype.

(A) Serum creatine phosphokinase (units per liter, U/l) from 49-d-old wt and mdx mice fed with low-fat diet (LFD) or HFD (wt LFD n = 6; wt HFD n = 7; mdx LFD n = 6; mdx HFD n = 10). (B) Representative hematoxylin and eosin staining of TA cross-sections from 49-d-old wt and mdx mice fed with LFD or HFD (20× magnification; scale bar, 100 µm). (C) Percentage of centrally nucleated myofibers in TAs (wt LFD n = 8; wt HFD n = 6; mdx LFD n = 6; mdx HFD n = 8). (D) Representative hematoxylin and eosin staining of diaphragm cross-sections from 49-d-old wt and mdx mice fed with LFD or HFD (20× magnification; scale bar, 100 μm). (E) Percentage of centrally nucleated myofibers in diaphragms (wt LFD n = 3; wt HFD n = 3; mdx LFD n = 3; mdx HFD n = 3). Statistical significance was estimated by Two-way ANOVA. (F) Representative picrosirius red staining of TA cross-sections (20× magnification; scale bar, 100 ìm). (G) Bar plot showing the extent of picrosirius red area in TA cross-sections from 49-d-old wt and mdx mice fed with LFD or HFD (wt LFD n = 8; wt HFD n = 7; mdx LFD n = 6; mdx HFD n = 8). (H) Representative picrosirius red staining of diaphragm cross-sections from 49-d-old wt and mdx mice fed with LFD or HFD (20× magnification; scale bar, 100 μm). (I) Bar plot showing the extent of picrosirius red area in diaphragm cross-sections (wt LFD n = 3; wt HFD n = 3; mdx LFD n = 3; mdx HFD n = 3). Statistical significance was estimated by one-way ANOVA. (J) Representative confocal micrographs of laminin-stained (green) TA cross-sections from 49-d-old wt and mdx mice fed with LFD or HFD (20× magnification; scale bar, 70 μm), left panel. Pseudo-color representation of the myofiber caliber, ranging from 0 to 5,000 μm², right panel. Nuclei (blue) were revealed with Hoechst 33342. (K) Bar plot reporting the average cross-sectional area in μm² (wt LFD n = 8; wt HFD n = 6; mdx LFD n = 6; mdx HFD n = 6). Statistical significance was estimated by two-way ANOVA. (L) Immunoblot of TA lysates from wt and mdx mice fed with LFD and HFD. Samples were probed with anti-pmTOR (Ser2448), anti-pRPS6 (Ser240/244), anti-mTOR, anti-RPS6, and anti-vinculin antibodies. (M) Bar plot showing the densitometric analysis of the phosphorylation level of mTOR (posho-mTOR). (N) Bar plot showing the densitometric analysis of the phosphorylation level of RPS6 (posho-RPS6). Statistical significance was estimated by two-way ANOVA. (O) Quantitative PCR of Igf1 in fibro/adipogenic progenitors from wt and mdx mice fed with LFD or HFD (wt LFD n = 3; wt HFD n = 4; mdx LFD n = 5; mdx HFD n = 7). Statistical significance was estimated by two-way ANOVA. All data are represented as mean ± SEM and statistical significance is defined as *P < 0.05; **P < 0.01; ***P < 0.001.