Fig 1: Ingenuity Pathway Analysis (IPA) and western blotting validation of common differentially expressed proteins. (a) Top 15 canonical pathways according to IPA. The blue bars represent -log(P value), and the orange points denote the ratio of differentially expressed proteins. (b–d) Top 3 networks generated by IPA. Network 1 is involved in metabolic disease, developmental disorder, and hereditary disorder (b); Network 2 is involved in energy production, lipid metabolism, and small molecule biochemistry (c); Network 3 is involved in lipid metabolism, small molecule biochemistry, and vitamin and mineral metabolism (d). (e) Validation of differentially expressed proteins PON1, GPD2, and APOA1 by western blotting. Data are shown as mean ± SEM, n = 5-6. #P < 0.05 versus WT and *P < 0.05 versus ApoE-/-. WT: wild-type; BBR: berberine; PON1: paraoxonase 1; APOA1: apolipoprotein A I; GPD2: glycerol-3-phospate dehydrogenase 2.
Fig 2: mGPDH activation improves skeletal muscle regeneration in ob/ob mice A–DExperimental setup (A, upper panel) and qRT–PCR of mGPDH, myogenin, and myh3 gene expressions (A, bottom panel), H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (B), distribution of CSA (C), and percentage of fibers with central nuclei (D) in GA muscle from AAV-mGPDH-treated ob/ob mice at day 7 post-CTX.Data information: Data are presented as the mean ± s.e.m. Scale bars represent 100 µm (25 µm for magnification insets) in panel (B). In panels (A–D), n = 6 mice per group; in panels (B–D), three sections were obtained per mouse. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired t-test was used for all analyses except in panel (C), where the Kolmogorov–Smirnov test was used.
Fig 3: mGPDH is essential to skeletal muscle regeneration A, BqRT–PCR (A) and immunoblot (B) of mGPDH, myogenin, and developmental myosin heavy chain (myh8, myl4, and myh3) in gastrocnemius (GA) muscle from C57BL/6J mice at the indicated day after CTX intramuscular injection.CActivity assay of mGPDH in GA muscle from C57BL/6J mice at days 0 and 7 after CTX injection.D–GRepresentative images of the H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (D), distribution of the fiber cross-sectional area (CSA) (E), percentage of myofibers with central nuclei (F), and immunofluorescence staining of desmin (green) (G) in GA muscle from WT and mGPDH-/- mice at day 7 post-CTX injection.H, IMuscle weight (H) and trichrome staining (I) in GA muscle from WT and mGPDH-/- mice at day 14 post-CTX injection. Quantification represents the fibrotic areas.J, KqRT–PCR (J) and immunoblot (K) for mGPDH, myogenin, and myh3 in GA muscle from WT and mGPDH-/- mice at day 7 post-CTX injection.L–QqRT–PCR for mGPDH, myogenin, and myh3 (L), H&E staining (M), distribution of the fibers CSA (N), qRT–PCR (O), and immunofluorescence staining (P) for utrophin and trichrome staining (Q) in GA muscle from mdx mice 4 weeks after AAV-mGPDH intramuscular injection.RExercise capacity of mdx mice 6 weeks after AAV-mGPDH tail vein injection.Data information: Data are presented as the mean ± s.e.m. Scale bars represent 100 µm (25 µm for magnification insets) in panels (D, I, M, and Q) and 50 µm in panels (G, P). In panels (A–C), n = 3; in panels (D–R), n = 6 mice per group; in panels (D–F, M, and N), three sections were obtained per mouse. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired t-test was used for all analyses except in panels (E, N), where the Kolmogorov–Smirnov test was used.Source data are available online for this figure.
Fig 4: Effect of mGPDH on inflammatory signaling A–CQuantification of the indicated inflammatory cytokines by qRT–PCR in GA muscles of mGPDH-/- mice (A), HFD-fed and STZ-treated mice (B), and HFD-fed and STZ-treated mice intramuscularly injected with AAV-mGPDH (C) at day 7 post-CTX injury.Data information: Data are presented as the mean ± s.e.m. n = 6 mice per group. *P < 0.05, **P < 0.01. Unpaired t-test was used for all panels.
Fig 5: Rescuing mGPDH deficiency improves skeletal muscle regeneration during obesity and diabetes A–CImmunoblot (A, C) and IHC (B) of mGPDH and myogenin in GA muscles from obese patients (A, B) and the indicated mice (C).DqRT–PCR of mGPDH in GA muscle of the indicated mice at days 0 and 3 after CTX intramuscular injection.E–HExperimental setup (E, upper panel); qRT–PCR of mGPDH, myogenin, and myh3 (E, bottom panel); H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (F); distribution of the fiber CSA (G); and percentage of myofibers with central nuclei (H) in GA muscle from AAV-mGPDH-treated HFD-fed mice at day 7 after CTX intramuscular injection.I–MExperimental setup (I and M, upper panels); qRT–PCR of mGPDH, myogenin, and myh3 (I, bottom panel); H&E staining (arrowhead, necrotic myofibers; asterisks, regenerating fibers) (J); distribution of the fibers CSA (K); percentage of myofibers with central nuclei (L); and muscle weight (M, bottom panel) in GA muscle from AAV-mGPDH-treated STZ-treated mice at days 7 (I–L) and 14 (M) after CTX intramuscular injection.NImmunoblots of mGPDH, p-AMPK, p-ACC, PGC1a, and myogenin for the experiment described in (E).Data information: Data are presented as the mean ± s.e.m. Scale bars represent 200 µm in panel (B) and 100 µm (25 µm for magnification insets) in panels (F, J). In panels (A, B), obese patients (n = 11) and normal subjects (n = 18); in panels (C, D), n = 3 mice per group; in panels (E–L and N), n = 6 mice per group; in panel (M), n = 4 mice per group; in panels (B, F–H, and J–L), three sections were obtained per mouse. *P < 0.05, **P < 0.01, ***P < 0.001. Unpaired t-test was used in panels (D, E, H, I, L, and M); the Wilcoxon test was used in panel (B); and the Kolmogorov–Smirnov test was used in panels (G, K).Source data are available online for this figure.
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