Table 1.
The primer sequences for qRT-PCR.
Fig 1.
Estrogen deficiency induces PFM atrophy associated with ERα downregulation in OVX rats.
(A) Serum estrogen levels at 12 weeks after surgery. (B) Body weight monitoring from 5 to 12 weeks after surgery. (C) H&E staining results of the PFM of rats in the OVX and Sham groups. (D, E) MASSON staining results of the PFM of rats in the OVX and Sham groups. (F) Distribution of myofiber CSA in the PFM of rats in the OVX and Sham groups. (G, H) Fast (red) and slow (green) muscle staining results of the PFM of rats in the OVX and Sham groups. (I, J) Immunofluorescence results for ERα (red) and ERβ (green) of the PFM of rats in the OVX and Sham groups. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ns, no significance.
Fig 2.
Estrogen deficiency impairs C2C12 myoblast function and mitochondrial integrity.
(A) qRT-PCR results of Esr1 and Esr2 in C2C12 cells under EsD conditions. (B-C) Western blotting results of ERα and ERβ in C2C12 cells under EsD conditions. (D, E) EdU results in C2C12 cells under EsD conditions. (F) C2C12 cells differentiated into myotubes on Day 5 and Day 7. (G) qRT-PCR results of Myh7 and Myh2 in C2C12 cells under EsD conditions. (H, I) TUNEL results in C2C12 cells under EsD conditions. (J, K) Results of JC-1 assay in C2C12 cells under EsD conditions. (L,M) Flow sorting results for mitochondrial number in C2C12 cells under EsD conditions. (N,O) Western blotting results of MyHC, PGC1α and TFAM in C2C12 cells under EsD conditions. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ns, no significance.
Fig 3.
Effects of ERα agonist in estrogen-deficient C2C12 cells.
(A, B) EdU results in C2C12 cells under different culture conditions. (C-D) Cell cycle experiment results in C2C12 cells under different culture conditions. (E, F) TUNEL results in C2C12 cells under different culture conditions. (G) C2C12 cells differentiated into myotubes on Day 7. (H) Mitochondrial membrane potential levels of C2C12 cells under different culture conditions. (I, J) Mitochondrial quantity of C2C12 cells under different culture conditions. (K) qRT-PCR results of Myh7 and Ppargc1a in C2C12 cells under different culture conditions. (L, M) Western blotting results of MyHC, PGC1α and TFAM in C2C12 cells under different culture conditions. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ns, no significance.
Fig 4.
Identification and validation of GLUT4 as a key estrogen target in skeletal muscle metabolism.
(A) KEGG pathway enrichment analysis of DEGs in skeletal muscle transcriptomes from pre- versus post-menopausal women. (B) GSEA of KEGG pathways. Red and blue indicate overall up- or down-regulation in post-menopausal samples, respectively. NES, normalized enrichment score. (C) Correlation heatmap between estrogen receptor genes and glucose metabolism-related genes. Color and number in each cell represent the Pearson correlation coefficient. (D) Expression of Slc2a4 in the uterus of immature mice treated with estrogen. (E) Effect of estrogen on SLC2A4 expression in ER-negative breast cancer cells. (F, G) Immunohistochemical staining of GLUT4 in the PFM of Sham and OVX rats. (H) qRT-PCR results of Slc2a4 during C2C12 differentiation. (I, J) Glucose uptake assay in C2C12 cells on Day 3 of differentiation. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ns, no significance.
Fig 5.
ERα maintains myoblast function and mitochondrial integrity through GLUT4.
(A) qRT-PCR results of Slc2a4 and Rac1 in C2C12 cells under different culture conditions. (B, C) Western blotting results of GLUT4 and RAC1 in C2C12 cells under different culture conditions. (D-F) Immunofluorescence staining and co-localization analysis of GLUT4 (green) and RAC1 (red) in C2C12 cells under different culture conditions. Nuclei are counterstained with DAPI (blue). (G, H) Glucose uptake assay in C2C12 cells under different culture conditions. (I) Establishment of a GLUT4-overexpressing C2C12 cell line. (J) Myotube images of C2C12 cells under different conditions on Day 7 of differentiation. (K, L) Mitochondrial quantity of C2C12 cells under different culture conditions. (M) qRT-PCR results of Myh7 in C2C12 cells under different culture conditions. (N, O) Western blotting results of MyHC and PGC1α in C2C12 cells under different culture conditions. Data are presented as mean ± SD. *p < 0.05, **p < 0.01.
Fig 6.
In vivo experiments to validate the effect of ERα/GLUT4 axis on PFM.
(A, B) H&E staining results of the PFM of rats in different groups. (C-E) Fast (red) and slow (green) muscle staining results of the PFM of rats in different groups. (F, G) Immunohistochemical staining of GLUT4 in the PFM of rats in different groups. (H, I) Western blotting results of GLUT4, PGC1α and TFAM in the PFM of rats in different groups. Data are presented as mean ± SD. *p < 0.05, **p < 0.01.