Transcription Factor E2F1 Knockout Promotes Mice White Adipose Tissue Browning Through Autophagy Inhibition

FIGURE 1. Effects of E2F1 knockout on body weight and inguinal adipose tissue morphology in mice. (A) Gross view of mice, in situ subcutaneous WAT, and ex vivo dissected WAT in WT and E2F1–/– group. (B) The bodyweight curve of the WT and E2F1–/– mice (n = 10) for 8 weeks. (C) Representative H&E staining images of WAT in WT and E2F1–/– mice. Scale bar, 100 μm. Magnification, 400×. (D) Quantitative analysis of adipocyte diameter in WT and E2F1–/– WAT. ∗∗∗P < 0.001.

FIGURE 2. E2F1 knockout increased the mitochondrial number and the UCP-1 expression in WAT in vivo. (A) Representative TEM images of WAT ultrastructures in WT and E2F1–/– group. Scale bar, 10 μm (left) and 2 μm (right). (B) Representative IHC pictures of UCP-1 protein (brown stain) in WAT of WT and E2F1–/– mice. Scale bar, 100 μm. Magnification, 400×. (C) Representative Western Blot images of UCP-1 protein level in WT and E2F1–/– WAT, n = 3. (D) The UCP-1 mRNA levels in WT and E2F1–/– WAT in mice by qRT-PCR. (E) The PPARγ mRNA levels in WT and E2F1–/– WAT in mice by qRT-PCR. (F) Quantitative analysis of mitochondrial number per cell in WT and E2F1–/– WAT. (G) The expression level of UCP1 staining in WAT was indicated with a stained positive index (integrated density/area) in WT and E2F1–/– WAT. ∗P < 0.05, ∗∗P < 0.01.

FIGURE 3. E2F1 knockout affected the expression of autophagy-related proteins in WAT in mice. (A) Representative Western Blot image of E2F1, LC3-II/I, and p62 protein levels in WT and E2F1–/– WAT. (B) mRNA levels of E2F1, LC3-II, ATG5, and ATG7 genes in WT and E2F1–/– WAT by qRT-PCR. (C) Quantitative analysis of the protein expressions relative to β-actin, n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

FIGURE 4. Characterization of mice ADSCs. (A) Representative images of WT and E2F1–/– ADSCs (P3). Scale bar, 100 μm. (B) Adipogenic differentiation was analyzed by Oil Red O staining. Scale bar, 100 μm. (C) Flow cytometry of phenotypic markers of ADSCs. The WT and E2F1–/– ADSCs were positive for CD29, CD90, CD105, and CD44, and negative for CD31 and CD34.

FIGURE 5. E2F1 knockout increased UCP-1 expression and reduced autophagy gene expressions in adipocytes in vitro. (A) Representative Western Blot image showing the level of UCP-1 protein in adipocytes and E2F1–/– adipocytes. (B) qRT-PCR analyses of UCP-1 expression at the mRNA level in adipocytes and E2F1–/– adipocytes. (C) Representative Western Blot image of E2F1, LC3-II/I, and p62 protein levels in adipocytes and E2F1–/– adipocytes. (D) The qRT-PCR analyses of E2F1, and LC3-II expressions at the mRNA level in adipocytes and E2F1–/– adipocytes. (E) Quantitative analysis of the protein expressions relative to β-actin, n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

FIGURE 6. Autophagy inhibition increased the UCP-1 expression, without reduced E2F1 expression in adipocytes in vitro. (A) Representative Western Blot image showing the level of LC3-II/I and UCP-1 protein in 3T3-L1 adipocytes and 3T3-L1 + 3MA adipocytes. (B–D) qRT-PCR analyses of LC3-II, p62, ATG5, UCP-1, PPARγ, and E2F1 expression at the mRNA level in 3T3-L1 adipocytes and 3T3-L1 + 3MA adipocytes. (E) The qRT-PCR analysis of E2F1 expression at the mRNA level in ADSCs + siNC group, ADSCs + E2F1 siRNA group, and ADSCs + 3MA group. (F) Representative Western Blot image of LC3-II/I, and UCP-1 protein levels in ADSCs + siNC group, ADSCs + E2F1 siRNA group, and ADSCs + 3MA group. (G,H) The qRT-PCR analyses of p62, and UCP-1 expressions at the mRNA level in ADSCs + siNC group, ADSCs + E2F1 siRNA group, and ADSCs + 3MA group. (I) Quantitative analysis of the LC3-II/I and UCP-1 protein expressions relative to β-actin in 3T3-L1 adipocytes and 3T3-L1 + 3MA adipocytes, n = 3. (J) Quantitative analysis of the LC3-II/I and UCP-1 protein expressions relative to β-actin in ADSCs + siNC group, ADSCs + E2F1 siRNA group, and ADSCs + 3MA group, n = 3. ∗P < 0.05, ∗∗P < 0.01.

FIGURE 7. Graphical summary. E2F1–/– WAT exhibited multiple small lipid droplets, densely packed mitochondria, and highly UCP-1 expression, indicating the browning form. Mechanically, E2F1 knockout suppressed the autophagy activity to promote WAT browning in mice, which was beneficial to expend energy in thermogenic manner for combating obesity.