Angiogenesis–Browning Interplay Mediated by Asprosin-Knockout Contributes to Weight Loss in Mice with Obesity

Figure 1. ASP-knockout alleviates mouse obesity induced by HFD. Representative appearance photographs (A) of C57BL/6 mice. Body weight (B), length (C) and Lee’s index (D) were measured and calculated. Representative photographs (E) and weights (F) from eWAT in mice. Representative HE staining images from eWAT (E) and adipocyte sizes from eWAT (G). Data are presented as mean ± SEM from three independent experiments (n = 3). Two-way ANOVA was performed followed by an unpaired two-tailed Student’s t-tests. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. the WT-ND group; # p < 0.05, ## p < 0.01, vs. the WT-HFD group; & p < 0.05, && p < 0.01, vs. the CKO-ND group; ns = no significance. WT-ND: wild type mice fed with normal diet; WT-HFD: wild type mice fed with high fat diet; CKO-ND: ASP-conditional knockout mice fed with normal diet; CKO-HFD: ASP-conditional knockout mice fed with high fat diet; eWAT: epididymal white adipose tissue. Scale bars in the images of HE staining in (E) 20 μm. ASP: asprosin, HE staining: hematoxylin-eosin staining.

Figure 2. ASP-knockout promotes angiogenesis and browning of white adipose in vivo. Representative images (A) of immunohistochemistry and immunofluorescence. Relative expression levels in situ of UCP1 (B) and CD31 (C) in eWAT normalized by the adipocyte size. Expression levels of CD31 in eWAT by Western blot (D,E). Expression levels of the browning-related proteins, including PRDM16, PGC-1α and UCP-1 in eWAT (F,G). Data are presented as mean ± SEM from three independent experiments (n = 3). Two-way ANOVA was performed followed by an unpaired two-tailed Student’s t-tests. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. the WT-ND group; # p < 0.05, ## p < 0.01, vs. the WT-HFD group; & p < 0.05, && p < 0.01, &&& p < 0.001, vs. the CKO-ND group. WT-ND: wild type mice fed with normal diet; WT-HFD: wild type mice fed with high fat diet; CKO-ND: ASP-conditional knockout mice fed with normal diet; CKO-HFD: ASP-conditional knockout mice fed with high fat diet; eWAT: epididymal white adipose tissue. Scale bars in the images of (A) 20 μm. ASP: asprosin, UCP1: uncoupling protein 1, PGC1-α: PPAR gamma coactivator 1, PRDM16: PRD1-BF-1-RIZ1 homologus domain-containing protein-16.

Figure 3. ASP-knockout promotes the migration and angiogenesis of vascular endothelial cells in vitro. Identification of ASP-knockout in 3T3-L1 adipocytes by Western blot (A,B). Wound-healing assay in exposure of HUVECs to ASP−/−-CM or WT-CM for 24 h or 48 h (C,D). Angiogenesis assay of HUVECs in vitro by a Matrigel system (E–G). Data are presented as mean ± SEM from three independent experiments (n = 3). Two-way ANOVA was performed, followed by an unpaired two-tailed Student’s t-test for Figure C and D; an unpaired two-tailed Student’s t-test was used in other figures. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. the WT group or the WT-CM group; # p < 0.05, vs. 24 h. WT: wild type 3T3-L1 preadipocytes; ASP−/−: ASP-knockout of 3T3-L1 preadipocytes; WT-CM: conditional medium from WT-3T3-L1 adipocytes; ASP−/−-CM: conditional medium from ASP−/−-3T3-L1 adipocytes; Nb: numbers, Tot: total. Scale bars: 200 μm (C) and 100 μm (E). ASP: asprosin.

Figure 4. ASP-knockout enhances VEGF/VEGFR2 and PI3K/AKT/eNOS signaling in vitro. HUVECs were treated with ASP−/−-CM or WT-CM for 48 h and total proteins were extracted and quantified. Expression levels of VEGF/VEGFR2 pathway proteins (A,B) and PI3K/AKT/eNOS pathway proteins were analyzed by Western blots (C,D). Data are presented as mean ± SEM from three independent experiments (n = 3). An unpaired two-tailed Student’s t-test was used for the comparison between two groups. * p < 0.05, ** p < 0.01, vs. the WT-CM group. WT-CM: conditional medium from WT-3T3-L1 adipocytes; ASP−/−-CM: conditional medium from ASP−/−-3T3-L1 adipocytes. ASP: asprosin, VEGF: vascular endothelial growth factor, VEGFR2: vascular endothelial growth factor receptor 2; PI3K: Phosphatidylinositol 3-kinase, AKT: protein kinase B, eNOS: endothelial nitric oxide synthase.

Figure 5. Effect of EC-CM on lipid aggregation and browning in vitro, where 80% of confluent 3T3-L1 adipocytes were treated with EC-CM or NM for 48 h. Lipid accumulation was detected by oil red O staining (A,B). Expression levels of browning-related proteins, including UCP-1, PGC1-α and PRDM16 (C,D), were analyzed by Western blots. Data are presented as mean ± SEM from three independent experiments (n = 3). An unpaired two-tailed Student’s t-test was used. * p < 0.05, ** p < 0.01, vs. the NM group. EC-CM: conditional medium from endothelial cells; NM: normal high-glucose DMEM completed medium. Scale bar in the images of (A): 20 μm. ASP: asprosin, UCP1: uncoupling protein 1, PGC1-α: PPAR gamma coactivator 1, PRDM16: PRD1-BF-1-RIZ1 homologus domain-containing protein-16.

Figure 6. EC-CM treatment enhances the mitochondrial biogenesis and respiratory function of adipocytes in vitro. Mitochondrial contents were evaluated by a mitochondrial fluorescent probe dye under confocal microscopy (A,B). Mitochondria OCR curves (C) and their quantification analysis (D) under basal or stress conditions were prepared by a Seahorse XF Analyzer in 3T3-L1 adipocytes. Data are presented as mean ± SEM from three independent experiments (n = 3) and were analyzed by an unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, vs. NM group. EC-CM: conditional medium from endothelial cells; NM: normal high-glucose completed DMEM. Scale bar in the images of (A): 20 μm. OCR: oxygen consumption rate, FCCP: Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone.