Fig 2: Gab2 is increased in adipose tissue in HFD-fed mice and deletion of Gab2 protects against HFD-induced obesity.A Relative mRNA level of Gab2 in brown adipose tissue (BAT), inguinal (iWAT), and epididymal (eWAT) white adipose tissue from mice fed with a standard diet (SD) or a high-fat diet (HFD) for 12 weeks detected by RT-qPCR. The results were normalized with S18 mRNA levels. B Left panel: representative blots of Gab2 protein expression in BAT (top), iWAT (middle), and eWAT (bottom) of mice described in A detected by immunoblotting analysis; Right panel: the relative grayscale values of Gab2 protein blots, ß-Tubulin protein was measured as a loading control. C–F Bodyweight gain from weeks 6 to 18 (C), food intake (D), fat mass (E), and lean mass (F) of Gab2 KO and WT mice fed with SD and HFD diet for 12 weeks. The data are presented as mean ± SD from at least six mice in each group. Statistical difference is indicated: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Gab2 deficiency increases the expression of thermogenesis-related genes in HFD-fed mice and differentiated brown adipocytes.A Representative IHC staining images of Ucp1 in BAT from Gab2 KO and WT mice fed with HFD diet for 12 weeks. The scale bar is 50 µm. B Left panel, representative blots of Ucp1 and Gab2 protein expressions in BAT from Gab2 KO and WT mice fed with HFD diet for 12 weeks detected by immunoblotting analysis. Middle and right panel, the relative grayscale values of Ucp1 (middle) and Gab2 (right) protein blots were normalized with the loading control protein ß-Tubulin. C, D The mRNA level of thermogenesis-related genes (C) and mitochondrial function associated genes (D) in BAT from Gab2 KO and WT mice fed with HFD diet for 12 weeks detected by RT-qPCR. The results were normalized with S18 mRNA levels. E Oil Red O staining of completely differentiated brown adipocytes (Day 6). F Left panel, representative blots of Ucp1 and Gab2 protein expressions during the differentiation of brown preadipocytes detected by immunoblotting analysis. Middle and right panel, the relative grayscale values of Ucp1 (middle) and Gab2 (right) protein blots were normalized with the loading control protein ß-Tubulin. G The mRNA level of Gab2 (left panel) and Ucp1 (right panel) during the differentiation of brown preadipocytes detected by RT-qPCR. The results were normalized with S18 mRNA levels. H Ucp1-luciferase reporter activity in response to Gab2 overexpression (left panel) or Gab2 knockdown (right panel) analyzed in immortalized brown preadipocytes by using the Dual-Luciferase Reporter Assay System (Promega). Con, immortalized brown preadipocytes only transfected with Ucp1-luciferase reporter and renilla; si-con, siRNA of control protein. I Representative blots of Ucp1 and Gab2 protein expressions in undifferentiated and differentiated brown adipocytes detected by immunoblotting analysis. ß-Tubulin protein was measured as a loading control. Con, undifferentiated brown preadipocytes; Induced, differentiated brown adipocytes. J The mRNA level of common or brown fat-selective genes in differentiated brown adipocytes (Day 6) detected by RT-qPCR. The results were normalized with S18 mRNA levels. The data are presented as mean ± SD from at least six mice in each group or three independent experiments. Statistical difference is indicated: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: Deletion of Gab2 reduces the adipose tissue weight in mice fed with HFD.A, B Tissue weight (A) and tissue weight ratio to bodyweight (B) of Gab2 KO and WT mice fed with SD and HFD diet for 12 weeks. C–F Representative image (C), hematoxylin and eosin (H&E) staining and adipocyte area of iWAT (D), eWAT (E) and BAT (F) from Gab2 KO and WT mice fed with SD and HFD diet for 12 weeks, scale bar is 50 µm. The data are presented as mean ± SD from at least six mice in each group. Statistical difference is indicated: *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: Gab2 deficiency reduces macrophage infiltration and inflammation in adipose tissue in HFD-fed mice.A Representative immunohistochemistry (IHC) staining images of F4/80 protein in eWAT from Gab2 KO and WT mice fed with HFD diet for 12 weeks. Crown-like structures are indicated by the red arrows. Scale bars are 100 µm (top) and 50 µm (bottom). B Quantification of crown-like structure (CLS) in eWAT from Gab2 KO and WT mice fed with HFD diet for 12 weeks. C The mRNA level of F4/80 and Mcp-1 in eWAT from Gab2 KO and WT mice fed with SD or HFD diet for 12 weeks were detected by RT-qPCR. The results were normalized with S18 mRNA levels. D Representative picro-sirius staining images of collagen fibrils in eWAT from Gab2 KO and WT mice fed with HFD diet for 12 weeks. Collagen fibrils are indicated by the red arrows. Scale bars are 100 µm (top) and 50 µm (bottom). E The mRNA level of adiponectin (Adipoq), leptin, and inflammation cytokines (Tnf-a, Il-1ß, Il-6) in eWAT from Gab2 KO and WT mice fed with HFD diet for 12 weeks detected by RT-qPCR. The results were normalized with S18 mRNA levels. F–H The concentration of adiponectin (F), leptin (G), and inflammation cytokines (TNF-a, IL-1ß, IL-6, H) in blood plasma samples analyzed by ELISA assay. The data are presented as mean ± SD from at least 6 mice in each group. Statistical difference is indicated: *P < 0.05; **P < 0.01; ***P < 0.001.