Figure 4.

Ablation of GSK3β (glycogen synthase kinase-3 beta) in adipocytes stimulates the expansion of adipose tissue (AT) vasculature and upregulates the expression of genes associated with angiogenesis in the AT of obese mice. Male C57Bl/6J GSK3βfl/fl and GSK3βADKO mice were fed a high-fat diet to induce obesity and used for the experiments. A, Gene ontology (GO) analysis showing upregulated gene signatures in the epididymal white adipose tissue (eWAT) of obese GSK3β^ADKO vs obese GSK3β^fl/fl mice (n=2). B, Heat map of expression values (Z score) for angiogenesis-associated genes in eWAT from the indicated obese mice. C, Validation of angiogenesis-associated genes using reverse transcription polymerase chain reaction (n=6). D, Western blotting analysis of the indicated proteins in eWAT from the indicated obese mice. E, VEGF (vascular endothelial growth factor) protein level analysis using ELISA in eWAT lysates from the indicated obese mice (n=7). F through H, Western blotting images of the indicated proteins in adipocytes (F) and stromal vascular fraction (SVF; G) from the indicated obese mice, along with their protein quantification (H; n=6). I, Representative images of whole-mount staining of eWAT for CD31 and analysis for CD31-positive area, capillary branch density, and capillary diameter quantified using Image J software (n=7). J, Representative images of whole-mount staining of eWAT from obese mice injected with lectin and quantification of the lectin area (n=6). K, Serum glycerol levels in the indicated obese mice before or 30 minutes after injection with 0.75 U/kg insulin (n=6). L, In vitro tube-formation assay of 3B-11 mouse endothelial cells cocultured with primary adipocytes (Pad, passage 0 [P0]) isolated from the indicated obese mice. Analysis is for the number of meshes, number of junctions, and total segment length (n=4). Scale bar=100 µm. C, E, and H through K, Two-tailed unpaired t test; L, nonparametric test.