Figure 7. Leptin and insulin induce WAT browning.

a–c) 8 week-old mice were administered leptin IP and a) daily body weights recorded, b–c) ingWAT and BAT extracted for b) analysis of Ucp-1 gene expression, c) histology and immunohistochemistry; C57BL/6 mice were administered vehicle as a control. d–g) 8 week-old C57BL/6 mice were ICV infused vehicle, insulin (3 mU/day), leptin (4.8 μg/day) or insulin + leptin and d) body weights monitored, e–f) BAT and ingWAT extracted for e) gene expression analyses, f) histology and immunohistochemistry, and g) energy expenditure assessed. h–j) C57BL/6 mice were subjected to sham or ingWAT bilateral denervations (6-OHDA) and ICV infused with insulin + leptin and h) body weights, i) BAT and ingWAT Ucp-1 expression and j) energy expenditure monitored. k–m) C57BL/6 mice were implanted with bilateral intra-ARC cannulas and infused with vehicle, insulin (3 mU/day) + leptin (4.8 μg/day) or insulin + leptin + PI3K inhibitor (PI3Ki; LY294002, 5 μg/day). k) Daily body weights were recorded (insert: intra-ARC cannula placement) and ingWAT extracted for l) analysis of Ucp-1 gene expression, m) histology and UCP-1 immunohistochemistry. n–p) Pomc-Cre mice were bilaterally injected with rAAV-hSyn-DIO-hM3D(Gq)-mCherry into the ARC. Contralateral ingWAT depots were sham operated or denervated and the mice administered vehicle or CNO (1.5 mg/kg/day, IP) and ingWAT processed for n) in situ morphology, o) gene expression p) histology and immunohistochemistry. Data are means ± SEM and are representative of 3 independent experiments; significance determined using a, d, h, j) two-way ANOVA, or b, e, g, i, k, l, n, o) one-way ANOVA. d) * corresponds to leptin (L) or L/insulin (I) v/s vehicle (V); ^# L/I versus L. h) * corresponds to L/I v/s V; ^# L/I sham v/s denervated. k) * corresponds to L/I, or L/I + PI3Ki v/s V; ^# L/I v/s L/I + PI3Ki.