p<0.05 was considered statistically significant. neutralization in HFD-challenged wild-type mice shows reduced vascularization in sWAT, associated with impaired glucose tolerance and lipid clearance. Blocking ANG-2 causes significant pro-inflammatory and pro-fibrotic changes, hallmarks of an unhealthy AT expansion. In contrast to other pro-angiogenic factors, such as vascular endothelial growth factor-A (VEGF-A), this is achieved without any enhanced beiging of white AT. DOI: http://dx.doi.org/10.7554/eLife.24071.001 mice, ANG-1 expression is decreased in AT, while ANG-2 expression is increased (Voros et al., 2005). Other data suggest that ANG-2 induced by FOXC2 overexpression results in enhanced blood vessel sprouting in white?AT (Xue et al., 2008). Our own data show that under different metabolic conditions, such as exercise, cold exposure, HFD feeding and fasting, mRNA levels in the ATs are also subject to change (Figure 1figure supplement 1AC1E). The physiological regulation of ANG-2, therefore, further supports the idea that ANG-2 plays an important functional role in AT physiology. There is an ongoing debate whether ANG-2 leads to vascular sprouting or regression, depending on the tissue under study. However, the specific contributions of ANG-2 to AT are unknown. Therefore, we utilized a genetic approach with a mouse model that specifically Mlst8 overexpresses ANG-2 in AT to help us define PP2 the effects of ANG-2 in AT PP2 angiogenesis and AT expansion upon exposing PP2 mice to a metabolic challenge, such as HFD. Here, we report results from a doxycycline (Dox)-inducible AT-specific ANG-2 overexpression mouse model with substantial induction of ANG-2 in sWAT. ANG-2 overexpressing mice show increased vascularization and reduced inflammatory changes in sWAT, leading to healthy AT expansion. This results in resistance to HFD-induced weight gain and improvements in metabolic function, including enhanced glucose tolerance, insulin sensitivity and lipid disposal. Conversely, we also demonstrate that antagonizing endogenous ANG-2 by neutralizing antibodies has opposite effects. Blocking ANG-2 causes a significant decrease in vascular density in sWAT, associated with an unhealthy expansion of AT defined by a highly pro-inflammatory microenvironment and pro-fibrotic changes. ANG-2 neutralization leads to an exacerbation of HFD-induced metabolic changes. Both gain- and loss-of-function studies of ANG-2 in AT therefore highlight the important role this factor plays in AT physiology. Results Inducible overexpression of ANG-2 in WAT promotes new blood vessel formation To directly evaluate the role of ANG-2 in AT, we took advantage of a mouse model carrying a tetracycline responsive element (TRE)-driven ANG-2 transgenic cassette (TRE-ANG2), shown schematically in Figure 1A. To obtain adipocyte-specific overexpression, we used a reverse tetracycline-dependent transcriptional activator (rtTA) which is driven by the adiponectin promoter (Adipo-rtTA). rtTA can bind and activate the TRE in the presence of Dox. Thus, we obtained the double transgenic mouse model (Adipo-ANG2) by crossing the TRE-ANG2 mice to the Adipo-rtTA mice and achieved an inducible and tissue-specific overexpression model of ANG-2 in the AT. To confirm both inducibility and tissue-specificity, we fed the mice with a chow diet containing 600 mg/kg Dox. After 5 weeks of Dox feeding, we harvested different fat pads and other tissues from both Adipo-ANG2 and Adipo-rtTA mice (serving as controls) and assessed mRNA levels. While both subcutaneous and epididymal WAT (sWAT and eWAT, respectively) show a significant increase in transcription, there was no elevation of mRNA levels in the liver, kidney or heart (Figure 1B). Notably, we also noticed enhancement of transcription in brown adipose tissue (BAT). Since the most abundant expression of mRNA was observed in sWAT, we examined the protein levels of ANG-2 in sWAT by Western blotting. Compared to Adipo-rtTA control mice, ANG-2 protein levels are significantly elevated in Adipo-ANG2 transgenic mice (Figure 1C). In addition, immunofluorescence staining of ANG-2 in sWAT reveals a substantially higher ANG-2 signal in Adipo-ANG2 mice (Figure 1D). Open in a separate window Figure 1. Adipocyte-specific, Dox-inducible overexpression of ANG-2.(A)?Schematic illustration of the adipocyte-specific, Dox-inducible ANG-2 PP2 overexpression mouse model. The adiponectin promoter-driven reverse tetracycline-dependent transcriptional activator (rtTA) mice were bred to tetracycline responsive element (TRE)-driven ANG-2 (TRE-ANG2) to generate the double transgenic mouse model. rtTA activity is activated to induce ANG-2 overexpression in the presence PP2 of Dox. (B) transcriptional changes in different tissues from control (Adipo-rtTA) mice and overexpressing (Adipo-ANG2) mice, fed with HFD/Dox 600 mg/kg for five weeks. (C) Representative Western blot image (upper panel) and quantitative analysis (lower panel) of ANG-2 and -actin protein levels in sWAT from HFD/Dox 600 mg/kg fed Adipo-rtTA mice and Adipo-ANG2.