Angiopoietin-2 in white adipose tissue improves metabolic homeostasis through enhanced angiogenesis

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  Figure 1.. Adipocyte-specific, Dox-inducible overexpression of ANG-2. (A) Schematic illustration of the adipocyte-specific, Dox-inducible ANG-2 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 of Dox. (B) Angpt2 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 β-actin protein levels in sWAT from HFD/Dox 600 mg/kg fed Adipo-rtTA mice and Adipo-ANG2 mice. (D) Representative images of ANG-2 immunofluorescence staining (Red, Magnification = 200 × ) in both Adipo-rtTA control and Adipo-ANG2 double transgenic groups after Dox induction. Perilipin (Blue) was also stained to label adipocytes. Bar = 100 µm. For all the statistical graphs: n = 5 ~ 7 mice per group. Data are shown as mean ± s.e.m. *p<0.05. DOI:…

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  Figure 1—figure supplement 1.. Differential regulation of Angpt2 mRNA under variety of metabolic challenges. (A) Angpt2 mRNA levels in sWAT from mice under static conditions or subjected to a running exercise (6 hr daily) for five weeks.(B) Angpt2 transcriptional changes in BAT, subcutaneous WAT (SC WAT) and epididymal WAT (IP WAT) from mice exposed to cold (6°C) or room temperature (RT) for 72 hr.(C) Ang-2 mRNA expression in the tissues of sWAT, eWAT and liver from chow diet and HFD fed mice for 6 months.(D) Angpt2, Angpt1 and Vegfa mRNA levels under fed and fasting conditions (24 hr). (E) Angpt2 mRNA expression in adipocyte and stromal vascular fractions (SVF) of sWAT and eWAT from chow diet and HFD-fed mice for 6 months. For all figures: n = 6 mice per group. Data are shown as mean ± s.e.m. *p<0.05. DOI: http://dx.doi.org/10.7554/eLife.24071.003

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  Figure 2.. Vascular changes after overexpression of ANG-2. (A) Angiogenic factors and endothelial cell markers including Vegfa, Tek, Pecam1 and Antxr1 mRNA levels in the harvested sWAT from 5 week HFD/Dox 600 mg/kg induced Adipo-rtTA and Adipo-ANG2 mice.(B) Representative immunofluorescence staining image of endothelial marker endomucin (Green, Magnification = 100×) and (C) the statistical result of relative endomucin area calculated by the percentage of endomucin positive signals in the total area. SWAT section staining images from both groups are shown. Bar = 160 µm. For all the statistical graphs: n = 6 mice per group. Data are shown as mean ± s.e.m. *p<0.05, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.24071.004

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  Figure 2—figure supplement 1.. Hypoxic parameters after ANG-2 overexpression in sWAT. (A) Representative immunoblot image (upper panel) and quantitative analysis (lower panel) of HIF-1α expression in sWAT from both control and ANG-2 transgenic mice. (B and C). Hif1a and Slc2a1 (stands for GLUT1) transcriptional changes in sWAT of both groups. (D) Representative images of pimonidazole immunohistochemistry staining (brown, Magnification = 100 × ) in both Adipo-rtTA control and Adipo-ANG2 transgenic groups after Dox induction. Bar = 100 μm. For the western blot image, n = 3 mice per group. For all other statistical figures: n = 10 mice per group. Data are shown as mean ± s.e.m. **p<0.01, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.24071.005

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  Figure 3.. Metabolic effects in mice overexpressing ANG-2. After HFD/Dox 600 mg/kg induction for five weeks, Adipo-rtTA control and Adipo-ANG2 transgenic mice were subjected to the following metabolic analyses: (A) Body weight was continuously monitored for five weeks.(B) Body composition data obtained via MRI. Both the percentage of fat mass and lean mass are shown. Glucose levels at different time-points from (C) oral glucose tolerance tests (OGTT) and (D) Insulin tolerance tests (ITT).(E) Oxygen consumption (VO2, left panel) and Carbon dioxide generation (VCO2, right panel) data measured by metabolic cages. The values for both light and dark cycles are shown. (F) Non-esterified free fatty acid (NEFA), cholesterol and triglyceride levels in serum samples from both groups. (G) After an oral gavage of 20% Intralipid, the clearance of serum triglycerides is shown over time for a period of 8 hr. (H) Lipoprotein lipase (LPL) activity measured through the LPL activity assay kit in the plasma, sWAT and eWAT from both groups. (I) Angiopoietin-like protein family members Angptl3, Angptl4, Angptl8 mRNA levels in the harvested sWAT from 5-week HFD/Dox 600 mg/kg fed Adipo-rtTA and Adipo-ANG2 mice.…

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  Figure 3—figure supplement 1.. Insulin signaling in the ANG-2 overexpressing mice. (A) Representative immunoblot image of phosphorylated Akt (pAkt, Ser 473) and total Akt expression in sWAT from both control and ANG-2 overexpressing mice after saline or insulin injection (i.v.) for 5 min. For the Western blot image, n = 3 mice per group. DOI: http://dx.doi.org/10.7554/eLife.24071.007

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  Figure 3—figure supplement 2.. Prevention effect of ANG-2 induction against high-fat diet induced obesity. Mice in both control and ANG-2 overexpression groups were pre-induced with Dox 600 mg/kg for five weeks, and then Dox containing food was replaced with high-fat diet. After subjecting the mice onto high-fat diet, (A) body weight gains on Day 3, Day 7 and Day 10 are recorded; (B, C and D). Oral glucose tolerance test (OGTT) results on high-fat diet Day 0, Day 3 and Day 7 are shown, respectively. For all statistical figures: n = 6 mice per group. Data are shown as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.24071.008

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  Figure 4.. Inflammatory and histological changes after overexpression of ANG-2. (A) Representative H&E staining of sWAT sections (Magnification = 200×) from 5 week HFD/Dox 600 mg/kg fed Adipo-rtTA and Adipo-ANG2 mice. Bar = 60 µm. (B) Representative H&E staining of liver tissues (magnification = 200×) from both groups. Lipid droplets are evident in the liver sections. Bar = 60 µm.(C) Representative immunofluorescence staining picture of the macrophage marker F4/80 (Green, Magnification = 200×) in sWAT sections from both groups. Bar = 100 µm. (D and E). Inflammatory marker mRNA levels assessed by qPCR in sWAT of control and ANG-2 transgenic mice. General inflammatory markers: Adgre1 (F4/80), Il6 (IL-6), Csf1r (CD115), Itgam (CD11b); pro-inflammatory macrophage markers: Ly6c, Ifng, Tnfa, Nos2; anti-inflammatory macrophage markers: Mrc1 (gene symbol for CD206), Il10, Clec10a (gene symbol for CD301). (F) The relative content of anti-inflammatory macrophages (M2 type macrophages) in both sWAT (left panel) and eWAT (right panel) from control and ANG-2 overexpressing mice. For the flow cytometry analysis, anti-inflammatory macrophages are considered as the F4/80+/CD11b+/CD206+ population, and the…

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  Figure 4—figure supplement 1.. Inflammation in epididymal WAT (eWAT) from ANG-2 overexpressing mice. (A) Representative H and E stain of eWAT sections (Magnification = 100 × ) from 5-week HFD/Dox 600 mg/kg fed Adipo-rtTA and Adipo-ANG2 mice. Bar = 160 µm. (B and C). Inflammatory marker mRNA levels measured through qPCR in eWAT of control and ANG-2 transgenic mice. General inflammatory markers: Adgre1, Il6, Csf1r, Itgam; pro-inflammatory macrophage markers: Ly6c1, Ifng, Tnfa, Nos2; anti-inflammatory macrophage markers: Mrc1, Il10, Clec10a. For all statistical figures: n = 10 mice per group. Data are shown as mean ± s.e.m. **p<0.01. DOI: http://dx.doi.org/10.7554/eLife.24071.010

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  Figure 4—figure supplement 2.. Representative histogram of flow cytometry analysis for anti-inflammatory (M2) macrophages in ANG-2 transgenic mice. (A) Representative flow cytometry histogram demonstrating the relative percentage of anti-inflammatory macrophages in the total macrophage population, specific anti-inflammatory macrophage marker CD206 is labeled with PerCP-A, and CD206-positive (CD206+) as well as CD206-negative (CD206-) cells are shown in sWAT stromal vascular fractions from both Adipo-rtTA (left panel) and Adipo-ANG2 (right panel) mice. (B) Representative flow cytometry histogram demonstrating the relative percentage of anti-inflammatory macrophages in the total macrophage population for eWAT stromal vascular fractions from both Adipo-rtTA (left panel) and Adipo-ANG2 (right panel) mice. DOI: http://dx.doi.org/10.7554/eLife.24071.011

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  Figure 4—figure supplement 3.. Fibrotic changes after ANG-2 overexpression. (A) Representative Trichrome (TC) staining pictures of sWAT sections (Magnification = 100 × ) from both Adipo-rtTA and Adipo-ANG2 groups. Fibrotic changes are demonstrated as the blue staining. Bar = 160 µm. (B) Transcriptional levels of key pro-fibrotic molecules analyzed through qPCR in sWAT from both groups. Fibrotic molecules: Collagen 1α1 (Col1a1), Col3a1, Col6a1 and Lox. For all statistical figures: n = 10 mice per group. Data are shown as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.24071.012

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  Figure 4—figure supplement 4.. The maintenance of brown adipose tissue (BAT) in the ANG-2 overexpressing mice. (A) Transcriptional levels of angiogenic gene Vegfa and brown adipose tissue marker Ucp1 assessed via qPCR in the BAT from control and ANG-2 overexpressing mice. (B) Representative H.E. staining images of BAT sections (Magnification = 100 × ) from both Adipo-rtTA control and Adipo-ANG2 transgenic groups. Bar = 160 µm. For all figures: n = 10 mice per group. Data are shown as mean ± s.e.m. **p<0.01. DOI: http://dx.doi.org/10.7554/eLife.24071.013