Increased angiogenesis protects against adipose hypoxia and fibrosis in metabolic disease-resistant 11β-hydroxysteroid dehydrogenase type 1 (HSD1)-deficient mice

Abstract

In obesity, rapidly expanding adipose tissue becomes hypoxic, precipitating inflammation, fibrosis, and insulin resistance. Compensatory angiogenesis may prevent these events. Mice lacking the intracellular glucocorticoid-amplifying enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1(-/-)) have "healthier" adipose tissue distribution and resist metabolic disease with diet-induced obesity. Here we show that adipose tissues of 11βHSD1(-/-) mice exhibit attenuated hypoxia, induction of hypoxia-inducible factor (HIF-1α) activation of the TGF-β/Smad3/α-smooth muscle actin (α-SMA) signaling pathway, and fibrogenesis despite similar fat accretion with diet-induced obesity. Moreover, augmented 11βHSD1(-/-) adipose tissue angiogenesis is associated with enhanced peroxisome proliferator-activated receptor γ (PPARγ)-inducible expression of the potent angiogenic factors VEGF-A, apelin, and angiopoietin-like protein 4. Improved adipose angiogenesis and reduced fibrosis provide a novel mechanism whereby suppression of intracellular glucocorticoid regeneration promotes safer fat expansion with weight gain.

FIGURE 1.

Less adipose hypoxia in 11βHSD1^−/− mice. A and B, representative images of Hypoxyprobe staining of subcutaneous (A) and mesenteric (B) adipose tissue in chow-fed control (WT CD; quantification histogram: hatched bars), HF-fed control (WT HF; quantification histogram: white bars), and HF 11βHSD1^−/− (KO HF; quantification histogram: black bars) mice (n = 6). 30–40 high power fields (magnification ×200) per section were quantified. Brown adducts indicate areas of low oxygen availability. Note the unstained blood vessel that acts as an internal negative control. n = 6/group, *, p < 0.05, **, p < 0.01,***, p < 0.001 by ANOVA. HFD, high fat diet.

FIGURE 2.

Reduced adipose HIF-1α in 11βHSD1^−/− mice. A and B, representative immunoblots of HIF-1α and HIF-2α protein levels (A) and PHD 1–3 protein levels (B) in scAT. Right panels, quantification histograms showing the relative protein levels corrected for β-actin control mice on chow diet (hatched bars, n = 6), control HF (white bars, n = 6), and 11βHSD1^−/− HF mice (black bars, n = 6). *, p < 0.05, ***, p < 0.001 by ANOVA. AU, arbitrary units; WT CD, chow-fed control; wt HFD, HF-fed control; KO HFD, HF 11βHSD1^−/−.

FIGURE 3.

11βHSD1^−/− show similar HIF response after acute hypoxia in vitro. A–E, SVF from HF control at 21% oxygen (white) and 1% oxygen (diagonal hatched) and HF 11βHSD1^−/− at 21% oxygen (black) and 1% oxygen (vertical hatched). Cells were kept in 1% oxygen for 6 h. Data are presented as means ± S.E. of SVF from four individual mice. mRNA levels of HIF-1α (A), HIF-2α (B), PHD1 (C), PHD2 (D), and PHD3 (E) were determined. *, p < 0.05, ***, p < 0.001, by two-way ANOVA. AU, arbitrary units.

FIGURE 4.

Attenuated adipose tissue fibrosis in HF 11βHSD1^−/−. A, representative images of picrosirius red (S RED) staining in subcutaneous (sc) and mesenteric (mes) adipose. Bold arrowheads show collagen deposition surrounding the adipocytes, and arrows point to the streaks of collagen between adipocytes. WT CD, chow-fed control; wt HF, HF-fed control; KO CD, chow-fed 11βHSD1^−/−; KO HF, HF 11βHSD1^−/−. B, α-SMA immunohistochemistry in mesenteric adipose; brown stained myofibroblasts are indicated by arrows. 30–40 fields (magnification ×200) per section were assessed in each group. Right, quantification histograms (n = 6/genotype) showing WT HF (white bars) versus KO HF (black bars) *, p < 0.05.

FIGURE 5.

Reduced profibrotic signaling in 11βHSD1^−/−. A and B, representative adipose Western blots of phosphorylated Smad3 (Smad3^P) (A) and unphosphorylated Smad2/3 protein pro-enzyme and active MMP14 (B). Lower panels, protein quantification histograms for control mice on chow diet (WT CD, hatched bars, n = 4), control HF (WT HFD, white bars, n = 4), and 11βHSD1^−/− HF mice (KO HFD, black bars, n = 4). AU, arbitrary units. C, mRNA of a panel of profibrotic genes (n = 8/genotype). White and black bars, differences between control on HF (white bars) and 11βHSD1^−/− on HF (black bars) by Student's t test. *, p < 0.05, **, p < 0.01.

FIGURE 6.

Higher vessel-to-adipocyte ratio in 11βHSD1^−/− in obesity. Top panels, representative pictures (×200 magnification) showing vessel staining using a CD31 antibody. Arrows indicate individual CD31-positive cells, and bold arrowheads indicate stained vessels. 30–40 fields per section were randomly selected, and the number of vessels positively staining for CD31 normalized to the total number of adipocytes was quantified blind to genotype. CD, chow-fed. A–C, quantification of total vessels to adipocyte ratio (A), total vessel number per area (mm²) (B), and total adipocyte number per area (mm²) (C). n = 4, *, p < 0.05, **, p < 0.01.

FIGURE 7.

Higher capacity of adipose-mediated tube-like structure formation in 11βHSD1^−/− mice. The number of tube-like structures formed from mouse aortic rings after the addition of vehicle (VEH, hatched bars), wild type periaortic fat conditioned media (WT PACM, white bars), or 11βHSD1^−/− periaortic fat conditioned media (KO PACM, black bars) was assessed. n = 4, *, p < 0.05, **, p < 0.01.

FIGURE 8.

Higher expression of proangiogenic genes in 11βHSD1^−/− in obesity. A, adipose mRNA levels of a panel of potent angiogenic factors, corrected for β-actin (n = 8). The bars indicate the following: SVF from HF control at 21% oxygen (white bars), 1% oxygen (diagonal hatched bars), HF 11βHSD1^−/− 21% oxygen (black bars) and 1% oxygen (vertical hatched bars). Cells were kept in 1% oxygen for 6 h. Data are presented as means ± S.E. SVF was prepared from four individual mice. AU, arbitrary units. B and C, mRNA levels of VEGF-A (B) and ANGPTL4 (C). *, p < 0.05, **, p < 0.01, and ***, p < 0.001.

FIGURE 9.

11βHSD1^−/− adipocytes are more sensitive to PPARγ-mediated angiogenic gene induction. A and B, VEGF-A (A) and ANGPTL4 (B) mRNA levels in adipocytes from HF-fed control mice (white bars, n = 4), rosiglitazone-treated mice (diagonal hatched bars, n = 6), 11βHSD1^−/−-untreated mice (black bars, n = 4) and rosiglitazone-treated mice (vertical hatched bars, n = 6). Two-way ANOVA, *, p < 0.05, **, p < 0.01. AU, arbitrary units.