Female Mice Have Higher Angiogenesis in Perigonadal Adipose Tissue Than Males in Response to High-Fat Diet

Background: Impaired capillary growth (angiogenesis) in skeletal muscle and adipose tissue contributes to the development of metabolic disorders in obese males. This association remains unexplored in females, despite mounting evidence that endothelial cells have sex-specific transcriptional profiles. Therefore, herein we assessed whether males and females show distinct angiogenic capacities in response to diet-induced obesity.Methods: Age-matched male and female mice were fed normal chow or high-fat obesogenic diets for 16 weeks. At the end of diet period, systemic glucose disposal was assessed as well as insulin sensitivity of skeletal muscle and visceral adipose tissue. Capillary content and the expression of angiogenic regulators were also evaluated in these tissues.Results: When placed on a high-fat diet, female mice gained less weight than males and showed a metabolic phenotype similar to NC-fed mice, contrasting with the impaired whole-body glucose metabolism observed in high-fat-fed males. However, high-fat-feeding elevated serum lipid levels similarly in male and female mice. Although skeletal muscle of high-fat–fed female mice had higher insulin sensitivity than male counterparts, no sex difference was detected in muscle capillarization. Metabolic functions of perigonadal white adipose tissue (pgWAT) were retained in high-fat-fed females, as evidenced by smaller adipocytes with preserved insulin sensitivity, greater responsiveness to isoproterenol, higher expression of Adiponectin and a lower ratio of Leptin:Adiponectin mRNA. An enhanced browning phenotype was detected in HF-fed female adipocytes with upregulation of Ucp1 expression. PgWAT from high-fat-fed females also showed augmented capillary number and expression of endothelial cell markers, which was associated with elevated mRNA levels of pro-angiogenic mediators, including vascular endothelial growth factor A (Vegfa) and its receptor (Vegfr2), the Notch ligand Jagged-1 (Jag1) and Angiopoietin-2 (Angpt2).Conclusion: Taken together, our findings provide novel evidence that visceral adipose tissue of female mice display greater levels of pro-angiogenic factors and vascularity than males in response to high-fat diet. This phenotype is associated with preserved metabolic homeostasis at both tissue and systemic levels. Our study discloses that a thus-far-unappreciated sex-specific difference in the regulation of adipose angiogenesis may contribute to an individual’s susceptibility to developing adipose dysfunction and obesity-related metabolic disturbances

Prazosin Can Prevent Glucocorticoid Mediated Capillary Rarefaction.

Glucocorticoids (GC) elicit skeletal muscle capillary rarefaction, which can subsequently impair blood distribution and muscle function; however, the mechanisms have not been established. We hypothesized that CORT would inhibit endothelial cell survival signals but that treatment with the alpha-1 adrenergic receptor inhibitor prazosin, which leads to angiogenesis in skeletal muscle of healthy rats, would reverse these effects and induce angiogenesis within the skeletal muscle of corticosterone (CORT)-treated rats. Male Sprague Dawley rats were implanted subcutaneously with CORT pellets (400 mg/rat), with or without concurrent prazosin treatment (50mg/L in drinking water), for 1 or 2 weeks. Skeletal muscle capillary rarefaction, as indicated by a significant reduction in capillary-to-fiber ratio (C:F), occurred after 2 weeks of CORT treatment. Concurrent prazosin administration prevented this capillary rarefaction in CORT-treated animals but did not induce angiogenesis or arteriogenesis as was observed with prazosin treatment in control rats. CORT treatment reduced the mRNA level of Angiopoietin-1 (Ang-1), which was partially offset in the muscles of rats that received 2 weeks of co-treatment with prazosin. In 2W CORT animals, prazosin treatment elicited a significant increase in vascular endothelial growth factor-A (VEGF-A) mRNA and protein. Conversely prazosin did not rescue CORT-induced reductions in transforming growth factor beta-1 (TGFβ1 and matrix metalloproteinase-2 (MMP-2) mRNA. To determine if CORT impaired shear stress dependent signaling, cultured rat skeletal muscle endothelial cells were pre-treated with CORT (600nM) for 48 hours, then exposed to 15 dynes/cm2 shear stress or maintained with no flow. CORT blunted the shear stress-induced increase in pSer473 Akt, while pThr308 Akt, ERK1/2 and p38 phosphorylation and nitric oxide (NO) production were unaffected. This study demonstrates that GC-mediated capillary rarefaction is associated with a reduction in Ang-1 mRNA within the skeletal muscle microenvironment and that concurrent prazosin treatment effectively increases VEGF-A levels and prevents capillary loss

Alterations to VEGF-A and TSP-1 with elevated CORT and concomitant prazosin treatment.

<p>RNA or protein was isolated from the TA muscle after 1W or 2W of CORT with or without concurrent prazosin treatment. Taqman qPCR was used to assess the mRNA levels of VEGF-A (A,B) and TSP-1 (D,E), while VEGF-A protein was assessed by ELISA (C) and TSP-1 protein by Western blot (F). (A) VEGF-A mRNA was not altered in response to 1W CORT and/or prazosin. (B) VEGF-A mRNA was not affected by 2W CORT (<i>P</i> = 0.08), while a significant prazosin effect was detected within the 2W CORT-prazosin group (*<i>P</i><0.05). (C) At the 2W time point, VEGF-A protein displayed a tendency for a CORT effect (<i>P</i> = 0.08), and was significantly increased in the CORT-prazosin cohort compared to water CORT animals (*<i>P</i><0.05). (D, E) TSP-1 mRNA was not altered by 1W or 2W CORT and/or prazosin. (F) TSP-1 protein level was significantly reduced with 2W CORT treatment (^#<i>P</i><0.05), and there was a trend (<i>P</i> = 0.09) for an increase in TSP-1 after 2W of prazosin treatment within control animals (n = 4–9).</p

Corticosterone-induced capillary rarefaction is abrogated by continuous prazosin treatment.

<p>TA muscle were sectioned and stained for capillaries using fluorescein<i>-Griffonia Simplicifolia</i> isolectin and Cy3-anti-α smooth muscle actin. (A) Representative images of isolectin staining after 1W and 2W of CORT-treatment. Inverted grey scale images of isolectin staining are displayed to enhance visualization of individual muscle fibers. Scale bar represents 100 μm. (B) C:F at the 1W and (C) 2W time points was calculated from the average of 5 non-overlapping fields of view, ^#<i>P</i><0.05 1W CORT vs. corresponding control group; ***, ^###<i>P</i><0.001 2W CORT vs. corresponding water or control group respectively, n = 6–9.</p

Influence of corticosterone on endothelial specific shear stress responsiveness.

<p>Cultured rat skeletal muscle endothelial cells were pre-treated with corticosterone (600 nM) for 48 hours prior to shear stress (15dynes/cm²), or were maintained in static conditions (C), for 2 hours. (A-D): Whole cell lysates were analyzed by Western blotting. (A) Phospho-ERK1,2 protein level relative to β-actin. (B) Phospho-p38 protein level relative to total p38. (C) Phospho-Ser473 Akt protein level relative to total Akt. (D) Phospho-Thr308 Akt protein level relative to total Akt. Two-way ANOVA indicated a significant shear effect for all kinases (pERK1,2: <i>P</i> = 0.007; pp38: <i>P</i> = 0.02; pSerAkt: <i>P</i> = 0.02; pThrAkt: <i>P</i> = 0.0003, n = 3–4). *,** <i>P</i><0.05, <i>P</i><0.01 compared to respective static control, post hoc analysis. A significant interaction between shear stress and CORT was detected only for pAktSer473 (^#<i>P</i> = 0.05). (E) Nitric oxide level was assessed indirectly by Griess assay. A main effect of shear stress was detected (<i>P</i> = 0.0006; n = 6). (*,**<i>P</i><0.05 and <i>P</i><0.01, relative to static controls as assessed via post hoc analysis).</p

Plasma CORT concentration.

<p>Plasma CORT concentration.</p

CORT influence on Ang-1 mRNA and Akt phosphorylation.

<p>RNA was isolated from the TA muscle after 1W or 2W of CORT treatment with or without concurrent prazosin or from cultured endothelial or C2C12 cell extracts after 48 hours of CORT-treatment. (A,B) Ang-1 mRNA, assessed by Taqman qPCR and represented as 2^-ΔCt relative to the housekeeping gene HPRT1, was significantly reduced with CORT treatment (^#<i>P</i> = 0.02; main effect of 1W CORT; ^##<i>P</i><0.05 2W CORT vs. control water, n = 4–8). Ang-1 mRNA was unaltered within cultured endothelial cells (C) or in differentiated C2C12 myotubes (D) after 48 hours of CORT-treatment (<i>P</i> = 0.85 and <i>P</i> = 0.18 respectively, n = 5–6). Protein was extracted from the TA muscle of control or 1W CORT treated rats. Changes in phosphorylated Ser473-Akt (E) and Thr308-Akt (F) were assessed using Western blot and normalized to levels of total Akt. (E) A significant interaction between prazosin and CORT treatment was detected for pSer473-Akt (^#<i>P</i><0.05). Post hoc analysis indicated a significant difference between water and prazosin in the control-treated animals (*<i>P</i><0.05). (F) No significant changes were detected in pThr308-Akt (n = 4–8).</p

CORT-induced inhibition of flow induced arteriolar remodeling is not reversed with prazosin treatment.

<p>Histological analysis of SMA⁺ vessels within the TA muscle at the 2W time point was calculated from 5 non-overlapping fields of view per rat. (A) Representative images of SMA⁺ staining at the 2W timepoint. Grey scale images are displayed to enhance visualization of individual muscle fibers. Arrows point to SMA⁺ vessels in each condition. Scale bar represents 100 μm. (B) Average SMA⁺ density (per mm²) was unaltered by CORT and/or prazosin treatment. (C) 2W of concurrent prazosin treatment caused a significant increase in average SMA⁺ vessel diameter in control, but not in CORT-treated, animals (*<i>P</i><0.05 vs. corresponding water group; n = 5–9). The mRNA levels of key arteriogenic factors within the TA muscle was assessed by Taqman qPCR and expressed as 2^-ΔCt relative to HPRT1. (D, E) TGFβ1 mRNA was repressed after 1W CORT (^#<i>P</i><0.05), which was not alleviated by concurrent prazosin treatment. Neither CORT nor prazosin influenced TGFβ1 mRNA at the 2W time-point. (F) MMP-2 mRNA level was repressed by 2W CORT (^#<i>P</i><0.05). (G) TIMP1 mRNA was unaltered by 2W CORT or prazosin (n = 5–9).</p