ROS, OAT3, AhR, and NF-κB p65 are involved in IS-induced prorenin expression in vascular smooth muscle cells.

<p>Serum starved HASMCs were pretreated with NAC (2.5 mmol/L) and DPI (10 µmol/L) for 30 min before incubation with IS (250 µmol/L) for 24 h (<b>A, B</b>). HASMCs were transfected with or without OAT3 siRNA (10 nmol/L), AhR siRNA (30 nmol/L) or p65 siRNA (10 nmol/L), and serum starved for 24 h, followed by incubation with IS (250 µmol/L) for 24 h. Cell lysates were immunoblotted using anti-OAT3, anti-AhR, anti-p65 and anti-prorenin antibodies (<b>C–F</b>). Mean±SE (n = 3). *p<0.05, **p<0.01 vs untreated group, #p<0.01 vs IS-treated group. Ctrl: control.</p

Immunohistochemistry of PRR in rat aorta.

<p><b>A</b>. Immonohistochemical localization of PRR in the aortas of normal, CKD and AST-120-treated CKD rats. <b>B</b>. Quantitative data of PRR in the aortas of normal (n = 9), CKD (n = 8) and AST-120-treated CKD rats (n = 8) (mean±SE). ***p<0.001 vs normal, ##p<0.001 vs CKD. <b>C</b>. Immonohistochemical localization of PRR in the aortas of DN, DN+IS, DH and DH+IS rats. <b>D</b>. Quantitative data of PRR-positive area in the aorta of DN, DN+IS, DH and DH+IS rats (mean±SE, n = 8). ***p<0.001vs DN, #p<0.05 vs DH.</p

Immunohistochemistry of renin/prorenin in rat aorta.

<p><b>A</b>. Immonohistochemical localization of renin/prorenin in the aortas of normal, CKD and AST-120-treated CKD rats. <b>B</b>. Quantitative data of renin/prorenin-positive area in the aortas of normal (n = 9), CKD (n = 8) and AST-120-treated CKD rats (n = 8) (Mean±SE). ***p<0.001 vs normal, #p<0.05 vs CKD. <b>C</b>. Immonohistochemical localization of renin/prorenin in the aortas of DN, DN+IS, DH and DH+IS rats. <b>D</b>. Quantitative data of renin/prorenin-positive area in the aortas of DN, DN+IS, DH and DH+IS rats (mean±SE, n = 8). ***p<0.001vs DN, ##p<0.01 vs DH.</p

IS-induced PRR activation is involved in cell proliferation and tissue factor expression in vascular smooth muscle cells.

<p>Serum-starved HASMCs (5×10³ cells/well) in a 24-well plate were stimulated with or without IS (250 µmol/L) or prorenin (20 nmol/L) for 24 h (<b>A, B</b>). HASMCs were transfected with siPRR (20 nmol/L) for 48 h, before stimulation with IS or prorenin for 24 h. Thereafter, the cell proliferation reagent MTS (50 µL) was added to each well, and cells were incubated for 4 h. The absorbance was measured at 492 nm using a microplate reader (mean±SE, n = 3). **p<0.01 vs untreated group, #p<0.01 vs IS-treated group. HASMCs were transfected with or without PRR siRNA (20 nmol/L), and then serum starved for 24 h, followed by incubation with IS (250 µmol/L) or prorenin (20 nmol/L) for 24 h. Cell lysates were immunoblotted using anti-PRR and anti-tissue factor antibodies (<b>C–F</b>). Mean±SE (n = 3). *p<0.05, **p<0.01 vs untreated group, #p<0.05 vs IS-treated group. Ctrl: control.</p

ROS, OAT3, AhR and NF-κB p65 are involved in IS-induced PRR expression in vascular smooth muscle cells.

<p>Serum-starved HASMCs were pretreated with NAC (2.5 mmol/L) and DPI (10 µmol/L) for 30 min before incubation with IS (250 µmol/L) for 24 h (A,B). HASMCs were transfected with or without OAT3 siRNA (10 nmol/L), AhR siRNA (30 nmol/L) or p65 siRNA (10 nmol/L), and serum starved for 24 h, followed by incubation with IS (250 µmol/L) for 24 h. Cell lysates were immunoblotted using anti-OAT3, anti-AhR, anti-p65 and anti-PRR antibodies (<b>C–F</b>). Mean±SE (n = 3). *p<0.05 vs control, #p<0.05 vs IS-treated group. Ctrl: control.</p

IS induces PRR expression in vascular smooth muscle cells.

<p>Serum-starved HASMCs were treated with IS (250 µmol/L). Incubation with IS increased PRR mRNA and protein expression in HASMCs time- (<b>A</b>, <b>C</b>) and dose- (<b>B</b>, <b>D</b>) dependently. Mean±SE (n = 3). *p<0.05, **p<0.01, ***p<0.001 vs control.</p

Angiotensin II Receptor Blocker Ameliorates Stress-Induced Adipose Tissue Inflammation and Insulin Resistance

<div><p>A strong causal link exists between psychological stress and insulin resistance as well with hypertension. Meanwhile, stress-related responses play critical roles in glucose metabolism in hypertensive patients. As clinical trials suggest that angiotensin-receptor blocker delays the onset of diabetes in hypertensive patients, we investigated the effects of irbesartan on stress-induced adipose tissue inflammation and insulin resistance. C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle, 3 and 10 mg/kg/day irbesartan. The plasma concentrations of lipid and proinflammatory cytokines [Monocyte Chemoattractant Protein-1 (MCP-1), tumor necrosis factor-α, and interleukin-6] were assessed with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was observed with CD11b-positive cell counts and mRNA expressions of CD68 and F4/80 using immunohistochemistry and RT-PCR methods respectively. The mRNA levels of angiotensinogen, proinflammatory cytokines shown above, and adiponectin in WAT were also assessed with RT-PCR method. Glucose metabolism was assessed by glucose tolerance tests (GTTs) and insulin tolerance tests, and mRNA expression of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in WAT. Restraint stress increased monocyte accumulation, plasma free fatty acids, expression of angiotensinogen and proinflammatory cytokines including MCP-1, and reduced adiponectin. Irbesartan reduced stress-induced monocyte accumulation in WAT in a dose dependent manner. Irbesartan treatment also suppressed induction of adipose angiotensinogen and proinflammatory cytokines in WAT and blood, and reversed changes in adiponectin expression. Notably, irbesartan suppressed stress-induced reduction in adipose tissue weight and free fatty acid release, and improved insulin tolerance with restoration of IRS-1 and GLUT4 mRNA expressions in WAT. The results indicate that irbesartan improves stress-induced adipose tissue inflammation and insulin resistance. Our results suggests that irbesartan treatment exerts additive benefits for glucose metabolism in hypertensive patients with mental stress.</p></div

Irbesartan rescued stress-induced decline in insulin sensitivity.

<p><b>A:</b> Glucose tolerance was comparable between the stressed mice treated with vehicle and irbesartan (10 mg/kg/day) after stress. Insulin tolerance showed significant recovery in the irbesartan-treated and stressed mice (lower panel). Data are mean ± SD of 10 mice per group. *<i>P<</i>0.05, and **<i>P<</i>0.02, compared with the vehicle-treated and stressed mice. <b>B:</b> Quantitative analysis of IRS-1 and GLUT4 expression in inguinal adipose tissue and skeletal muscle (adductor muscle) of the stressed mice treated with vehicle or irbesartan (10 mg/kg/day). Data are mean ± SD of 10 mice per group. *<i>P<</i>0.05, compared with the vehicle-treated and stressed mice.</p

Accumulation of monocytes in inguinal adipose of stressed mice.

<p>Stressed mice were individually subjected to 2 h/day of immobilization stress for two weeks. Animals received oral vehicle, 3, or 10 mg/kg/day of irbesartan during the same period. Inguinal adipose tissues from stressed and control (non-stressed) mice were analyzed by H&E staining (A), CD11b immunostaining (B and C), and quantitative RT-PCR for CD68 and F4/80 (D and E). <b>A:</b> Accumulation of mononuclear cells in inguinal adipose tissues following the 2-week restraint stress. Top panel, ×40 magnification, bar = 250 µm. Inset, ×200 magnification, bar = 50 µm. <b>B:</b> Increased accumulation of CD11b-positive cells (monocytes) in adipose tissue of stressed mice (×200 magnification, bar = 50 µm). <b>C:</b> Quantitative analysis of CD11b-positive cells relative to total nuclear number. Data are mean±SD. n = 10 for all the groups.*<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.001, compared with the vehicle-treated and stressed mice. <b>D</b> and <b>E</b>: Quantitative analysis of F4/80 (D) and CD68 (E) expression levels in adipose tissue. Data are mean±SD. n = 10 for all the groups. Values are expressed relative to the vehicle-treated control mice. (<b>D</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.001, compared with the vehicle-treated and stressed mice, respectively. (<b>E</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, ^†<i>P<</i>0.012, compared with vehicle-treated and stressed mice, ^#<i>P<</i>0.02, compared with the stressed mice treated with a lower dose of irbesartan (3 mg/kg/day), respectively.</p

Irbesartan restored stress-induced decrease in weight gain and reduced adipose tissue weight.

<p>Body weight and inguinal adipose tissue of the control and stressed mice were weighed before and after the stress period, and the cell size in the collected adipose tissue was estimated under a microscope at×200 magnification using image analysis software. <b>A:</b> Body weight gain in the control mice with or without irbesartan treatment (10 mg/kg/day) and stressed mice with or without irbesartan treatment (3 or 10 mg/kg/day). *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.01, compared with the vehicle-treated and stressed mice, ^†<i>P<</i>0.001, compared with the vehicle-treated and stressed mice. <b>B:</b> Plasma fat and fatty acid composition in the control mice with or without irbesartan treatment (10 mg/kg/day) and stressed mice with or without irbesartan treatment (3 or 10 mg/kg/day). *<i>P<</i>0.01, compared with the vehicle-treated control mice, **<i>P<</i>0.05, compared with the vehicle-treated and stressed mice. <b>C:</b> Inguinal adipose tissue weight in the control mice with or without irbesartan treatment (10 mg/kg/day) and stressed mice with or without irbesartan treatment (3 or 10 mg/kg/day). * <i>P</i><0.03, compared with the vehicle-treated control mice. <b>D:</b> Subcutaneous and inguinal fat pad. Circle dot line: adipose tissue. <b>E:</b> Distribution of adipocyte size in inguinal adipose tissues of stressed mice with or without irbesartan (10 mg/kg/day) treatment. Data are mean ± SD of 10 mice per group.</p