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

BAY 60–2770 reduces myocardial infarction in a rat IR injury model.

<p>Seven days after reperfusion, the infarct area was visualized using TTC staining from IR-injured and BAY 60–2770 pre-treated hearts (A). Representative TTC images from Nor (n = 10), IR (n = 5), BAY (n = 6) (B). *p < 0.05 between IR and BAY groups. Echocardiographic analysis is shown as percent of EF (C), FS (D), and LVDD (mm, E). *p <0.05, **p <0.005 vs. IR.</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

BAY 60–2770 activates PKG in isolated cardiac tissue.

<p>BAY 60–2770 was perfused at 5 nM or 5 μM for 10 min in a Langendorff system. cGMP (A) and cAMP (B) concentrations in tissue homogenates were measured. Western blot analysis was used to determine VASP phosphorylation at ser239 (C), and the ratio of phospho-VASP to VASP was analyzed (D). PKG activity was examined using the CycLex cGK/PKG Assay (E). All the experiments were performed duplicated or triplicated in 3 animals from each group. *p <0.05, **p <0.005 vs. IR.</p

Irbesartan reduced the expression of stress-induced proinflammatory adipokines and restored adiponectin expression in adipose tissue.

<p>Inguinal adipose tissues from control mice treated with vehicle or irbesartan (10 mg/kg/day), and stressed mice treated with vehicle or irbesartan (3 or 10 mg/kg/day) were analyzed by quantitative RT-PCR for angiotensinogen (<b>A</b>), MCP-1 (<b>B</b>), TNF-α (<b>C</b>), IL-6 (<b>D</b>), and adiponectin (<b>E</b>). Values are expressed relative to the vehicle-treated control mice. Plasma levels of MCP-1, TNF-α, and IL-6 from these groups were also measured. Data are mean ± SD of 10 mice for RT-PCR, 6 mice for ELISA per group. (<b>A</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.046, compared with the vehicle-treated and stressed mice, ^†<i>P<</i>0.042, compared with the stressed mice treated with a lower dose of irbesartan (3 mg/kg/day), respectively. (<b>B</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, ^†<i>P<</i>0.003, compared with the vehicle-treated and stressed mice, respectively. (<b>C</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, ^†<i>P<</i>0.004, compared with the stressed mice treated with a lower dose of irbesartan (3 mg/kg/day), ^††<i>P<</i>0.05, compared with the vehicle-treated and stressed mice, respectively. (<b>D</b>) *<i>P<</i>0.001, compared with the vehicle-treated control mice, **<i>P<</i>0.003, compared with the vehicle-treated and stressed mice, ^†<i>P<</i>0.004, compared with stressed mice treated with a lower dose of irbesartan (3 mg/kg/day), ^††<i>P<</i>0.002, compared with the vehicle-treated control mice, ^#<i>P<</i>0.02, 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>0.05, compared with the vehicle-treated and stressed mice, respectively.</p

Echocardiographic analysis.

<p>Echocardiographic analysis.</p

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

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

The effects of gemigliptin on mesangial matrix expansion and urinary albumin/creatinine ratios in db/db mice.

<p>Mesangial expansion was explored by PAS staining of the kidneys of a) db/m, b) db/db, c) db/db + 0.04% (w/w) gemigliptin-, and d) db/db + 0.04% (w/w) gemigliptin-treated mice (A). Quantitative analysis of glomerular matrix scores (B). Urinary albumin/creatinine ratios (C). The results are expressed as means ± SEMs. **p < 0.01, ***p < 0.001 vs. db/m; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. db/db. Original magnification ×400.</p

The effect of gemigliptin on myocardial capillary and arteriole density in db/db mice.

<p>Representative immunohistochemical stain showing myocardial capillary (CD31) and arteriole (α-SMA) density (A and B). Quantitative analysis of the capillary and arteriole density in myocardium was shown (C and D). Data were analyzed by one-way ANOVA using Newman-Keuls post-hoc testing, and the results are expressed as means ± SEMs. **p < 0.01 vs. db/m; ***p < 0.001 vs. db/m; ###p < 0.001 vs. db/db.</p