Detection of function of vesicle uptake in secretory vesicles.

<p>(a) FFN206 was added to RBL-2H3 cells and the uptake process was observed under an ImageXpress Micro microscope (×400). Scale bar: 20 μm. (b) Total positive area (left axis) and integrated intensity (right axis) to loading time were quantified based on the captured images. Results are expressed as the mean ± S.E.M. (c) Representative uptake images of FFN206 in non-treated and reserpine-treated cells 90 min after FFN206 application (×400). Scale bar: 20 μm. (d) Blockade of functional vesicle uptake by the administration of reserpine in mast cells. The cells were pre-treated with reserpine at the indicated doses and FFN206 loading was performed. The maximal positive area (left axis) and integrated intensity (right axis) at different doses of reserpine were quantified based on the captured images. Results are expressed as the mean ± S.E.M.</p

Observation of exocytotic trafficking in mast cells.

<p>FFN206-loaded RBL-2H3 cells were stimulated with different concentrations of thapsigargin (0.05, 0.5 and 5 μM). Exocytotic processes were monitored. (a) Representative images of TG-activated mast cells captured by an ImageXpress Micro microscope (×400). Scale bar: 20 μm. (b) Rate of exocytosis quantified using the total positive area based on the captured images. Results are expressed as the mean ± S.E.M. (c) The rate of exocytosis was quantified using the integrated intensity based on the captured images. Results are expressed as the mean ± S.E.M. (d) TG-dependent histamine release was measured 30 min after stimulation. Results are expressed as the mean ± S.E.M. *<i>p</i> < 0.05; ***<i>p</i> < 0.001.</p

Localization of FFN206-labeled vesicles in mast cells.

<p>Primary differentiated bone marrow-derived mast cells (BMMC) derived from <i>+/+</i>, <i>bg/+</i>, and <i>bg/bg</i> mice were used for the detection of secretory vesicles. (a) The expressions of FcεRI receptor and c-kit receptor in BMMC after 4 weeks of culture were analyzed using a BD FACSCalibur flow cytometer. (b) BMMC were stained with Alcian blue after 4 weeks of culture and observed under a light microscope (×400). Scale bar: 5 μm. (c) Visualization of secretory vesicles using FFN206 in BMMC. The cells were observed by confocal microscopy (×1000) at room temperature. Scale bar: 5 μm. (d) Colocalization of FFN206 and Alexa568-dextran in the secretory vesicles of RBL-2H3 cells. The cells were loaded using FFN206 and Alexa568-dextran and were observed by confocal microscopy (×1000) at room temperature. The small images were cropped from original images (gray square) and are present on the upper right of each original image. Scale bar for original image: 10 μm; scale bar for cropped image: 2 μm. (e) Representative image of FFN206-labeled RBL-2H3 cells captured by an ImageXpress Micro microscope at 37°C in a CO₂ incubator (×200). Scale bar: 100 μm. (f) Imaging map of vesicle analysis in RBL-2H3 cells. Sixteen powered fields were captured for each well. The imaging map was acquired from 32 wells (A9: H12). (g) Representative captured image and (h) segmented vesicles from the background in FFN206-labeled RBL-2H3 cells. Red area: segmented positive area. Scale bar: 20 μm.</p

Effect of Angiotensin II Type 2 Receptor-Interacting Protein on Adipose Tissue Function via Modulation of Macrophage Polarization

<div><p>We demonstrated that angiotensin II type 2 (AT₂) receptor-interacting protein (ATIP) 1 ameliorates inflammation-mediated vascular remodeling independent of the AT₂ receptor, leading us to explore the possibility of whether ATIP1 could exert anti-inflammatory effects and play a role in other pathophysiological conditions. We examined the possible anti-inflammatory effects of ATIP1 in adipose tissue associated with amelioration of insulin resistance. In mice fed a high-cholesterol diet, adipose tissue macrophage (ATM) infiltration and M1-to-M2 ratio were decreased in ATIP1 transgenic mice (ATIP1-Tg) compared with wild-type mice (WT), with decreased expression of inflammatory cytokines such as tumor necrosis factor-α and monocyte chemoattractant protein-1 in white adipose tissue (WAT), but an increase in interleukin-10, an anti-inflammatory cytokine. Moreover, 2-[³H]deoxy-d-glucose (2-[³H]DG) uptake was significantly increased in ATIP1-Tg compared with WT. Next, we examined the roles of ATIP1 in BM-derived hematopoietic cells, employing chimeric mice produced by BM transplantation into irradiated type 2 diabetic mice with obesity, KKAy, as recipients. ATM infiltration and M1-to-M2 ratio were decreased in ATIP1 chimera (ATIP1-tg as BM donor), with improvement of insulin-mediated 2-[³H]DG uptake and amelioration of inflammation in WAT. Moreover, serum adiponectin concentration in ATIP1 chimera was significantly higher than that in WT chimera (WT as BM donor) and KKAy chimera (KKAy as BM donor). These results indicate that ATIP1 could exert anti-inflammatory effects in adipose tissue via macrophage polarization associated with improvement of insulin resistance, and ATIP1 in hematopoietic cells may contribute to these beneficial effects on adipose tissue functions in type 2 diabetes.</p> </div

WAT in ATIP1-Tg and WT after treatment with high-cholesterol diet for 18 weeks.

<p>(<b>A</b>) Ratio of WAT weight to body weight in epididymal and retroperitoneal tissue. (<b>B</b>) Morphological comparison of epididymal WAT. Representative photomicrographs at ×100 magnification and histogram of adipocyte number per field. n = 7–8 for each group.</p

Macrophage infiltration and polarization in ATIP1-Tg and WT after treatment with high-cholesterol diet.

<p>Cells in the stromal vascular fraction (SVF) of the epididymal fat pad from each mouse were analyzed by flow cytometry as described in “Methods”. (<b>A</b>) Representative results of flow cytometry. F4/80-positive cells were further analyzed with anti-CD11c and anti-CD206 antibodies. Blue dots show M1 macrophages and purple dots show M2 macrophages. Red dots represent both CD11c- and CD206-negative fraction evaluated using isotype controls. (<b>B</b>) Percentage of F4/80-positive cells in SVF. n = 5 for each group. (<b>C</b>) Ratio of M1 to M2 fraction in F4/80-positive cells. Light gray squares; M1 fraction, dark gray squares; M2 fraction. n = 5 for each group.</p

Cytokine levels and glucose uptake in WAT of ATIP1-Tg and WT after treatment with high-cholesterol diet.

<p>(<b>A</b>) Expression of TNF-α, MCP-1 and IL-10 in epididymal (Epi) and retroperitoneal (Retro) WAT. Open squares; WT, closed squares; ATIP1-Tg. n = 7–8 for each group. *p<0.05 vs. WT. (<b>B</b>) Rate constant of 2-[³H] DG uptake in epididymal and retroperitoneal WAT was determined with and without insulin (1.0 U/kg) injection. Open squares; WT, closed squares; ATIP1-Tg. n = 6 for each group.</p

Glucose uptake and cytokines levels in white adipose tissue and serum levels of TNF-α and adiponectin in each chimeric mouse.

<p>(A) Rate constant of 2-[³H] DG uptake in epididymal (Epi) and retroperitoneal (Retro) WAT were determined with and without insulin (1.0 U/kg) injection. n = 6 for each group. (B) Expression of TNF-α, MCP-1 and IL-10 in epididymal and retroperitoneal WAT. n = 6 for each group. (C) Serum TNF-α and adiponectin concentrations measured by ELISA. n = 8–10 for each group. *p<0.05 vs. KKAy chimera and WT chimera.</p

Change in mRNA expression of inflammatory cytokines and NADPH oxidase subunits in hippocampus of AD model mice by CH-3 treatment.

<p>mRNA expression of MCP-1 (<b>A</b>), TNF-α (<b>B</b>), IL-6 (<b>C</b>), iNOS (<b>D</b>), eNOS (<b>E</b>), p22^phox (<b>F</b>), p47^phox (<b>G</b>), p67^phox (<b>H</b>) and gp91^phox (I) in hippocampus. n = 8–12 mice in each group. †<i>P</i><0.05 vs. control, *<i>P</i><0.05 or **<i>P</i><0.01 vs. Aβ1–42 (+).</p

Mouse characteristics in early training stage.

<p>Mouse characteristics in early training stage.</p