Administration of EP4 agonist alters adipose tissue macrophage polarization.

<p>(A) Relative expression of marker genes for M1 (<i>Cd11c</i>) and M2 (<i>MR</i>, <i>Cd163</i>) macrophages in epididymal fat tissues from <i>db/db</i> mice administered EP4 agonist (black bar) or vehicle (white bar). All values are mean ± SEM (n = 4–5 each). ♯ p<0.01 vs. vehicle. (B) Epididymal adipose tissues of EP4 agonist–or vehicle–treated <i>db/db</i> mice were double stained with anti-F4/80 (green), and anti-CD11c (red, upper panel) or anti-CD163 (red, lower panel) antibodies. Arrows indicate double-positive cells. Scale bar: 100 μm. MR, Mannose Receptor.</p

Adipose tissue morphology.

<p>(A) Representative images of epididymal adipose tissue stained with anti-F4/80 antibody (upper panel; the arrows indicate F4/80-positive cells) and H&E (lower panel) in <i>db/db</i> mice administered EP4 agonist (right column) or vehicle (left column). Scale bars: 100 μm. (B) Percent of F4/80⁺ CLS areas in mouse epididymal adipose tissue (n = 7 each). (C) Quantification of adipocyte size (n = 7 each). All values are mean ± SEM. ♯ p<0.01 vs. vehicle. CLS, crown-like structures.</p

Cytokine and chemokine gene expression in SVF.

<p>Relative expression of <i>Tnfα</i>, <i>Il-6</i>, <i>Mcp-1</i>, and <i>Ip-10</i> mRNA in SVF isolated from <i>db/db</i> mice administered EP4 agonist (black bar) or vehicle (white bar). All values are mean ± SEM (n = 3 each). * p<0.05; ♯ p<0.01 vs. vehicle. SVF, stromal vascular fraction; Tnfα, tumor necrosis factor α; Il-6, interleukin-6; Mcp-1, monocyte chemotactic protein-1; Ip-10, interferon gamma–induced protein 10.</p

Effects of EP4 agonist on metabolic parameters.

<p>Values are mean ± SEM (n = 3–8 each). T-Cho, total cholesterol; TG, triglyceride; HbA1c, Hemoglobin A1c; WAT, white adipose tissue.</p><p>Effects of EP4 agonist on metabolic parameters.</p

Impact of EP4 activation on adiponectin expression.

<p>Relative adiponectin mRNA levels in epididymal fat tissue (A) and plasma adiponectin concentrations (B) were measured in EP4 agonist–(black bar) or vehicle–treated (white bar) <i>db/db</i> mice. All values are mean ± SEM (n = 6 each). ♯ p<0.01 vs. vehicle.</p

EP4 signaling is important in macrophage polarization.

<p><i>In vitro</i> M1/M2 polarization assays were performed using peritoneal macrophages freshly isolated from 12 to 15-week-old male WT (white bar) or EP4-deficient mice (black bar). To determine M1 or M2 polarization, cells were incubated with 1 μg/ml of LPS (A, C), or with 20 ng/ml of IL-4 and IL-13 (B, D), as well as with 1 μM of EP4 agonist or vehicle. Eight hours later, marker gene expression for M1 (<i>Tnfα</i> and <i>Il-6</i>) (A, C) or M2 (<i>MR</i> and <i>Cd163</i>) (B, D) polarity was measured. To clarify the role of PPARs in EP4-dependent M2 polarization, peritoneal macrophages were pretreated with GSK3787 (PPARδ antagonist) (E), GW9662 (PPARγ antagonist) (F) or vehicle prior to the administration of IL-4/IL-13 and the EP4 agonist. Results are expressed as the fold induction of M2 genes compared with unantagonized, vehicle-treated cells. All values are mean ± SEM (n = 6 each). * p<0.05; ♯ p<0.01. LPS, lipopolysaccharide.</p

The Prostaglandin E2 Receptor EP4 Regulates Obesity-Related Inflammation and Insulin Sensitivity

<div><p>With increasing body weight, macrophages accumulate in adipose tissue. There, activated macrophages secrete numerous proinflammatory cytokines and chemokines, giving rise to chronic inflammation and insulin resistance. Prostaglandin E₂ suppresses macrophage activation via EP4; however, the role of EP4 signaling in insulin resistance and type 2 diabetes mellitus remains unknown. In this study, we treated <i>db/db</i> mice with an EP4-selective agonist, ONO-AE1-329, for 4 weeks to explore the role of EP4 signaling in obesity-related inflammation <i>in vivo</i>. Administration of the EP4 agonist did not affect body weight gain or food intake; however, in the EP4 agonist–treated group, glucose tolerance and insulin resistance were significantly improved over that of the vehicle–treated group. Additionally, administration of the EP4 agonist inhibited the accumulation of F4/80-positive macrophages and the formation of crown-like structures in white adipose tissue, and the adipocytes were significantly smaller. The treatment of the EP4 agonist increased the number of anti-inflammatory M2 macrophages, and in the stromal vascular fraction of white adipose tissue, which includes macrophages, it markedly decreased the levels of proinflammatory cytokines and chemokines. Further, EP4 activation increased the expression of adiponectin and peroxidase proliferator–activated receptors in white adipose tissue. Next, we examined <i>in vitro</i> M1/M2 polarization assay to investigate the impact of EP4 signaling on determining the functional phenotypes of macrophages. Treatment with EP4 agonist enhanced M2 polarization in wild-type peritoneal macrophages, whereas EP4-deficient macrophages were less susceptible to M2 polarization. Notably, antagonizing peroxidase proliferator–activated receptor δ activity suppressed EP4 signaling-mediated shift toward M2 macrophage polarization. Thus, our results demonstrate that EP4 signaling plays a critical role in obesity-related adipose tissue inflammation and insulin resistance by regulating macrophage recruitment and polarization. The activation of EP4 signaling holds promise for treating obesity and type 2 diabetes mellitus.</p></div

Effects of EP4 activation on glucose tolerance and insulin resistance.

<p>Seven-week-old male <i>db/db</i> mice subcutaneously received either the EP4-selective agonist ONO-AE1-329 (0.3 mg/kg) or vehicle twice a day for 4 weeks. (A) Change in body weight during the study period (n = 8 each). (B) Amount of food intake (n = 8 each). (C) Results of glucose tolerance test: left, time-course of blood glucose levels; right, area under curve (AUC) (n = 7–8 each). (D) Results of insulin tolerance test: left, time-course changes of blood glucose levels; right, the inverse AUC (n = 13 each). All values are mean ± SEM. * p<0.05; ♯ p<0.01 vs. vehicle.</p

EPRAP in bone marrow–derived cells attenuates colitis and colitis-induced tumorigenesis.

<p>Four kinds of chimeric mice were generated by adoptive bone marrow transplantation: <b>(A)</b> Representative photographs of colons with adoptive bone marrow transplantation. <b>(B)</b> Percent changes in colon length expressed relative to DSS-free water controls in each group. (n = 4–8 each). <b>(C)</b> Histological colitis scores (n = 4–8 each). <b>(D)</b> Representative H&E staining of rectal sections. Scale bars: 100 μm. <b>(E)</b> Representative photographs of colons at the end of AOM/DSS treatment. <b>(F)</b> The number of colonic tumors per mouse, with the size distribution (left) and the total number (right) in each group (n = 4–9 each). <b>(G)</b> EPRAP did not directly affect cell proliferation. DLD-1 cell proliferation was determined by MTS assay (over expression of EPRAP, left; knockdown of EPRAP, right). Data represent fold induction of absorbance compared with mock or negative control. All values represent means ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

The colons of ulcerative colitis (UC) showed accumulation of EPRAP-positive macrophages.

<p><b>(A)</b> Immunohistochemical staining for EPRAP in the sections of normal human and UC colons: the insets show higher magnifications of selected regions (indicated by dash boxes). Scale bars: 100 μm. <b>(B)</b> Double-color immunofluorescence analyses using sections of normal and UC colons were performed with anti-CD68 (green) and anti–EPRAP (red) antibodies (left). Scale bars: 20 μm. The percentage of EPRAP-positive cells in CD68-positive cells (right) (n = 20 [normal]; n = 20 [UC]). **<i>P</i> < 0.01 vs. normal colons. <b>(C)</b> Relationship between the percentage of EPRAP positive cells in CD68-positive macrophages and the clinical severity of UC patients (mild to moderate vs. severe) (n = 22 [mild to moderate]; n = 16 [severe]). *<i>P</i> < 0.05.</p