Additional file 1: Table S1. of What is the best adjustment of appendicular lean mass for predicting mortality or disability among Japanese community dwellers?

Coincidence rate of the lowest quintile among each variable (XLSX 10 kb

Additional file 3: Figure S1. of What is the best adjustment of appendicular lean mass for predicting mortality or disability among Japanese community dwellers?

A. Estimated linear changes in ALM over 12 years by 4-year age groups at baseline. B. Estimated linear changes in ALM/leg length over 12 years by 4-year age groups at baseline. C. Estimated linear changes in ALM/height over 12 years by 4-year age groups at baseline. D. Estimated linear changes in ALM/height2 over 12 years by 4-year age groups at baseline. E. Estimated linear changes in ALM/weight over 12 years by 4-year age groups at baseline. F. Estimated linear changes in ALM/BMI over 12 years by 4-year age groups at baseline. (XLSX 181 kb

Additional file 1: Table S1. of What is the best adjustment of appendicular lean mass for predicting mortality or disability among Japanese community dwellers?

Coincidence rate of the lowest quintile among each variable (XLSX 10 kb

Additional file 1 of Dietary amino acid intake and sleep duration are additively involved in future cognitive decline in Japanese adults aged 60 years or over: a community-based longitudinal study

Supplementary Material

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

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

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

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