1. FIGURE 1. CL 316,243 administration induces lipolysis and neutrophil accumulation in adipose tissue. Mice were injected with 0.5 mg/kg CL316,243 or PBS intraperitoneally and adipose tissue was harvested 30 min to 24 h later as indicated. A, plasma was drawn from the retroorbital sinus and nonesterified fatty acids (NEFA) were measured (n=3, *; p<0.05). B, visceral adipose tissue was isolated, fixed in 10% formalin, embedded in paraffin, and stained with H&E. Slides are representative of 3–6 animals at 100 ×magnification. C, mice were injected with 1 mg/kg CL 316,243 or PBS. After 18 h, adipose tissue SVF was isolated and FACS analysis was performed. Upper panels, PBS-treated animals. Lower panels, CL-treated animals. Left panels, Siglec F-negative, CD11b- and F4/80-positive cells. Right panels, GR-1-positive cells. Panels are representative of 6–11 animals per group. D, GR-1 positive cells (from 1C, lower right panel) were stained additionally forCD11b(left), Ly6c and F4/80 (right). Panels are representative of 6–11 animals per group. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 4, pp. 2882–2892, January 25, 2013  3 -Adrenergic Receptor Stimulation Induces E-Selectin-mediated Adipose Tissue Inflammation Rachel J. Roth Flach, Anouch Matevossian, Thomas E. Akie, Kimberly A. Negrin, Marina T. Paul, and Michael P. Czech CL316243 による浸潤した細胞はマクロファージではない可能性が高いことが考えられる。 2015.12.21 M1 松村明導 FIGURE 2. Temporal regulation of inflammatory cellgeneexpressionbyCL316,243. Mice were injected with 0.5mg/kgCL316,243 or PBS intraperitoneally for30 min to 24h as indicated. Visceral adipose tissue was isolated, RNA was extracted and quantitative RT-PCR was performed forA, Itgam (CD11b),B,CD68,C, Itgax (CD11c), D, EMR1 (F4/80), E, Il1, F, CCL2 (MCP-1), G, TNF, and H, IL- 6. Data represent average gene expression as compared with 36b4±S.E. (n=3–7, *; p<0.05). CL316243 の腹腔内への注射で血中において NEFA の増加が、 組織において好中球とみられる細胞の浸潤が認められた。

2. FIGURE 3. Endothelial cell activation by3-adrenergic stimulation of adipose tissue in vivo, but not in vitro CL 316,243 treatment. Mice were injected with 0.5 mg/kg CL 316,243 or PBS intraperitoneally for 30 min to 24 h as indicated. Visceral adipose tissue was isolated, mRNA was extracted, and qRT-PCR was performed for A, Icam-1, B, Vcam-1, C, Sele (E-selectin), and D, Selp (P-selectin). Data represent average gene expression as compared with 36b4S.E. (n=3–10, *; p<0.05). E, human umbilical vein endothelial cells (HUVEC) were treated for 6 h with various doses of CL316,243 or 10 ng/ml TNF. mRNA was extracted and qRT-PCR was performed for ICAM-1, VCAM-1, and SELE (E-selectin). Data represent average gene expression as compared with RPLP0±S.E. (n=3–5, *; p<0.05,**; p <0.005). 内臓脂肪組織に浸潤した細胞がマクロファージでない可能性が高く、 CL316243 は直接的には 血管内皮細胞に影響を与えないことが考えられる。 FIGURE 4. Fasting-induced lipolysis causes macrophage infiltration and reduces cytokine expression in adipose tissue. Mice were fed ad libidum or fasted as indicated. A, plasma was drawn from the retroorbital sinus and NEFA were measured (n6, #; p0.0005). B, visceral adipose tissue was isolated, RNA was extracted, and quantitative RT-PCR was performed for the indicated genes. Data represent average gene expression as compared with 36b4 S.E. (n=6–7, *; p<0.05, **; p<0.005). C–F, adipose tissue SVF was isolated and FACS analysis was performed. C, ad lib fed animals. D, fasted animals. Left panels, Siglec F-negative, CD11b- and F4/80-positive cells (p0.005). Right panels, GR-1-positive cells. Panels are representative of 9–11 animals per group. E and F, CD11b, F4/80- positive cells from C-D were stained additionally for Galectin 3, Ly6c, and CD11c. E, ad lib fed animals. F, fasted animals. Left, Galectin 3, Ly6c- positive cells. Right, CD11b, CD11c- positive cells. Panels are representative of 9–11 animals per group. A. 絶食状態の NEFA の増加は CL316243 での結果と違いサイトカインの発現を抑制 し、不断給餌と比較して M2 マクロファージの浸潤が増加することが認められ た。

3. FIGURE 5. E-selectin KO mice are protected from CL 316,243-mediated adipose tissue immunecell infiltration. Wild type, E-sel KO, or P-sel KO mice were injected with 0.5 mg/kg CL 316,243 or PBS intraperitoneally for 30 min to 24 h as indicated. A, plasma was drawn from the retroorbital sinus and NEFA were measured (n=3-7, **; p<0.005). B, visceral adipose tissue was isolated, fixed in 10% formalin, embedded in paraffin, and stained with H&E. Slides are representative of 3–6 animals at 50 magnification. FIGURE 6. E-selectin KO mice are protected from CL 316,243-mediated adipose tissue inflammation. A, wild type, E-sel KO, or P- sel KO mice were injected with 0.5 mg/kg CL 316,243 or PBS intraperitoneally for 18 h. Visceral adipose tissue was isolated, mRNA was extracted, and qRT-PCR was performed for the indicated genes. Data represent average gene expression as compared with 36b4S.E. (n=3–9, *; p<0.05, **; p<0.005). B–D, wild type or E-sel KO mice were injected with 1 mg/kg CL 316,243 or PBS for 18 h. Adipose tissue SVF was isolated and FACS analysis was performed. B, wild type mice. C, E-sel KO mice. Upper panels, PBS- treated animals. Lower panels, CL-treated animals. Left panels, Siglec F-negative, CD11b and F4/80-positive cells. Right panels, GR-1-positive cells. Panels are representative of 5–14 animals per group. D, quantitation of B-C. Left panel, Siglec F-negative, CD11b and F4/80-positive cells. Right panel, GR-1- positive cells. (n =5–14, ; p <0.06, #; p <0.0005). E- セレクチン欠損マウスは、 CL316243 の注射によって起こる脂肪組織のサイトカインの発現を抑制し 好中球の浸潤も抑制した。 E- セレクチン欠損マウスは、他のマウスと比べて脂肪組織への CL316243 による免疫細胞の浸潤が抑制さ れた。

4. まとめ  3 アドレナリン受容体の刺激は、正常な脂肪組織の炎症を引き起こし好中球の浸潤を促 進させることが示唆された。 絶食での血中 NEFA の増加は  3 アドレナリン受容体の刺激とは全く違う免疫反応であるこ とが示唆された。  3 アドレナリン受容体の刺激から連なる脂肪組織の炎症には E- セレクチンが必要であるこ とが示唆された。