Transplanted circulating MPs from ob/ob mouse lead to monocyte activation in the blood and macrophage infiltration in the adipose tissue.

<p>(A) Scheme of transplanted experiment; ob/ob platelet free plasma was collected, circulating MPs were purified and injected into the WT mouse. (B) Dot plot analysis of the entire leukocyte population in blood resulting from the mock, ob ctrl (control) MP, and ob/ob MP injections, respectively. X-axis indicates CD11b intensity and Y-axis indicates Ly6C intensity. (C-D) Flow cytometry analysis of monocyte (CD11b⁺-Ly6C^high) percentage (C) and activated monocyte (CD11b⁺-Ly6C^high-CD204⁺) percentage (D) in blood resulting from the mock, ob ctrl MP, or ob/ob MP injections (n = 4 each group). (E) Flow cytometry analysis of infiltrated monocytes (CD11b⁺-Ly6C^high) percentage in epididymal (Epi) adipose tissue from the mock, ob ctrl MP, or ob/ob MP injections. P<0.4. Values represent mean ± S.E.M. *P < 0.05; **P < 0.01 compared to ob ctrl MPs as a control.</p

Obesity is associated with increased levels of circulating MPs and increased adipose activity of Rho associated kinase and caspase 3

<p>Annexin V positive MPs from (A) ob control or ob/ob mice. Blood was collected by cardiac puncture and PFP was obtained as detailed in methods section. Annexin V positive MPs were analyzed by flow cytometry (n = 5 each group). (B) Dynamic light scattering analysis and Transmission Electron Microscopy of isolated circulating MPs. (C) Western blot analysis of perilipin A levels in MPs isolated from mouse PFP in WT mice on a regular chow diet and ob/ob mice (n = 3 each group). Perilipin A abundance in MPs isolated from mouse PFP correlated to: (D) total mouse body weights and (E) weight of extracted mouse epididymal fat pads. (F) Western blots of p-MYPT1, total MYPT1/2, cleaved (active) caspase 3, total caspase 3, and actin levels in subcutaneous and epididymal adipose tissue lysates from ob control or ob/ob mice. Quantification of Western blots of (G) cleaved caspase 3 and (H) p-MYPT in ob control and ob/ob mice adipose tissues normalized to actin levels. Values represent mean ± S.E.M. *P < 0.05; ***P < 0.001 compared to respective controls.</p

Adipocyte-derived MPs mediates attraction of macrophages in vitro and in vivo.

<p>(A) In vitro chemotaxis assay of MPs-free supernatant and MPs from adipocytes treated with palmitic acid. Values represent mean ± S.D. ***P < 0.001 compared to controls. (B-F) C57BL/6 mice were injected intraperitoneally with 1x10⁶ adipocyte-derived MPs (isolated from 3T3-L1 adipocytes treated with 0.5 mM palmitic acid or without palmitic acid) or controls (n = 3 each group). (B) Four days post injection infiltrated cells were isolated from peritoneal cavity by lavage and counted. (C-F) flow cytometry analysis of infiltrated cells. The infiltrated cells were stained by CD45 (leukocyte common antigen) (C), CD11b (monocytes) (D), F4/80 (macrophages) (E), or Ly6G (neutrophils) (F). Values represent mean ± S.E.M. (D) In vivo macrophages migration. C57BL/6 mice were injected intraperitoneally with 1x10⁶ palmitic acid-derived MPs or vehicle alone (n = 5 per group). Three days post injection; macrophages were isolated from peritoneal cavity by lavage. Number of macrophages present in peritoneal cavity was counted. Values represent mean ± S.E.M.</p

Microparticles Release by Adipocytes Act as “Find-Me” Signals to Promote Macrophage Migration

<div><p>Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical “find-me” signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel “find me” signals that contributes to macrophage infiltration associated with obesity.</p></div

Caspase 3 activation is required for attraction of macrophages to stressed adipocytes.

<p>Caspase-3 activity assay in adipocytes treated with (A) a range of doses of palmitic acid (0.1 to 1 mM) in the absence or presence of a selective caspase 3 inhibitor (Ac-DEVD-CHO). Differentiated adipocytes were plated on black 96-well plate for 2 hrs followed by the different treatments for additional 4 hrs. Caspase-3 activity assay was determined by the Apo-One Homogeneous Caspase 3/7 fluorescent assay as described under Experimental Procedures. Assays were performed on three replicates for each treatment. Values represent as mean ± S.D. ** P < 0.01; ***P < 0.001 compared to controls. Transmigration of primary mouse macrophages to supernatants from differentiated adipocytes treated with (C) palmitic acid, in the absence or presence of increasing doses of the caspase-3 inhibitor was assessed. Values represent as mean ± S.D. ***P < 0.001 compared to stressor alone (no caspase inhibitor).</p

MPs are released by stressed adipocytes in a caspase 3 and Rho-associated kinase dependent manner.

<p>(A-D) Characterization of adipocyte-derived MPs. (A) Morphology of the differentiated 3T3-L1 adipocytes treated with control, 0.5 mM palmitic acid. (B) The number of Annexin V positive MPs was quantitated by flow cytometry. (C) Dynamic light scattering analysis and Transmission Electron Microscopy of isolated MPs. Isolated MPs were measured by Zetasizer and analyzed using intensity. (D) Western Blot analysis of MPs released by adipocytes. Isolated MPs were fractionated by SDS-PAGE and probed with FABP4, MCP-1, Chemerin, Adiponectin and Perilipin antibody. (E-F) Differentiated adipocytes were incubated with or without palmitic acid in the absence or presence of a selective caspase-3 inhibitor (E), or a range of doses of two different Rho associated kinase inhibitors (Y27632 and fasudil) (F) for up to 12 hrs. Supernatants were then collected and MPs isolated by ultracentrifugation as detailed in methods section. (G) Morphology of isolated mouse primary adipocytes. (H) Number of annexin V positive mouse primary adipocyte-derived MPs assessed by flow cytometry. Values represent mean ± S.D. * P < 0.5; ** P < 0.01; ***P < 0.001 compared to controls.</p

Attraction of monocytes and macrophages to supernatants of mature adipocytes exposed to palmitic acid.

<p>Migration of (A) RAW264.7 cells or (B) primary mouse macrophages through a transwell (8 μm pore size) to supernatants from untreated (control) or treated differentiated mature adipocytes with 0.5mM palmitic acid. MCP-1 (50 ng/ml) was used as positive control. (C) Adipocytes were treated with 0.5 mM palmitic acid in the absence or presence of 0.025 U/ml apyrase, 0.5 U/ml phospholipase-D or 50 μg/ml control IgG or MCP-1 neutralizing antibody. Macrophages that migrated to the lower chamber were stained with DAPI and the number of cells was counted under fluorescence microscopy. Values represent as mean ± S.D. of representative experiment. * P < 0.05; ** P < 0.01; ***P < 0.001 compared to controls.</p

RYGB surgery improves body weight (A) and glycemic control (B) for up to 90 days in SD rats fed a high fat diet.

<p>(A) Records of body weight for Sham control vs RYGB animals. (B and C) Plasma Glucose response (left) and glucose AUC (right) during OGTT for Sham (B) and RYGB (C), before (gray) and after (black) surgery. Data are mean ± SE, asterisk sign denotes a significant difference between Sham control vs. RYGB with P<0.05.</p

Correlation analysis between gene expression, and changes in body weight (left) and absolute change in insulin resistance (right) in subcutaneous adipose tissue.

<p>* Threshold of significance set at P<0.05</p><p>Correlation analysis between gene expression, and changes in body weight (left) and absolute change in insulin resistance (right) in subcutaneous adipose tissue.</p

The expression of IL–6 (A), MCP–1 (B), IL–1β (C), NLRP3 (D), IL–18 (E), caspase–1 (F) and ASC (G) genes in mesenteric adipose tissue between sham and RYGB groups.

<p>N = 8 (control) and N = 8 (RYGB). Data are mean ± SE. * denotes a significant difference between Sham control vs. RYGB.</p