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Microparticles release by adipocytes act as "find-me" signals to promote macrophage migration.

Eguchi A, Mulya A, Lazic M, Radhakrishnan D, Berk MP, Povero D, Gornicka A, Feldstein AE - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of California San Diego (UCSD), La Jolla, California, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

MPs are released by stressed adipocytes in a caspase 3 and Rho-associated kinase dependent manner.(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.
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pone.0123110.g003: MPs are released by stressed adipocytes in a caspase 3 and Rho-associated kinase dependent manner.(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.

Mentions: Our findings demonstrating the regulated release of attraction signals by stressed adipocytes occurring mostly through distinct mechanisms from the previously described “find me” signals led us to further explore the potential identity of alternative recruitment signals. Subsequent, several lines of evidence identified adipocyte-derived MPs as a possible key “find me” signal release by stressed adipocytes. Exposure of adipocyte to palmitic acid that induces chemoattractant activity resulted in hypertrophy of adipocytes (Fig 3A) and in marked production and release into the supernatants of annexin V positive MPs (Fig 3B). Notably, the production of annexin V positive MPs in 3T3-L1 cells (undifferentiated) was not increased (S2A Fig). To establish the characteristics of the adipocyte-derived MPs, we next performed a series of studies including dynamic light scattering analysis and transmission electron microscopy (Fig 3C). Adipocyte-derived MPs have a diameter ranging between 30 and 500 nm (mean diameter 142 nm for palmitic acid-treated adipocytes) corresponding to MP size [30] (Fig 3C). Moreover, western blot analysis of MPs from palmitic acid treated adipocytes expressed a variety of adipocyte-specific markers including FABP4, adiponectin, and perilipin A/B (Fig 3D), and none expressed MCP-1 (Fig 3D). The release of MPs was abolished by co-incubation with a selective caspase 3 inhibitor (Fig 3E), as well as by Rho-associated kinase inhibitors (Fig 3F). Furthermore, to investigate whether stressed primary adipocytes increase MP release, we isolated mouse primary adipocytes (Fig 3G) and SVF from epididymal AT of obese mice and treated them with palmitic acid. Here we demonstrated that the production of annexin V positive MPs was also significantly increased in palmitic acid-stressed mouse primary adipocytes as compared to control treatment (Fig 3H). On the other hand, the production of annexin V positive MPs were not increased in SVF with palmitic acid treatment compared to control treatment (S2B Fig).


Microparticles release by adipocytes act as "find-me" signals to promote macrophage migration.

Eguchi A, Mulya A, Lazic M, Radhakrishnan D, Berk MP, Povero D, Gornicka A, Feldstein AE - PLoS ONE (2015)

MPs are released by stressed adipocytes in a caspase 3 and Rho-associated kinase dependent manner.(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.
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Related In: Results  -  Collection

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pone.0123110.g003: MPs are released by stressed adipocytes in a caspase 3 and Rho-associated kinase dependent manner.(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.
Mentions: Our findings demonstrating the regulated release of attraction signals by stressed adipocytes occurring mostly through distinct mechanisms from the previously described “find me” signals led us to further explore the potential identity of alternative recruitment signals. Subsequent, several lines of evidence identified adipocyte-derived MPs as a possible key “find me” signal release by stressed adipocytes. Exposure of adipocyte to palmitic acid that induces chemoattractant activity resulted in hypertrophy of adipocytes (Fig 3A) and in marked production and release into the supernatants of annexin V positive MPs (Fig 3B). Notably, the production of annexin V positive MPs in 3T3-L1 cells (undifferentiated) was not increased (S2A Fig). To establish the characteristics of the adipocyte-derived MPs, we next performed a series of studies including dynamic light scattering analysis and transmission electron microscopy (Fig 3C). Adipocyte-derived MPs have a diameter ranging between 30 and 500 nm (mean diameter 142 nm for palmitic acid-treated adipocytes) corresponding to MP size [30] (Fig 3C). Moreover, western blot analysis of MPs from palmitic acid treated adipocytes expressed a variety of adipocyte-specific markers including FABP4, adiponectin, and perilipin A/B (Fig 3D), and none expressed MCP-1 (Fig 3D). The release of MPs was abolished by co-incubation with a selective caspase 3 inhibitor (Fig 3E), as well as by Rho-associated kinase inhibitors (Fig 3F). Furthermore, to investigate whether stressed primary adipocytes increase MP release, we isolated mouse primary adipocytes (Fig 3G) and SVF from epididymal AT of obese mice and treated them with palmitic acid. Here we demonstrated that the production of annexin V positive MPs was also significantly increased in palmitic acid-stressed mouse primary adipocytes as compared to control treatment (Fig 3H). On the other hand, the production of annexin V positive MPs were not increased in SVF with palmitic acid treatment compared to control treatment (S2B Fig).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of California San Diego (UCSD), La Jolla, California, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus