Adipose tissue corticosterone concentration and inflammation. A, corticosterone was directly quantified from serum of mice fed a normal chow (NCD), HFD, or HFD + Dex. B, corticosterone was extracted and quantified from frozen eWAT of mice from the indicated groups. Bars, average values ± S.D. of duplicates from at least three independent experiments. A above the bars shows statistical groups (p < 0.05). N.D., not detected.

Studies of macrophages CM. A, chemotaxis assays were performed with Raw264.7 cells prepared from Raw264.7 conditioned media incubated for 6 or 24 h with vehicle (Un), Dex (100 nm), and/or LPS (100 ng/ml) in serum-free DMEM. B, QPCR was performed on Raw264.7 cells treated with LPS (100 ng/ml final) in serum-free DMEM for the indicated times. C, QPCR was performed on Raw264.7 cells treated for the indicated times and reagents as described above. D, primary peritoneal macrophages (IP-MØ) elicited from mice fed a normal chow (NCD) or HFD were tested for migration toward adipocyte CM or control medium (DMEM). Bars, average values ± S.D. of duplicates from at least three independent experiments. A–D above the bars show statistical groups (p < 0.05). A.U., arbitrary units; Rel, relative.

Macrophages chemotaxis correlates with adipocyte chemokine expression and secretion. A, Raw264.7 cells were used for a chemotaxis assay on 3T3L1 media conditioned for the indicated times and treated with either vehicle (Un) or TNFα (10 ng/ml final) or dexamethasone (100 nm final). B, QPCR was performed on 3T3L1 cells treated with TNFα (10 ng/ml final) for the indicated times. C, QPCR was performed on 3T3L1 cells treated for 24 h with either vehicle (Veh.; DMSO), rosiglitazone (Rosi; 5 μm final), or Dex (100 nm final) in the presence (+) or absence (−) of TNFα (10 ng/ml final) for the indicated times. D, QPCR was performed on 3T3L1 cells treated for 24 h with vehicle (DMSO) and TNFα (10 ng/ml final) alone or in combination with corticosterone (Cort.; 100 nm) or dexamethasone (100 nm). E, MCP1 secretion was quantified from 24-h 3T3L1 mature adipocyte conditioned media. The FFA mixture was used at 500 μm, corticosterone (Cort.) was used at 100 nm, and TNFα was used at 10 ng/ml. Bars, average values ± S.D. of duplicates from at least three independent experiments. A–E above the bars show statistical groups (p < 0.05). AVG, average; A.U., arbitrary units; KC, keratinocyte-derived chemokine.

In vivo dexamethasone inhibits ATM accumulation. A, adipocyte size areas were calculated from paraffin-embedded epididymal fat tissue sections. Data are presented as the percentage of cells counted falling within the indicated ranges of sizes for mice either fed an HFD or fed an HFD and treated with dexamethasone (HFD + Dex). B, FACS sorting was performed on epididymal fat stromal vascular cells sorted for F4/80, CD11b, and CD11c. Data represent relative cell numbers of the reported macrophage subtypes for HFD versus HFD + Dex mice. C, dot plot representation of CD11b versus CD11c relative expression for FACS data obtained from F4/80 gated macrophages. D, MAC2 immunostaining on paraffin-embedded epididymal fat tissue sections from HFD versus HFD + Dex mice. E, QPCR analysis of CD68, RANTES, CCR2, and CCR5 expression on epididymal fat from normal chow diet, HFD, and HFD + Dex mice. Bars, average values ± S.D. of duplicates from at least three independent experiments. A–D above the bars show statistical groups (p < 0.05). NCD, normal chow diet; A.U., arbitrary units.

The GR mediates the repressive effects of glucocorticoids on chemokine expression and macrophage chemotaxis. A, QPCR was performed on 3T3L1 cells treated with TNFα (10 ng/ml final) for the indicated times. B, a chemotaxis assay with Raw264.7 cells was performed on 24 h 3T3L1 mature adipocyte conditioned media treated with vehicle (un) or the indicated agents (final concentrations: TNFα, 10 ng/ml; dexamethasone (dex; 100 nm; aldosterone (aldo), 100 nm; RU4868 (ru), 1 μm; spironolactone (spiro), 1 μm). C, QPCR was performed on 3T3L1 cells (day 6 postdifferentiation) 36 h postelectroporation with siRNAs and 24 h incubation in serum-free DMEM. D, a chemotaxis assay with Raw264.7 cells was performed on 3T3L1 cell conditioned media, prepared from electroporated cells that were subsequently treated with either TNFα (10 ng/ml) alone or in the presence of dexamethasone (100 nm) for 24 h. E, QPCR was performed on 3T3L1 cells (day 6 postdifferentiation) 36 h postelectroporation and 24 h subsequent to incubation in serum-free DMEM with the indicated drugs (TNFα, 10 ng/ml; dexamethasone, 100 nm). Bars, average values ± S.D. of duplicates from at least three independent experiments. A–C above the bars show statistical groups (p < 0.05). A.U., arbitrary units.

Effects of CM on signaling pathways in macrophages and adipocytes. A, Western blots were performed on protein extracts from Raw264.7 cells treated for the indicated times with serum-free DMEM or mature 3T3L1 adipocyte conditioned media that were pretreated (CM-TNFα) or not (CM) with 10 ng/ml TNFα for 24 h prior to washes and resuspension for another 24 h in serum-free DMEM. B, a chemotaxis assay toward either serum-free DMEM or 24-h mature 3T3L1 adipocyte conditioned media (CM) was performed with Raw264.7. Akt inhibitor was used at the indicated concentrations as pretreatment (30 min prior to migration) and/or throughout the migration. C, Western blots were performed on protein extracts from mature adipocytes (3T3L1) that were pretreated with vehicle or corticosterone (100 nm, 24 h) and subsequently exposed to TNFα (10 ng/ml, 5 min). D, mature adipocytes (3T3L1) were pretreated with vehicle, corticosterone, or dexamethasone (100 nm, 24 h) and subsequently exposed to TNFα (10 ng/ml, 15 min) prior to protein extraction and Western blotting. E, Western blots were performed on 3T3L1 mature adipocytes treated for 24 h with the indicated drugs or vehicle in serum-free DMEM. F, QPCR was performed on 3T3L1 mature adipocytes RNA that were co-treated for 24 h with the indicated drugs (TNFα, 10 ng/ml; corticosterone and dexamethasone, 100 nm), and relative expression of MKP1 is normalized to glyceraldehyde-3-phosphate dehydrogenase expression. Bars, average values ± S.D. of duplicates from at least three independent experiments. A–E above the bars show statistical groups (p < 0.05). AVG, average; A.U., arbitrary units; p-, phosphorylated.

Adipocyte secreted factors induce macrophage migration. A, mature 3T3L1 adipocytes were pretreated for 24 h with an FFA mixture (500 μm final) and or TNFα (10 ng/ml final). Cells were subsequently washed and resuspended in serum-free medium for another 24 h. 3T3L1 conditioned media were then collected, and chemotaxis assays were performed with Raw264.7 macrophage cells. The graph represents the average (AVG) number of Raw264.7 cells counted per microscope field as having performed chemotaxis. B, chemotaxis assays were performed with THP1 cells in response to serum-free DMEM (Un), MCP1 (100 μg/ml), MCP1 and a MCP1 neutralizing antibody (2H5; 50 μg/ml), MCP1 and a control antibody (IgG; 50 μg/ml), 3T3L1 mature adipocyte CM, CM and an MCP1-neutralizing antibody (2H5; 50 μg/ml), CM and a control antibody (IgG; 50 μg/ml), and DMEM containing 10% fetal bovine serum (FBS). C, chemotaxis assays were performed with Raw264.7 cells on 3T3L1 mature adipocyte conditioned medium (serum-free), treated for 24 h with the indicated drugs (final concentrations: TNFα, 10 ng/ml; Wy14643, 10 μm; GW501516, 1 μm; T0901317, 5 μm; rosiglitazone (Rosi), 5 μm; corticosterone, 100 nm; dexamethasone, 100 nm). The left part of the graph represents the migration of Raw264.7 in response to non-conditioned medium, prior to incubation with 3T3L1 cells. The graph represents the relative (Rel) average number of Raw264.7 cells counted per microscope field as having performed chemotaxis. D, chemotaxis assays were performed with bone marrow-derived macrophage on 3T3L1 mature adipocyte conditioned media (serum-free), treated for 24 h with the indicated drugs (final concentrations: TNFα, 10 ng/ml; dexamethasone, 100 nm). E, chemotaxis assay was performed with Raw264.7 cells on 3T3L1 mature adipocyte conditioned media prepared from 24-h pretreated cells with vehicle (Veh) or an FFA mixture (500 μm final) in the presence or absence of 100 nm corticosterone (Cort). Conditioned media were collected 24 h after resuspension in serum-free media. Bars, average values ± S.D. of duplicates from at least three independent experiments. A–D above the bars show statistical groups (p < 0.05). BMDM, bone marrow-derived macrophages.