Figure 4.
Figure 4. DIO promotes a gene signature enriched in anti-inflammatory genes in eWAT macrophages. (A–C) WT C57BL/6J male mice (4–6-wk-old) were placed on the HFD OpenSource D12492i for 20 wk or kept on ND. (B) Flow cytometry analysis of eWAT macrophage distribution. Representative dot plot; n = 3–5 per group, representative of three independent experiments. (C) Myeloid cell distribution of WAT macrophages per gram of adipose tissue. (D) Epididymal adipose tissue weight. (E) eWAT SVF and eWAT CD45+ cell distribution of ND and HFD-fed animals. Data representative of three independent experiments (n = 3–5 per group). (F) DP macrophages can be generated from monocytes. Ly6CHIGH monocytes from bone marrow and spleen from CD45.1 WT lean mice were labeled with the Cell Proliferation Dye eFluor 450 and injected i.v. into CD45.2 HFD-fed WT mice. 80 h after injection, the transferred cells were quantified and characterized by flow cytometry. Representative data of two independent experiments (n = 3 each experiment). Data shown as mean ± SEM. (G) PCA of the transcriptional signature obtained by RNAseq data from the populations VAM2, VAM1, PreVAM, and DPs of ND- and HFD-fed animals. Each dot represents data from one animal’s epididymal fat pads. (H) Volcano plot comparing the top 7,000 genes expressed by VAM2 of ND- and HFD-fed animals. Very similar genes arise when the comparison is made with DP macrophages from HFD-fed mice (data not shown). Consolidated data from at least three biological replicates. (I) Heat maps of genes differentially expressed by eWAT macrophages in HFD-fed mice. Each column represents consolidated data from three biological replicates per cell type. The z-score of the gene expression profiles gives a scale to measure the differential expression. Each column represents consolidated data from three biological replicates. (J) Representative confocal images of epididymal adipose tissue full-mounted sections of lean (ND) and obese (HFD) male mice showing the distribution of CD206+CD11b+ (VAM) macrophages contouring adipocytes. Bars, 10 µm. 25× magnification; n = 3, representative of two independent experiments. (K) Heat maps of genes encoding cytoskeletal proteins. Each column represents consolidated data from three biological replicates per cell type. The z-score of the gene expression profiles gives a scale to measure the differential expression. Each column represents consolidated data from three biological replicates. (L) VAM endocytic activity in vivo after chronic HFD feeding. Dextran-rhodamine 70 kD was injected i.v., and 20 min after injection, animals were anesthetized and perfused intracardially. The endocytic activity of the indicated eWAT populations was quantified by the fluorescence intensity of rhodamine, as determined by flow cytometry. Representative histogram; n = 4, representative of two independent experiments. (M) Intravital microscopy analysis of eWAT dextran-rhodamine uptake in mice fed ND or HFD (Video 4). qDots (Qtracker 655), Hoechst, and dextran-rhodamine were i.v. injected, and uptake of rhodamine was observed for 30 min. 25× magnification. Bars, 20 µm. Arrows in the ND-fed samples indicate VAMs uptaking rhodamine. Arrows in the HFD samples point to nucleated cells in tight proximity with blood vessels, which did not uptake rhodamine. Red labeling traces the blood vasculature. Representative image; n = 3, representative of two independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001. Statistical analysis was performed using unpaired t test. AT, adipose tissue.

DIO promotes a gene signature enriched in anti-inflammatory genes in eWAT macrophages. (A–C) WT C57BL/6J male mice (4–6-wk-old) were placed on the HFD OpenSource D12492i for 20 wk or kept on ND. (B) Flow cytometry analysis of eWAT macrophage distribution. Representative dot plot; n = 3–5 per group, representative of three independent experiments. (C) Myeloid cell distribution of WAT macrophages per gram of adipose tissue. (D) Epididymal adipose tissue weight. (E) eWAT SVF and eWAT CD45+ cell distribution of ND and HFD-fed animals. Data representative of three independent experiments (n = 3–5 per group). (F) DP macrophages can be generated from monocytes. Ly6CHIGH monocytes from bone marrow and spleen from CD45.1 WT lean mice were labeled with the Cell Proliferation Dye eFluor 450 and injected i.v. into CD45.2 HFD-fed WT mice. 80 h after injection, the transferred cells were quantified and characterized by flow cytometry. Representative data of two independent experiments (n = 3 each experiment). Data shown as mean ± SEM. (G) PCA of the transcriptional signature obtained by RNAseq data from the populations VAM2, VAM1, PreVAM, and DPs of ND- and HFD-fed animals. Each dot represents data from one animal’s epididymal fat pads. (H) Volcano plot comparing the top 7,000 genes expressed by VAM2 of ND- and HFD-fed animals. Very similar genes arise when the comparison is made with DP macrophages from HFD-fed mice (data not shown). Consolidated data from at least three biological replicates. (I) Heat maps of genes differentially expressed by eWAT macrophages in HFD-fed mice. Each column represents consolidated data from three biological replicates per cell type. The z-score of the gene expression profiles gives a scale to measure the differential expression. Each column represents consolidated data from three biological replicates. (J) Representative confocal images of epididymal adipose tissue full-mounted sections of lean (ND) and obese (HFD) male mice showing the distribution of CD206+CD11b+ (VAM) macrophages contouring adipocytes. Bars, 10 µm. 25× magnification; n = 3, representative of two independent experiments. (K) Heat maps of genes encoding cytoskeletal proteins. Each column represents consolidated data from three biological replicates per cell type. The z-score of the gene expression profiles gives a scale to measure the differential expression. Each column represents consolidated data from three biological replicates. (L) VAM endocytic activity in vivo after chronic HFD feeding. Dextran-rhodamine 70 kD was injected i.v., and 20 min after injection, animals were anesthetized and perfused intracardially. The endocytic activity of the indicated eWAT populations was quantified by the fluorescence intensity of rhodamine, as determined by flow cytometry. Representative histogram; n = 4, representative of two independent experiments. (M) Intravital microscopy analysis of eWAT dextran-rhodamine uptake in mice fed ND or HFD (Video 4). qDots (Qtracker 655), Hoechst, and dextran-rhodamine were i.v. injected, and uptake of rhodamine was observed for 30 min. 25× magnification. Bars, 20 µm. Arrows in the ND-fed samples indicate VAMs uptaking rhodamine. Arrows in the HFD samples point to nucleated cells in tight proximity with blood vessels, which did not uptake rhodamine. Red labeling traces the blood vasculature. Representative image; n = 3, representative of two independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001. Statistical analysis was performed using unpaired t test. AT, adipose tissue.

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