Physiological measurements of sham and OVX of WT and MCP-1-KO mice 12 weeks after operation.

<p>Data are expressed as mean ± SEM. ND, non-detectible. Differences between groups were analyzed by two-way ANOVA, followed by Bonferroni post-tests (Increased body weight, subcutaneous fat, visceral fat, serum H₂O₂, and blood insulin; <i>P</i><0.001, serum M-CSF; <i>P</i><0.01, blood glucose; <i>P</i><0.05, effect of surgery. Increased body weight, subcutaneous fat, and visceral fat; <i>P</i><0.001, serum M-CSF and blood insulin; <i>P</i><0.01, serum ROS; <i>P</i><0.05, effect of MCP-1). WT OVX vs. MCP-1-KO OVX;</p>*<p><i>P</i><0.05,</p>**<p><i>P</i><0.01,</p>***<p><i>P</i><0.001.</p

MCP-1-deficiency decreased OVX-induced immune cell infiltration in AT.

<p>SVCs from visceral fat were extracted from WT (open bar) and MCP-1-KO mice (oblique-lined bar) 12 weeks after sham or OVX surgery. SVCs were labeled with conjugated Abs to CD11bF4/80 (A), CD11cF4/80 (B), CD4 (C), and CD8 (D) and quantified by flow cytometry. Data are expressed as mean ± SEM. Differences between groups were analyzed by two-way ANOVA, followed by Bonferroni post-tests (CD11bF4/80, CD11cF4/80, CD4; <i>P</i><0.01, CD8; <i>P</i><0.05, effect of surgery. CD11cF4/80; <i>P</i><0.05, effect of MCP-1). *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001 compared with WT OVX mice. Similar results were obtained in three independent experiments.</p

MCP-1-deficiency decreased CD11c-expressing cells via impairing the production of ROS and decreased activation of PLCγ2, Akt, and ERK upon M-CSF stimulation in BMM.

<p>BMMs from WT (open bar) and MCP-1-KO mice (oblique-lined bar) were incubated in the presence of M-CSF (30 ng/ml) with U73122 (10 µM), Akt inhibitor IV (0.3 µM), PD098059 (5 µM), DPI (50 nM), NAC (3 mM), or H₂O₂ (300 µM) for 4 d (A). *, <i>P</i><0.05; ***, <i>P</i><0.001 compared with vehicle-treated WT cells. There was no significant difference between WT and MCP-1-KO cells except upon vehicle treatment. Treatment with U73122, Akt inhibitor IV, PD98059, DPI, NAC, or H₂O₂ abolished the decrease in CD11cF4/80 observed in MCP-1-KO cells. BMMs were serum-starved for 8 h and stimulated with M-CSF for 1, 2, or 3 d (B). Phosphorylation of PLCγ2 was determined by Western blotting. Total protein level served as the loading control. Relative ratios of phosphorylated forms to total forms were plotted. **, <i>P</i><0.01; ***, <i>P</i><0.001 compared with WT cells. Intracellular levels of ROS upon stimulation in the presence of M-CSF (M) or/and MCP-1 with control IgG (3 µg/ml) or anti-MCP-1 Ab (3 µg/ml) for 2 d were determined in WT cells and MCP-1-KO cells using H2DCFDA (C, D). ROS levels were quantified by flow cytometry. *, <i>P</i><0.05; **, <i>P</i><0.01 compared with M-stimulated WT cells. No significant difference between WT and MCP-1-KO cells stimulated with M+MCP-1 (C). *, <i>P</i><0.05; ***, <i>P</i><0.001 compared with IgG-treated WT cells. No significant difference between IgG- and anti-MCP-1 Ab-treated MCP-1-KO cells (D). BMMs were transfected with sip47^phox or scRNA. Downregulation of p47^phox by siRNA was confirmed by RT-PCR and qPCR (E). The expression level obtained from scRNA-treated cells was set to be 1. After 24 h of transfection with siRNA, cells were stimulated with M-CSF for 2 d (mRNA) or 4 d (FACS) in order to determine CD11c (F) and for 2 d to measure ROS (G). *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001 compared with scRNA-transfected WT cells. No significant difference was found in MCP-1-KO cells (F, G). Similar results were obtained in three independent experiments.</p

The absence of MCP-1 reduced fat mass and improved metabolic perturbation induced by OVX.

<p>WT mice (open bar) and MCP-1-KO (oblique-lined bar) mice were subjected to OVX or sham surgery, and then held for 12 weeks. Whole body weight change up to 12 weeks after surgery (A) and average daily food intake (B) were measured. *, <i>P</i><0.05; WT OVX vs. MCP-1-KO OVX mice. Adipocyte volume was calculated from photograph of hematoxylin-eosin staining of visceral fat, assuming that an adipocyte is a sphere (magnification, ×200). Scale bar, 100 µm (C). Glucose clearance (D) and insulin sensitivity (E) were determined 12 weeks after sham or OVX, following an intraperitoneal injection of glucose (1 mg/kg) and insulin (0.75 munits/kg), respectively. AUC was measured for each group for D and E. *, <i>P</i><0.05, ***, <i>P</i><0.001; WT OVX vs. MCP-1-KO OVX. Data are expressed as mean ± SEM. Differences between groups were analyzed by two-way ANOVA, followed by Bonferroni post-tests (C, D) (adipocyte volume; <i>P</i><0.001, AUC for glucose tolerance; <i>P</i><0.01, effect of surgery. adipocyte volume; <i>P</i><0.001, AUC for glucose tolerance; <i>P</i><0.05, effect of MCP-1). *, <i>P</i><0.05; ***, <i>P</i><0.001 compared with WT OVX mice. Similar results were obtained in three independent experiments.</p