Figure 1.
Effect of HDF and BVT.2733 on adiposity and metabolic parameters in C57BL/6J mice.
A, Percentage change in body weight. B–C, Glucose tolerance and plasma insulin level. D–G, Changes in adipose gene mRNA expression. H–I, Serum adiponectin and leptin concentration. The results are shown as the means ± SEM. *, P<0.05; **, P<0.01 compared with NC group; #, P<0.05; ##, P<0.01 compared with HFD group. n = 5−10 animals per group.
Figure 2.
Effect of HFD and BVT.2733 treatment on the abundance of macrophage in adipose tissue.
A–B, Representative immunohistochemical staining of white adipose tissue using the specific macrophage marker F4/80 and quantification of the of macrophages present in adipose tissue. C–E, Changes in the expression of macrophage marker genes determined by real time PCR. The results are shown as the means ± SEM. *, P< 0.05; **, P< 0.01 compared with NC group; #, P<0.05; ##, P< 0.01 compared with HFD group. n = 6−8 animals per group. Bar = 50 µm.
Figure 3.
Effect of HFD and BVT.2733 treatment on the number of macrophages in SVF of epididymal fat tissue.
Cells in the SVF of epididymal fat tissue from three groups of mice were analyzed using flow cytometry as described in METHODS section. A, Representative flow cytometric profiles of cells in the SVF of epdidymal fat tissue derived from NC, HFD, and HFD+BVT mice individually. B, The cell number in F4/80-positive fraction. C, F4/80-positive/CD11b-positive/CD11c- negetive fraction. D, F4/80-positive/CD11b-positive/CD11c- positive fraction. The results are shown as the means ± SEM. *, P<0.05; **, P<0.01 compared with NC group; #, P<0.05; ##, P<0.01 compared with HFD group. n = 3−4 animals per group.
Figure 4.
Effect of HFD and BVT.2733 treatment on the inflammation gene expression in the adipose tissue and the level of circulating inflammation markers.
A–C, The mRNA expression of IL-6, TNF-α and MCP-1 in adipose tissue. D–E, Plasma concentrations of IL-6, TNF-α and MCP-1. The results are shown as the means ± SEM. *, P<0.05; **, P< 0.01 compared with NC group; #, P<0.05; ##, P<0.01 compared with HFD group. n = 5−6 animals per group.
Figure 5.
Effect of 11β-HSD1 on the inflammation gene expression in PA or LPS-activated J774A.1 macrophages in vitro.
A, J774.1 macrophages were treated with palmitic acid (PA) (50−200 µmol/L) or LPS (100 ng/ml) for 24 h. B–C, J774.1 macrophages were activated by PA (200 µmol/L) or LPS (100 ng/ml) and co-treated with 11β-HSD1 inhibitor BVT.2733 (25−100 µmol/L) for 24 h. D–G, J774.1 macrophages were transfected with either sh-RNA for mouse 11β-HSD1 (sh-HSD1) or a negative control (sh-con) by Lentivirus. After 72 h incubation, cells were treated with PA (200 µmol/L) or LPS (100 ng/ml) for 24 h. Efficiency of 11β-HSD1 knockdown on mRNA level (D) and protein level (E). Effects of knockdown of 11β-HSD1 on MCP-1, IL-6, and TNF-α expression in PA (F) or LPS (G) treated macrophages. H–K, J774.1 macrophages were transfected with the expression vector for 11β-HSD1 (HSD1) or a corresponding empty vector (emp) using Lentivirus. After 72 h incubation, cells were treated with PA (100 µmol/L) or LPS (50 ng/ml) and co-treated with 11β-HSD1 inhibitor BVT.2733 for 24 h. Efficiency of 11β-HSD1 overexpression on mRNA level (H) and protein level (I). Effects of overexpression of 11β-HSD1 on MCP-1, IL-6, and TNF-α expression in PA (J) or LPS (K) treated macrophages. mRNA for IL-6, MCP-1 and TNF-α were determined by real-time PCR, protein of 11β-HSD1 were determined by Western blot. The results are shown as the means ± SEM of three individual experiments. *P<0.05; **P<0.01 vs con (B, C, F, G) or sh-con (D) or emp (H) or emp+PA (J) or emp+LPS (K). # P<0.05; ## P<0.01 vs PA (B) or LPS (C) or sh-con+PA (F) or sh-con+LPS (G) or HSD1+PA (J) or HSD1+LPS (K).
Figure 6.
Effect of 11β-HSD1 on the inflammation gene expression in PA or LPS-activated 3T3-L1 preadipocytes in vitro.
3T3-L1 preadipocytes were activated by PA (200 µmol/L) (A) or LPS (200 ng/ml) (D) and co-treated with 11β-HSD1 inhibitor BVT.2733 (50−100 µmol/L) for 24 h. 3T3-L1 preadipocytes were transfected with either sh-RNA for mouse 11β-HSD1 (sh-HSD1) or a negative control (sh-con) by Lentivirus. Cells were treated with PA (200 µmol/L) (B) or LPS (200 ng/ml) (E) for 24 h. 3T3-L1 preadipocytes were transfected with the expression vector for 11β-HSD1 (HSD1) or a corresponding empty vector (emp) using Lentivirus. Cells were treated with PA (200 µmol/L) (C) or LPS (200 ng/ml) (F) for 24 h. mRNA for IL-6, MCP-1 and TNF-α were determined by real-time PCR. The results are shown as the means ± SEM of three individual experiments. *P<0.05; **P<0.01 vs con (A, B, D, E) or or emp+PA (C) or emp+LPS (F). # P<0.05 vs PA (A) or LPS (D) or sh-con+PA (B) or sh-con+LPS (E) or HSD1+PA (C) or HSD1+LPS (F).
Figure 7.
Effect of glucocorticoid receptor (GR) on proinflammatory properties of 11β-HSD1 in J774A.1 macrophages.
A, J774A.1 macrophages were transfected with the expression vector for 11β-HSD1 (HSD1) or a corresponding empty vector (emp) using Lentivirus. Cells were treated with PA (100 µmol/L) or co-treated with glucocorticoid antagonist RU486 for 24 h. B, J774A.1 macrophages were activated by LPS(100 ng/ml) in the absence (con) or presence of increasing amounts of corticosterone (10−10 M to 10−6 M) for 24 h in steroid hormones free media. C, J774A.1 macrophages were activated by LPS (100 ng/ml) in the absence (con) or presence of increasing amounts of 11-dehydro corticosterone (10−10 M to 10−5 M) for 24 h in Charcoal Dextran Stripped Serum media. D, J774A.1 macrophages were transfected with the expression vector for 11β-HSD1 (HSD1) or a corresponding empty vector (emp) using Lentivirus. Cells were activated by LPS(100 ng/ml) in the absence (con) or presence of increasing amounts of 11-dehydro corticosterone (10−11 M to 10−5 M) for 24 h in Charcoal Dextran Stripped Serum media. mRNA for IL-6, MCP-1 and TNF-α were determined by real-time PCR. The results are shown as the means ± SEM of three individual experiments. *P<0.05; **P<0.01 vs emp+PA (A) or con (B–C) or emp (D), # P<0.05 vs HSD1+PA (A).