All animal and tissue sample experiments were performed in accordance with the guidelines of the National Institutes of Health and Nanjing Medical University with procedures(ID:2082101) approved by the Institutional Animal Care and Use Committee of the university.
BVT.2733, an 11β-HSD1 selective inhibitor was synthesized according to the patent information by China Pharmaceutical University. Anti-F4/80 antibody was purchased form Serotec (Oxford, UK). Phycoerythrin-Cy5 conjugated anti-mouse F4/80 antibody was purchased form eBioscience (San Diego,CA, USA). Phycoerythrin anti-mouse CD11b and Alexa Fluor488 anti-mouse CD11c antibodies were purchased from BioLegend (San Diego,CA). RU486, corticotisone, LPS and PA were purchased from (Sigma-Aldrich, St. Louis, MO). M-MLV, dNTP, RNase inhibitor and other reverse transcription reagents were purchased from Promega Corp (Madison, WI, USA). Trizol were purchased from Invitrogen (Carlsbad, CA, USA).
Male C57BL/6J mice at age of 18 days were purchased from Slac Laboratories (Shanghai, China). In all experiments, mice were housed three or four per cage in a room kept at 23±1°C with a 12-h light, 12-h dark cycle and were allowed free access to water and food. From 3 weeks of age, all mice were fed with a normal chow diet containing 10% calory from fat (NC mice) (Collaborative Bio-Engineering Corporation, Nanjing, China), or a high fat diet (HFD) containing 50% calorie from fat (Collaborative Bio-Engineering Corporation, Nanjing, China) for 24 weeks. During the last four weeks the HFD-fed mice were dosed (09.00 and 17.00 h) with BVT.2733 (100 mg/kg, orally) (HFD+BVT mice) or vehicle (HFD mice). Body weight and food intake were determined weekly. At the time of sacrifice, trunk blood was collected, centrifuged, and stored at −20°C. Adipose tissues were weighed, frozen in liquid nitrogen, and stored at −80°C.
Intraperitoneal Glucose Tolerance Test
The mice were fasted overnight and then injected intraperitoneally with 2 mg/g D-glucose (25% stock solutionin saline). Blood samples were taken by tail venesection at 0 min (before injection and within 1 min of disturbing the cage) and at 15-, 30-, 60-, and 120-min intervals after the glucose load. Glucose was measured with the Accu-Chek Aviva system (Roche, Germany) at 0-, 15-, 30-, 60-, and 120-min post glucose load. Insulin was measured with the Ultra Sensitive Mouse Insulin ELISA kits (Millipore Corporation, MA, USA) at 0-, 15-, 30-, 60-min post glucose load.
Immunohistochemistry of Macrophages in White Adipose Tissues
Portions of epididymal adipose tissue were removed and fixed with 10% formaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin. For the detection of macrophage infiltration in adipose tissue, immunohistochemistry was performed with F4/80 antibody, which detected macrophage-specific protein. The number of F4/80-positive cells was counted in a blinded fashion under a microscope with a 400× objective. More than 50 serial fields were examined in each mouse, and five mice were analyzed per group.
Isolation of Adipocytes and Stromal-vascular Fractions
Epididymal adipose tissues from mice were rinsed in saline, minced into fine pieces, and digested with collagenase (Sigma-Aldrich, St. Louis, MO) with Krebs-Hense-leit-HEPES buffer, pH 7.4, supplemented with 20 mg/ml of BSA and 2 mmol/l glucose at 37°C using a shaker for 45 min. Then, the samples were passed through a mesh and fractionated by brief centrifugation (1,000 rpm). The pellets or the floating cells were collected as the stromal-vascular fraction (SVF) or as the adipocyte fraction, respectively. Cells in each fraction were used for flow cytometry analysis.
Flow Cytometry Analysis
Cells in the SVF were incubated with Fc Block (BD Biosciences, San Jose, CA) for 20 min at 4°C prior to staining with fluorescently labeled primary antibodies or control IgGs for 25 min at 4°C. Then cells were gently washed twice and resuspended in FACS buffer with propidiumiodide (Sigma-Aldrich). SVF were analyzed using FACS Calibur and FACS Aria flow cytometers (BD Biosciences). Unstained, single stains, and fluorescence minus one controls were used for compensation and gates settings. The data analysis was performed using FlowJo (Tree Star, Ashland, OR). Macrophages were identified as F4/80-positive, F4/80-positive/CD11b-positive or F4/80-positive/CD11b-positive/CD11c-positive cells, respectively. The numbers of macrophages were shown according to the percentage of each fraction per 10000 cells.
Serum levels of adiponectin and leptin were determined using commercially available ELSA kits from Millipore (Millipore Corporation, MA) and Linco (LINCO Research Inc., St. Charles, MO) individually. Proinflammatory cytokines interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and tumour necrosis factor α (TNF α) were determined using the mouse commercially available kit from Bender (Bender MedSystems, Austria). All assays were performed according to the manufacturer’s protocol.
The mouse reticulum cell sarcoma-derived cell, J774.1, was obtained from the American Type Culture Collection (ATCC, Manassas, VA), and maintained in RPMI 1640 supplemented with 10% heat-inactivated FBS(Life Technologies Corporation, USA)in a humidified 37°C/5% CO2 incubator. Some J774.1 was maintained in RPMI 1640 supplemented with 10% Charcoal Dextran Stripped Serum (Life Technologies Corporation, USA) as indicated. The mouse fibroblast 3T3-L1 preadipocytes was obtained from ATCC and maintained in DMEM culture medium supplemented with 10% fetal calf serum and 4 mM glutamine.
Plasmids and Lentiviral Vectors
Full-length mouse cDNA sequence for 11β-HSD1 was cloned into pLL3.7-BSD lentiviral vector. The RNAi construct for 11β-HSD1 was generated using one sequence in the coding region of 11β-HSD1: 5′ -GTACGGAACTGCATAAGCA-3′ (Sigma). Oligonucleotides containing this sequence or random sequence were subcloned into the lentiviral vector.
Lentivirus Production and Infection of J774A.1 Macrophage and 3T3-L1 Preadipocyte
Recombinant lentiviruses were produced by cotransfecting 293T cells with the lentivirus expression plasmid and packaging plasmids using Lipofectamine 2000 (Invitrogen) according to themanufacturer’s instruction. Infectious lentiviruses were harvested at 48 after transfection and filtered through 0.45 m cellulose acetate filters. The infection of 3T3-L1 preadipocytes and J774A.1 macrophages was carried out by adding lentivirus into the cell culture; the controls were infected with scrambled shRNA or vector. 12 hours after the incubation, the medium was changed. Infected cells were selected with BSD (3 mg/ml).
Preparation of PA
To avoid bacterial LPS contamination, 
PA were reconstituted to 10 mmol/L in ethanol, and diluted directly into warmed culture medium for experiments.
RNA Preparation and Quantitative Real-time PCR
Total RNA was extracted from epididymal adipose tissues using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. Two micrograms of total RNA were reverse-transcribed with 200 U Moloney murine leukemia virus reverse transcriptase (M-MLV, Promega, Madison, WI, USA), and in the presence of 0.5 mmol/L deoxynucleotide triphosphate, 25 U RNase inhibitor, and 0.5 µg N15 random primers, in a total volume of 25 µl. PCR primers were designed by Primer5 software. The sequences of the primers used are available upon request. Each quantitative real-time PCR was carried out in triplicate, in a 25 µl volume of SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan). The PCR program was designed as follows: 60 s at 95°C, followed by 40 cycles of 15 s at 95°C, 15 s at 60°C, 45 s at 72°C, and 5 s at 80°C on the plate reader (Rotor Gene-3000, Corbett Research, Sydney, Australia). All data were analyzed using the expression of the gene encoding β-actin as an internal reference. Expression for each gene is arbitrarily set at 1 to facilitate comparison between several groups.
Western Blot Analysis
For western blot analysis, cells or tissues were lysed in RIPA buffer (0.5% NP-40, 0.1% SDS, 150 mM NaCl, 50 mM Tris-Cl [pH 7.5]). Proteins were separated by SDS-PAGE, transferred to PVDF membrane (Millipore) and probed with anti-11β-HSD1 (Abcam), and anti-Tubulin (Abcam) antibodies.
Comparisons were performed using the Student’s t test for two groups, or ANOVA for multiple groups. Results are presented as means ± SEM. A p value of less than 0.05 was considered statistically significant.