levels in vehicle, 10 or 30 mg/ kg/d of AS-605240–treated ob/ob mice , and vehicle-treated C57BL/6J mice. Glucose levels during ITT or GTT in vehicle or AS-605240–treated ob/ob mice were determined at the indicated Alvespimycin 467214-21-7 time points after i.p. injection with a bolus of insulin for ITT or glucose for GTT. Immunohistochemical analysis of adipose tissue macrophage. eWAT of ob/ob mice treated with vehicle or AS-605240 were stained with antibody against F4/80. Expression levels of genes encoded macrophage-related protein in eWAT of vehicle or AS-605240–treated ob/ob mice. Serum MCP-1 levels in vehicle or AS-605240–treated ob/ob mice and vehicle-treated C57BL/6J mice.. #P 0.05 for vehicle-treated ob/ob compared with vehicle-treated C57BL/6J mice. *P 0.05 and **P 0.01 for AS-605240–treated ob/ob compared with vehicletreated ob/ob control.
Kobayashi et al. PNAS | April 5, 2011 | vol.108 | no.14 | 5757 MEDICAL SCIENCES Gene Expression Analysis. TRIzol reagent was used to prepare total RNA from tissues. The reverse-transcription reaction was carried out with a high-capacity cDNA reverse transcription kit. Quantitative PCR analyses using TaqMan assays were performed as previously ABT-888 912444-00-9 described. The relative expression levels were normalized by measurement of the amount of cyclophilin in each sample. Histological Analysis. Tissue samples for histology were fixed in 4% paraformaldehyde in PBS overnight, and paraffin-embedded sections were prepared. Sections of liver were stained with H&E, and adipose tissues were stained hematoxylin and incubated with anti-F4/80 overnight at 4 C, followed by incubation with the Vectastain Elite ABC Rat IgG Kit and visualization with the ImmPACT DAB Substrate Kit , as previously described.
Adipose Tissue Fractionation and FACS Analysis. Adipose tissue fractionation into the stromal vascular fraction was performed as previously described. Briefly, epididymal adipose tissue pads were minced into fine pieces and centrifuged at 3,000 × g to remove erythrocytes and free leukocytes. Tissues were incubated with 2 mg/mL of collagenase type 2 at 37 C with gentle agitation for 15�?0 min. Digested tissues were filtered through nylon mesh , and the filtrate was centrifuged at 1,200 × g. Pelleted cells were collected as the SVF. For isolation of mRNA, the erythrocytedepleted SVF was resuspended in TRIzol reagent.
For flow cytometric analysis, after removing red blood cells, the SVF was incubated with either labeled monoclonal antibody or isotype control antibody and analyzed by flow cytometry using a FACS Calibur. Data acquisition and analysis were performed using CellQuest Pro software. Propidium iodide was used to exclude dead cells. Plasma MCP-1 and Hepatic Triglyceride Content. Plasma levels for MCP-1 were measured by ELISA. Hepatic triglyceride was extracted from the liver homogenate with Folchsolutioin. An aliquot of the organic phase was collected and resuspended in ethanol containing 1% Triton X-100 and then measured by enzyme-based measurement kits. Bone Marrow Transplantation. For BM transplant studies, bone marrow cells were prepared from the femur and tibia of Pik3cg+/+ and Pik3cg�?�?mice and injected i.v.
into lethally irradiated ob/ob mice or C57BL/6J mice as recipients, as described previously. Treatment with a PI3Kγ Inhibitor. A PI3Kγ selective inhibitor, AS-605240, which was synthesized by Discovery Research Laboratories, Kyorin Pharmaceutical, was used as described previously. Vehicle or AS-605240 was administered intraperitoneally to ob/ob mice twice a day from 6 wk of age. Statistical Analysis. Values of the data are expressed as mean _ SEM. Differences between two groups wer