Substantial expression of ATRAP protein in tissues of WT Agtrap+/+ mice, whereas the protein expression of ATRAP was not detected in tissues of homozygous Agtrap??mice (Figure 1D). All experiments within this study have been performed together with the Agtrap??mice and their Agtrap+/+ littermates.Biochemical AssayBlood samples have been obtained by cardiac puncture at the time mice had been sacrificed within the fed state, unless otherwise stated. Enzymatic assay kits were applied for the determination of plasma glucose, glycoalbumin, absolutely free fatty acids, triglycerides, and total cholesterol (Wako Pure Chemical). Plasma insulin concentrations were measured using a commercially out there ELISA kit (Morinaga).also stained with an Wnt8b Protein Biological Activity Antibody against F4/80 (rat monoclonal; Abcam). Briefly, antigen retrieval was performed by microwave heating and endogenous reactive LY6G6D Protein web molecules were quenched by peroxidase blocking reagent (DAKO). Then, the sections were incubated with monoclonal anti-F4/80 antibody (diluted 1:ten) at area temperature for two hours, followed by Histofine Straightforward Stain Max PO (Nichirei Bioscience Inc). Antibody binding was visualized with three,30 -diaminobenzidine (DAB) making use of a detection kit (Nichirei Bioscience Inc), and all sections had been counterstained with hematoxylin. The adipocyte diameter and location had been quantified applying Image-Pro Plus computer software, and F4/80-positive nuclei have been counted in low-powered fields.Fat TransplantationIn the fat transplantation experiments,13 6-week-old male Agtrap??mice were applied as recipients. Donor epididymal fat pads have been removed from sex-matched Agtrap?? WT Agtrap+/+, or Agtrap transgenic (Tg19) mice (six to 11 weeks of age). The generation and characterization of Agtrap transgenic (Tg64 and Tg19) mice carrying the hemagglutinin (HA)-tagged mouse ATRAP cDNA have been described previously.14 The donor fat pads were reduce into 100- to 200-mg pieces and kept in saline till transplantation. Tiny incisions were made on the back of every single anesthetized recipient mouse, and a total of 900 mg of fat pad tissue (5 pieces from the donor fat pads 3 cm aside from one particular an additional) was implanted subcutaneously (ie, beneath the skin around the back of recipient mouse). 1 week right after transplantation surgery, the recipient mice had been fed an HF diet program (5.6 kcal/g; 60.0 energy as fat; Oriental MF, Oriental Yeast Co Ltd) for 6 weeks, and also the endogenous epididymal adipose tissues with the recipient mice have been harvested for analysis of adipose tissue weight.Glucose Tolerance Test and Insulin Tolerance Test (ITT)Glucose tolerance test (GTT) was performed in 13-week-old male mice soon after 16-hour fasting. Blood glucose concentrations have been measured using a blood glucose test meter (Glutest Neo Super; Sanwa-Kagaku) using blood samples taken from the tail tip at baseline and at 30, 60, and 120 minutes following the intraperitoneal injection of glucose (1 g/kg body weight). For insulin tolerance test (ITT), insulin (0.7 U/kg physique weight in 0.1 BSA; Humulin R-Insulin; Eli Lilly Co, Kobe, Japan) was administered by means of intraperitoneal injection immediately after 1-hour fasting. Blood glucose concentrations had been measured 0 minutes before and 30 and 60 minutes after the injection. GTT and ITT had been performed 7 days apart.Real-time Quantitative RT-PCR AnalysisTotal RNA was extracted from epididymal adipose tissue with ISOGEN (Nippon Gene), and cDNA was synthesized using the SuperScript III First-Strand System (Invitrogen). Real-time quantitative RT-PCR was performed with an ABI PRISM 7000 Sequence Detection Method by.
