, USA). Protein extracted from epididymal adipose tissue was quantified by the method of Bradford [6]. Adipocytes were isolated from epididymal fat pads by the method of Rodbell [22]. Digestion
was carried out at 37 °C with constant shaking for 45 min. Cells were filtered through nylon mesh and washed three times with buffer containing (mM): 137 NaCl, 5 KCl, 4.2 NaHCO3, 1.3 CaCl2, 0.5 MgCl2, 0.5 MgSO4, 0.5 KH2PO4, 20 mM HEPES (pH 7.4), plus 1% BSA. Glycerol release was measured and used as lipolytic index, as previously described [11]. After isolation, adipocytes were incubated at 37 °C in a water bath for 60 min, in basal conditions or in the presence of 0.1 μM isoproterenol (ISO), a non-specific beta-receptor agonist. The effects of 25 ng/mL insulin on isoproterenol-stimulated lipolysis were also determined. Vorinostat solubility dmso Total RNA from adipose tissue was prepared using Tri-Phasis reagent (BioAgency, São Paulo, SP, Brazil), treated with DNAse. Strand cDNA this website was generated from 2 μg of RNA using M-MuLV Reverse Transcriptase (Fermentas, Thermo Fisher Scientific Inc., USA). The hypoxanthine guanine phosphoribosyltransferase (HPRT-endogenous control), peroxisome proliferator-activated receptor gamma (PPARγ) and acetyl-CoA carboxylase (ACC) cDNA were amplified using specific primers and SYBER green reagent (Applied Biosystems) in
an ABI Prism 7000 platform (Applied Biosystems). The following primer pairs were used: HPRT reverse 5′-gattcaacttgcgctcatcttaggc-3′; HPRT forward 5′-gttggatacaggccagactttgtt-3′; PPARγ reverse 5′-aggaactccctggtcatgaatcct-3′; PPARγ forward 5′-agatcatctacaccatgctggcct-3′; ACC reverse 5′-aatccactcgaagaccactg-3′; ACC forward 5′-cggcttgcacctagtaaaac-3′. Protein was extracted from epididymal adipose tissue and 30 μg of protein were resolved on SDS-PAGE (10%) and then transferred onto nitrocellulose membranes. For immunoblotting, the membranes were probed with a polyclonal rabbit anti-FAS antibody (1:1000; Abcam Inc., USA). The blots were then incubated with HRP-conjugated anti-rabbit IgG (1:1000; Sigma-Aldrich) and β-actin was used as endogenous
control. The protein abundance was detected by chemiluminescence (Immobilon Western, Millipore Corporation) and immuno-reactive bands were visualized by densitometry CYTH4 using an Image J program, National Institute of Health, USA. The results were expressed by the relationship FAS/β-actin in units of relative density. The data are reported as mean ± SEM. Differences between groups were evaluated using unpaired Student’s test. To analyze lipolytic activity analysis of variance (ANOVA) was used, followed by Newman–Keuls test. Significance level was set at p < 0.05. The absence of Mas receptor induced 60% decrease in the gene expression of PPARγ (WT = 1.6 ± 0.13 arbitrary unit vs. Mas-KO = 0.65 ± 0.08 arbitrary unit, p < 0.05) and a 51% decrease in the gene expression of ACC (WT = 1.5 ± 0.031 arbitrary unit vs. Mas-KO = 0.73 ± 0.012 arbitrary unit, p < 0.05) ( Fig.
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