[14]. Lipolysis was measured by the rate of glycerol released from adipocytes. To isolate adipocytes, the fat pads of epididymus were digested EPZ-6438 with collagenase at 37 °C in a shaking water bath for 45 min. Next, cells were filtered through nylon mesh and washed three times with a HEPES buffer (pH 7.4) containing: 137 mM NaCl, 5 mM KCl, 4.2 mM NaHCO3, 1.3 mM CaCl2, 0.5 mM MgCl2, 0.5 mM MgSO4, 0.5 mM KH2PO4, 20 mM HEPES and 1% BSA. After a period of incubation, an aliquot of the infranatant was removed for enzymatic determination of glycerol released into the medium. Glycerol levels were measured before and after isoproterenol (0.1 μmol/L) or isoproterenol

followed by insulin (12.5 ng/mL) incubation. At eighth week of treatment, fasting glucose was evaluated selleck in tail blood sample using an Accu-Check glucometer (Roche Diagnostics, Indianapolis, IN). Glucose uptake was also measured in isolated adipocytes from epididymal fat tissue. The isolated adipocytes were incubated for 45 min at 37 °C in the presence or absence of insulin (50 ng/mL). The uptake of 2-deoxy-[3H]glucose (2DOG) was used to determine the insulin sensitivity in glucose uptake, as described by Green [7]. The assay was initiated by the addition of 2DOG (0.2 μCi/tube) for 3 min. Next, cells were separated

by centrifugation through silicone oil and cell-associated radioactivity was determined by scintillation counting. Nonspecific association of 2DOG was determined

by performing parallel incubations in the presence of 15 mmol/l phloretin, and this value was subtracted from glucose transport activity in each condition. Epididymal fat pads were homogenized in buffer Tris–HCl (pH 8.3) containing detergents. LPL activity was measured using a [9,10-3H]triolein containing substrate with lecithin [16] and 24 h fasted rat plasma as a source of apo CII. The reaction was stopped with a mixture of extraction Bacterial neuraminidase [1], and the released 3H-free fatty acids (FFAs) were quantified by liquid scintillation. The enzyme activity was expressed as nanomoles of FFA released per minute. Data were presented as mean ± SE. Differences between before and after were assessed by Student t test for paired observations. Differences among groups were analyzed by one-way ANOVA followed by Newman–Keuls post hoc or two-way ANOVA followed by Bonferroni post hoc. Statistical analysis was performed using the GraphPad Prism Software (version 5.0). At the beginning of treatment, animals presented similar body weight (averaged of all animals = 322 ± 5 g, n = 40; Fig. 1A). After 14 weeks of oral treatment, animals of all groups had significantly increased body weight and the values obtained were not different among the groups (averaged of all animals = 391 ± 5 g, n = 40; Fig. 1A).