Radioimmunoassay kit

Insulin secretion was measured in static batch incubations described previously [25 (link)]. Briefly islets were pre-incubated in 1 mM glucose for 30 minutes followed by 1 h incubation in extracellular solution with variable glucose concentration. Incubation was 15 min when 50 mM KCl was used as stimulator. The extracellular solution was supplemented with 10 μM FSK, 0.1 μM GLP-1 (Bachem, Weil am Rhein, Switzerland), 100 μM Tolbutamide (Sigma Aldrich, Stockholm, Sweden), 200 μM DIDS, 50 μM TMinh-AO1, 40 μM CFTRinh-172 and/or 50 μM GlyH-101 as indicated. Insulin secretion was measured using radioimmunoassay kit (mouse: Linco Research Inc.,; human: Millipore, Billerica, MA, USA).

Blood and urine parameters were measured as follows. Blood glucose (GLU) level was detected with a HemoCue B-Glucose kit (HemoCue AB, Angelholm, Sweden). Insulin (INS) levels were detected with a radioimmunoassay kit (Linco Research, St Charles, MO, USA). Total cholesterol (TC) and triglycerides (TG) levels were detected by an auto-analyzer (Wako, Osaka, Japan). Blood urea nitrogen (BUN) was measured with a iStat-Kit (HESKA, Fort Collins, MO, USA). Serum and urine creatinine concentrations were detected by HPLC (Beckman Instruments, Fullerton, CA, USA). Urine albumin concentration was detected by an immunoassay (Bayer, Elkhart, IN, USA). Urine albumin-to-creatinine ratio (UACR) was derived as urine albumin/urine creatinine (μg/mg). Serum creatinine and alanine aminotransferase (ALT) amounts were examined with an automatic biochemical analyzer (Olympus AU480, Japan). Urinary podocalyxin levels in mice were measured by enzyme linked immunosorbent assay (ELISA) (Exocell, Philadelphia, PA, USA). The above assays followed the directions of the respective manufacturers.

Adiponectin was assayed by a sandwich ELISA employing an adiponectin-specific antibody. The intra- and inter-assay CV were 3.3% and 7.5%, respectively (Otsuka Pharmaceutical Co., Ltd., Tokushima City, Japan). Leptin was assessed by a radioimmunoassay kit purchased from LINCO Research (St. Charles, MO, interassay CV = 4.9%).

10 ml of venous blood was taken from subjects in the morning after overnight fast. Glucose, TG, HDL-C levels were measured using enzymatic methods by DADE Dimension AR®. Leptin and insulin levels were measured using a radioimmunoassay kit from Linco Research (St Louis, USA). Serum BDNF levels were determined using an ELISA protocol, according to the manufacturer’s instructions (DBD00; R & D Systems, Europe).

Fasting blood samples were obtained (10 ml), centrifuged, and serum samples were stored at − 80 °C until further processing for measuring circulating metabolites and hormones. Samples were analyzed at the Nutrition Laboratory of the Catholic University Medical Center (Santiago, Chile). This laboratory conducts daily assessments of the accuracy of the measurements using quality control softwares (Bio-Rad Laboratories Inc., Hercules, CA), and has a Certificate of Traceability periodically updated by the Centers for Disease Control and Prevention (CDC). Serum glucose was measured using enzymatic colorimetric techniques (HUMAN; Gesellschaft für Diagnose und Biochemical, Wiesbaden, Germany) and serum insulin was measured with a radioimmunoassay kit (Linco Research Inc., St. Charles, MO). Total cholesterol (T-Chol) and triglycerides (TG) were measured using enzymatic colorimetric techniques (HUMAN). High-density lipoprotein cholesterol (HDL) was isolated by precipitation with a solution of sodium phosphotungstate magnesium chloride^{29 (link)}. LDL cholesterol was calculated using the Friedewald formula (i.e., all concentrations of triglycerides were < 400 mg/dl)^{30 (link)}. Serum leptin and adiponectin were measured using commercial radioimmunoassays (Millipore) and C-reactive protein (CRP) was assessed with a high-sensitivity (hs-) enzyme immunoassay kit (BIOMERICA, Inc.).