Ezglp1t 36k

Stool samples were collected by subjects at home within maximum 48 h prior to the study visits. Stool samples were directly frozen at subjects’ home after collection (within 10 min) and returned to study site in a frozen state. The stool biomarkers SCFAs (acetate, butyrate, propionate) were analyzed by gas chromatography at Enterosan Labor LS SE & Co. KG, an accredited lab (Bad Bocklet-Großenbrach, Germany). Blood sampling was performed at each visit after at least 10 h overnight fast. For the assessment of total GLP-1 and PYY, DPP-IV and AEBSF inhibitor was added to the EDTA-plasma tube prior to blood collection. Total GLP-1 and PYY were analyzed in plasma utilizing ELISA technique with technical duplicates for each sample according to the manufacturers protocol (Merck Millipore EZGLP1T-36K for total GLP-1 and EZHPYYT66K for PYY). Lipid status (triglycerides, total cholesterol, HDL- and LDL-cholesterol) and further safety blood routine measures were determined in an accredited routine lab at Synlab Medizinisches Versorgungszentrum Leinfelden (Leinfelden, Germany). Stool frequency and consistency by means of the Bristol stool scale were reported daily in a diary. Fiber intake was estimated with a 3-days food protocol and evaluated with EBISpro (Dr. Ehrhardt, Willstätt-Legelshurst, Germany) based on the German Nutrient Data Base.

Urine, drink and serum samples were analysed in duplicate for osmolality by freezing point depression (Gonotec Osmomat 030, Gonotec, Berlin, Germany). To prevent the degradation of acylated ghrelin, 50 µL of Pefabloc (Roche Diagnostics Limited, Burgess Hill, UK) was immediately added to blood samples. Blood samples were centrifuged at 1500× g for 15 min at 4 °C and the serum aliquoted and stored at −80 °C until analysis was performed. Serum glucose, lactate, and triglyceride concentrations were determined in duplicate using a clinical chemistry analyser (Randox Daytona, Crumlin, UK). Serum fructose concentration was determined using a colorimetric assay (EnzyChrom™ EFRU-100; BioAssay Systems, Hayward, CA, USA). Circulating concentrations of acylated ghrelin, insulin, GIP, and leptin were determined using multiplex analysis (Luminex 200, Luminex Corporation, Austin, TX, USA) with kits purchased from Merck-Millipore (HMHMAG-34K, Milliplex MAP, Merck Millipore Ltd., Feltham, UK). Circulating concentrations of total GLP-1 were determined in duplicate using Enzyme Linked Immunoassay (EZGLP1T-36K, Merck Millipore Ltd., Feltham, UK).

NCI-h716 and HuTu-80 cells were cultivated in respectively RPMI and DMEM, both supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 50 IU ml⁻¹ penicillin and 50 µg ml⁻¹ streptomycin in humidified incubator at 37°C with 5% CO2. Oxygen consumption and secretion assays were performed in a secretion buffer composed of NaCl 140 mM, KCl 5 mM, MgCl₂ 2 mM, CaCl₂ 2 mM, Hepes 10 mM, pH adjusted to 7.3. Na₂S (from Sigma) was dissolved in the secretory buffer and pH adjusted to 7.3 with NaOH.
For secretion, 4 million cells per sample were rinsed in secretion buffer and resuspended in 2mL. Na₂S was added either every 90s or at once, all resulting in the same final volume of 2.1mL. After 15 minutes of experiment, cells were centrifuged at 300g at 4°C and supernatant was frozen at -20°C. GLP-1 was then measured using a GLP-1 Elisa (EZGLP1T-36K from Merck Millipore) and data analyzed using R. Experiments were performed in duplicate and replicated 4 times, resulting in 8 independent measures of GLP-1 in each condition. Data were normalized with the basal secretion of each experiment and differences between conditions assessed using a Kruskall-Wallis test followed by a Dunn test, excluding the positive control Forskolin/IBMX high glucose from the analysis. A p adjusted value below 0.05 was considered to indicate difference between groups.

The patients were subjected to pre-operative fasting and plasma samples were collected before surgery, placed immediately on ice and transferred to −80 °C. The total GLP-1 (7–36 and 9–36) (Cat. No.: EZGLP1T-36K, Merck, Darmstadt, Germany) was measured according to the manufacturer’s instructions.