Diprotin a ile pro ile

The cells were grown in the presence or absence of GLP-1 (100 nmol/l) for 72 hours and then incubated for two more hours in Krebs-Ringer buffer (KRB) containing 16.7 mmol/l glucose and 0.5% BSA (pH 7.4) in the absence of GLP-1 (100 nmol/l). The medium samples were then collected in vials containing aprotinin (0.1 mg/ml) and kept frozen at −20°C until the active GLP-1 ELISA analysis (Millipore Corp. Billerica, MA, U.S.A.) [32] (link). Secretion was normalized to the protein content. Some GLP-1 secretion experiments were performed on rat pancreatic islets cultured up to 72 hours in the presence or absence of Exendin-4 (100 nmol/l) a specific GLP-1 analogue. At the end of culture the islets were washed and incubated for two more hours in Krebs-Ringer buffer (KRB) 0.5% BSA (pH 7.4) in the presence or absence of Exendin-4 (100 nmol/l). All the secretion experiments were performed by incubating the cells in Krebs-Ringer Buffer containing 0,1% DiprotinA (Ile-Pro-Ile, Sigma-Aldrich) a specific dipeptidyl peptidase IV inhibitor, as previously reported [33] (link), [34] (link).

Porcine pancreatic α-amylase, dinitrosalicylic acid (DNSA), acarbose, p-nitrophenyl α-D-glucopyranoside, diprotin A (Ile-Pro-Ile), DPP-IV from porcine kidney, and Gly-pro-p-nitroanilide (GPPN) were purchased from Sigma, Bangalore, India. DPPH, butylated hydroxy toluene (BHT) was obtained from Himedia, Mumbai. α-Glucosidase and dimethyl sulphoxide (DMSO) were obtained from SRL, Mumbai.

High-performance liquid chromatography (HPLC) grade water was from VWR (Dublin, Ireland). Trifluoroacetic acid, formic acid (FA), tris(hydroxymethyl)aminomethane (Tris), Gly-Pro-pNA, diprotin A (Ile-Pro-Ile), porcine DPP-IV (≥10 U mg⁻¹ protein), mass spectrometry (MS) grade water, and acetonitrile were purchased from Sigma Aldrich (Dublin, Ireland). Synthetic peptides (Ala-Ile-Pro, Ala-Pro-Ala, Ala-Pro-Phe, Ala-Pro-Arg, Ala-Arg-Pro, Ile-Phe-Lys, Ile-Ile-Phe, Ile-Pro-Ala, Ile-Arg-Phe, Ile-Arg-Lys, Lys-Pro-Ala, Lys-Arg-Ile, Arg-Ile-Phe, Arg-Ile-Arg, Arg-Lys-Arg, Phe-Pro-Phe, Phe-Pro-Ile, Phe-Pro-Trp, Ile-Ala-Ile, Ile-Pro-Phe, Ile-Pro-Trp, Trp-Pro-Phe, Trp-Pro-Ile, Trp-Pro-Trp) with a purity > 95% (w/w) were obtained from Genscript (Piscataway, NJ, USA).