To assess the effects of NPY and Pancreatic Polypeptide on rodent BRIN-BD11 and human 1.1B4 beta-cell proliferation, cells are seeded at a density of 150,000 cells per well and cultured overnight in the presence of NPY or Pancreatic Polypeptide (1 μM), and compared to positive control GLP-1 (1 μM). Cells are washed with PBS and fixed using 4% paraformaldehyde. After antigen retrieval with citrate buffer at 95°C for 20 min, tissue is blocked using 2% BSA for 45 min. The slides are then incubated with rabbit anti-Ki-67 primary antibody, and subsequently with Alexa Fluor^? 594 secondary antibody. Slides are viewed using fluorescent microscope and photographed by DP70 camera adapter system. Proliferation frequency is determined in a blinded fashion and expressed as % of total cells analysed. Approximately 150 cells per replicate are analysed. For analysis of ability of NPY and Pancreatic Polypeptide to protect against streptozotocin-induced DNA damage, BRIN-BD11 and 1.1B4 cells are seeded. Cells are then exposed to streptozotocin (5 mM) in the presence or absence of NPY or Pancreatic Polypeptide (1 μM) for 2 h, with GLP-1 (1 μM) as positive control. Cells are then harvested and a comet assay is performed. Resulting gels are stained using DAPI (4′,6-diamidino-2-phenylindole) (100 μg/mL) and slides are viewed under appropriate filter. Comet score software is used for the analysis of % tail DNA (100 cells per gel) and olive tail moment^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
Plasma glucose and insulin responses are evaluated after intraperitoneal (i.p.) injection of glucose alone (18 mmol/kg body weight) or in combination with test peptides (Pancreatic Polypeptide, etc.; 25 nmol/kg body weight) in overnight (18 h) fasted C57BL/6 mice. In a second series of experiments, 18 h fasted mice are used to assess the effects of respective test peptides on food intake. Mice receive an i.p. injection of saline alone (0.9% (w/v) NaCl) or in combination with test peptides (25 nmol/kg body weight) and food intake measured at 30 min intervals for 180 min. A dose of 25 nmol/kg is chosen with other NPYR modulators on glucose homeostasis, insulin secretion and feeding at this dose^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Khan D, et al. Influence of neuropeptide Y and pancreatic polypeptide on islet function and beta-cell survival. Biochim Biophys Acta. 2017 Apr;1861(4):749-758.  [Content Brief]