Measurements of GLP-1 release in STC-1 cells.

STC-1 cells (10⁶ cells/ml) were cultured in a 24-well plate (1 ml each well) for 2 days to reach ∼70% confluence. The method for collecting cell culture medium was modified from a previous report (19). On the test day, cells were rinsed three times with Hanks' balanced salt solution, and then new Hanks' balanced salt solution (1 ml) containing LA with or without MA was added, and cells were incubated for 30 min at 37°C. After incubation, conditioned medium was collected, centrifuged, and stored at −80°C until GLP-1 determination. Cells were stored at −20°C for isolation of proteins. Concentration of GLP-1 was determined by enzyme immunoassay with a GLP-1 EIA Kit (RAB0201; Sigma-Aldrich). To test effects of FA and MA on GLP-1 secretion, we used optimal doses of LA (100 μM) and MA (0.1 mM) determined from pilot studies. MA was applied 2 min before LA treatment. Total protein in each well was measured using a BCA protein assays kit (ThermoFisher Scientific), and GLP-1 levels were normalized with the total protein in each well.