1. Cell cultures and reagents

The rat pancreatic β-cell line RIN-5F was obtained from the European Collection of Animal Cell Cultures (America Type Culture Collection, CRL-11605; ATCC, VA, USA). Cells were cultured in Roswell Park Memorial Institute medium (cat. A1049101, Gibco, Gaithersburg, MD, USA) containing L-glutamine (Gibco) and supplemented with fetal bovine serum (Gibco) and an antibiotic-antimycotic mixture (Gibco). Cell cultures were maintained at 37℃ in a humidified atmosphere containing 5% CO₂.

Sources of reagents were as follows: glucose and alloxan (Sigma-Aldrich, St. Louis, MO, USA); a specific inhibitor for extracellular signal-regulated kinase (ERK), PD98059 (Cell Signaling, Danvers, MA, USA); anti-S1P/EDG5, anti-pSPHK2, anti-SPHK2, anti-insulin, and anti-SREBP antibodies (Thermo Fisher Scientific, Waltham, MA, USA); anti-pERK1/2, anti-ERK1/2, anti-pAKT, anti-AKT, anti-β-actin, and horseradish peroxidase-conjugated secondary antibodies (Cell Signaling, Danvers, MA, USA). Western blots were detected using an enhanced chemiluminescence reagent (Amersham Bioscience, Amersham, UK).