2.1. Cell culture and measurement of insulin release

Rat insulinoma cell line, INS‐1 cells, were kindly provided by Dr. P. Maechler (Geneva) (Asfari et al., ¹⁹⁹²). INS‐1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen) including 10% fetal calf serum, and additions were as previously described (Lawrence et al., ²⁰⁰⁸; Merglen et al., ²⁰⁰⁴). Cells were harvested using a nonenzymatic cell dissociation liquid (Invitrogen). Cells were resuspended in the above media and were dissociated before being seeded into 96‐well plates (40,000 cells per well). The medium was replaced with defined serum‐free medium containing TZDs, and other inhibitors at the designated concentrations. Rosiglitazone was purchased from Sigma‐Aldrich, while pioglitazone and lobeglitazone were kind gifts from Chonggundang Co. After 24 h of treatment, cells were washed with glucose‐free Krebs (NaCl 140 mM, KCl 3.6 mM, CaCl₂ 1.5 mM, MgCl₂ 1.19 mM, KH₂PO₄ 1.19 mM, NaHCO₃ 2 mM, and HEPES (pH 7.4) 10 mM) containing 0.1% insulin‐free bovine serum albumin, and then incubated for an additional 60 min in 1 ml of KRBH buffer containing 3.3 or 16.7 mM of glucose. Insulin released in the medium was determined by enzyme‐linked immunosorbent assay (Mercodia). Insulin concentration in nanograms per milliliter (ng/ml) was normalized against total protein in micrograms.