Enzyme linked immunosorbent assay

Rat insulinoma cell line, INS‐1 cells, were kindly provided by Dr. P. Maechler (Geneva) (Asfari et al., 1992 (link)). 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 (link); Merglen et al., 2004 (link)). 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.

Glucose utilization was measured by following the conversion of [5‐³H]glucose into [³H]H₂O. as previously described¹⁰. Cells were seeded in 24‐well plates and subjected to dox treatment. The cells were pre‐incubated in HBKRBB⁹, ¹⁰ with 0.1% bovine serum albumin and 5 mmol/L glucose for 0.5 h, and then incubated with HBKRBB with 5, 12.5 or 20 mmol/L [5‐³H]glucose. After a 2‐h incubation period, a 0.1‐mL aliquot of the incubation medium was transferred to a microtube and then placed in plastic scintillation vials containing 0.6 mL of distilled water. The vials were stoppered and kept at 37°C for 36 h to allow the [³H]H₂O in the microtube to equilibrate with the water. Subsequently, the microtube was taken out and 10 mL of scintillation fluid were added.
For insulin secretion, cells were treated as aforementioned and then incubated in HBKRBB supplemented with varying concentrations of glucose, 5 mmol/L glucose (Glc) + 30 mmol/L KCl, 5 mmol/L Glc + 10 mmol/L leucine + 10 mmol/L glutamine or 5 mmol/L Glc + 0.1 μmol/L glimepiride for 1 h. The media were then collected and assayed for immunoreactive insulin by enzyme‐linked immunosorbent assay (Mercodia, Uppsala, Sweden). Protein contents were analyzed after extraction with 0.1N NaOH using the Pierce 660 nm protein assay kit (Thermo Fisher Scientific).

Glucose in whole blood was detected with the glucose oxidase method using AccuChek Aviva (Hoffman‐La Roche, Basel, Switzerland). Insulin was determined by enzyme‐linked immunosorbent assay (Mercodia, Uppsala, Sweden). The intra‐assay coefficient of variation of the method was 4% at both low and high levels, and the interassay coefficient of variation was 5% at both low and high levels. The lower limit of quantification of the assay was 6 pmoL/L. Total GIP levels were determined with a mouse GIP enzyme‐linked immunosorbent assay kit (Crystal Chem, Elk Grove Village, IL, USA). The intra‐ and interassay coefficient of variation was <10% at low and high levels, and the lower limit of quantification was 2.5 pmoL/L. The assay showed no cross‐reactivity with glucagon, glucagon‐like peptide (GLP)‐1 or GLP‐2.