Atorvastatin

Rat pancreatic INS-1 cells were kindly provided by Prof. Hai Qian (China Pharmaceutical University, Nanjing, China) [21 (link)]. The cells were routinely reseeded every 2-3 days and cultured in RPMI 1640 medium containing 11 mmol/L (mM) glucose, 2 mM L-glutamine supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 50 μmol/L (μΜ) β-mercaptoethanol, 10 mM N-2-hydroxyethyl piperazine-N-2-ethane-sulphonic acid (HEPES), 100 IU/mL penicillin, and 100 mg/mL streptomycin in a humidified cell incubator at 37°C in a humidified atmosphere (5% CO₂ and 95% air). Lek Pharmaceuticals d.d. generously provided us with atorvastatin (Ljubljana, Slovenija). Final concentrations of atorvastatin ranged from 0.2 μM to 20 μM. Pioglitazone hydrochloride, U-73122, and GW1100 were purchased from MedChem Express (NJ, USA). INS-1 cells were incubated with atorvastatin, pioglitazone, GW1100, FFA1 siRNA, and/or U-73122 for 24 h prior to insulin secretion and other experiments. atorvastatin, pioglitazone, GW1100, and U-73122 were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO was adjusted to 0.1% (v/v). The medium containing the same amount of DMSO was used as the control.

Trametinib (Selleckchem, Planegg, Germany) was dissolved in DMSO at a final concentration of 40 mM, aliquoted, and kept at −80 °C. Atorvastatin, simvastatin, cerivastatin, and verteporfin (MedChemExpress, Sollentuna, Sweden) powders were dissolved with DMSO at a final concentration of 10 mM, aliquoted, and kept at −20 °C. Valproic acid (Sigma-Aldrich, Milan, Italy) was dissolved in deionised water at a final concentration of 1 M and kept at −20 °C.

HCM, C2C12 and HUVEC were cultured in high-glucose DMEM (Gibco BRL, USA) supplemented with 5% fetal bovine serum (Gibco BRL, USA) without antibiotics in acceptable conditions (5% CO₂, 37°C). Ferrostatin-1 (Fer-1), deferoxamine mesylate (DFO), liproxstatin-1 (Lip-1), RSL3, Z-VAD-FMK and mitoTEMPO (MT) were purchased from Selleck (Selleck, China). Atorvastatin, lovastatin, pravastatin, and rosuvastatin were obtained from MCE (MedChemExpress, China). Fer-1, DFO, Lip-1, RSL3, Z-VAD-FMK and MT were dissolved in dimethylsulfoxide (DMSO) and diluted in a culture medium at ultimate concentrations (0.5 μM with RSL3; 1 μM with Fer-1; 80 μM with DFO; 1 μM with Lip-1; 10 mM with mitoTEMPO; and 2 μM with Z-VAD-FMK). Statins were stocked as 50 mM solutions in DMSO. Final concentrations ranged from 10 to 160 μM.