Cell culture

HUVECs were isolated by collagenase digestion (0.25 mg/ml Collagenase Type I from Clostridium histolyticum; Gibco Life Technologies, Grand Island, NY, USA) as previously described (González et al., 2004). In brief, cells were cultured (37°C, 5% CO₂) up to passage 3 in medium 199 (M199) (Gibco Life Technologies, Grand Island, NY, USA) containing 5 mM D-glucose, 10% newborn calf serum (NBCS), 10% fetal calf serum (FCS), 3.2 mM L-glutamine, and 100 U/ml penicillin-streptomycin (primary culture medium, PCM). Experiments were performed on cells incubated (0–30 min) in M199 in the absence or presence of insulin (1 nM). Cell viability estimated by Trypan blue exclusion was higher than 97% (not shown). Sixteen hours prior, the experimental incubation medium was changed to sera-free M199 (González et al., 2015).