In vitro chronic glucolipotoxicity impairs glucose uptake/metabolism. Rat islets or NIT1 cells were cultured under normal conditions (control) or with 16.7 mM glucose and 500 μM palmitate for 72 h (GL). Post 72 h, RNA was isolated from islets and the mRNA levels of Glut2/slc2a2 (A), GCK (C) and PC (E) were measured as described in the materials and methods. Protein levels of Glut2/slc2a2 (B) were measured in both islets and NIT-1 cells by western blotting using anti-Glut2/slc2a2 antibodies. Glucose uptake (D) was measured NIT-1 cells using 2-NBDG, a non-metabolized fluorescent analog as described in the materials and methods. (F) For insulin secretion in the presence of ATP citrate lyase (ACLY) inhibitor (Radicicol), post 72 h treatment islets were treated with 11 mM glucose (HG) with/without 50 μM inhibitor for 2 h and secreted insulin were measured. Data are expressed as mean±SEM and statistical analysis was performed using the unpaired Student’s t-test. (*P<0.05, **P<0.01 and ***P<0.001, n=4).