Chronic glucolipotoxicity in vitro reduces mitochondrial number and activity. Rat islets and 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, total DNA was isolated from islets and mitochondrial DNA copy number (A) was estimated by assessing mtCox1 (mitochondrial gene) copy number normalized to HPRT (nuclear gene). Post treatment, islet ATP levels (B) were estimated after 60min induction with 11 mM (HG) as described in the materials and methods. Both SDH activity in NIT-1 cells (C) and amino acids (5 mM of leucine and 5 mM of glutamine) mediated insulin secretion (D) in islets were measured as described in the materials and methods. VC is vehicle control with 2 mM glucose and without amino acids. 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).