H4IIE rat hepatoma cells were serum starved for 16 h in the glu- cose production buffer (glucose-free Dulbecco’s modified essential medium, pH 7.4, containing 20 mM sodium lactate and 2 mM so- dium pyruvate without phenol red) and subsequently treated for
8 h with 2 mM metformin or different concentrations of ANC or D3S5G. At the end of the incubation period, 0.5 mL of medium were taken out to measure the glucose concentration in the culture medium using the Amplex Red® glucose assay kit (Invitrogen) according to manufacturer instructions. All the experiments were performed with cells from passage ten or less. Corrections for cell number were made on the basis of the protein concentration mea- sured using the BCA Protein assay kit (Pierce Biotechnology, Rock- ford, IL). Results were expressed as folds control.