A Technique for the Isolation of Chicken Hepatocytes and Their Use in a Study of Gluconeogenesis

ABSTRACT A new technique was developed for the isolation of chicken liver parenchymal cells. Glucose produced from 10 mM lactate was proportional to the amount of cells present. In the time-course study, gluconeogenesis from lactate and fructose was linear up to 60 min. Fructose proved to be the best substrate. Fructose was converted to glucose at the highest rate; this was followed by lactate, pyruvate, and xylitol. Alanine, glycerol, propionate, alpha-ketoglutarate, and succinate proved to be poor substrates. There was no statistical difference between the results obtained with hepatocytes obtained from fed or fasted chickens. The isolated hepatocytes re-sponded to glucagon, dibutyryl-cAMP, and epinephrine. The dose-response for glucagon was a sigmoid-curve and the half-maximum stimulation was given by approximately 1 X 10 — 2 nM hormone. The same type of curve was obtained with dibutyryl-cAMP, but the half-maximum stimulation was achieved at around 1.0 jiiM. The response to epinephrine was marginal. In the time-course experiment, prior to glucagon stimulation, glucose accumulated at a linear rate (slope =.2484). After the addition of the hormone, the level of cAMP increased by about 30 % in the first minute and reached a peak (100%) in about 2 min; thereafter, it decreased to the level prior to the stimulation by the hormone. Two minutes after the addition of glucagon there was a signifi-cant increase in the rate of gluconeogenesis; this continued for another 3 min and then at a slower pace (slope =.2566). (Key words- Chicken hepatocytes, gluconeogenesis, isolation technique