A technique for the isolation of chicken hepatocytes and their use in a study of gluconeogenesis

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 responded 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) micrometers hormone. The same type of curve was obtained with dibutyryl-cAMP, but the half-maximum stimulation was achieved at around 1.0 micrometer. 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 significant increase in the rate of gluconeogenesis; this continued for another 3 min and then at a slower pace (slope = .2566).