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).

Gluconeogenesis in isolated chicken hepatocytes: effect of fatty acids, beta-hydroxybutrate, ethanol, and various pyruvate/lactate ratios.

The effect of fatty acids beta-hydroxybutyrate, ethanol, and different pyruvate/lactate ratios on gluconeogenesis in isolated chicken hepatocytes was investigated. Glucogenesis was significantly affected by a change in the oxidation-reduction (pyruvate/lactate) ratio, and this effect was greater than could be accounted for by the additive effects of these substrates. Substituting lactate with nongluconeogenic substrates, such as beta-hydroxybutyrate or ethanol, increased the formation of glucose by 80 and 200%, respectively, demonstrating the beneficial effect of the increased reducing equivalents in the hepatocytes. Oleic acid per se had no effect but when added complexed with albumin, it had a negative effect on gluconeogenesis.

Intracellular distribution of hepatic and renal gluconeogenic enzymes in embryonic and growing chickens.

The intracellular distribution of hepatic and renal gluconeogenic enzymes in 20-day-old chicken embryos and 4-week-old chickens (Gallus domesticus: New Hampshire male X Columbian female) has been studied. Pyruvate carboxylase, fructose-1,6-diphosphatase, and glucose-6-phosphatase were found primarily in the mitochondrial, cytosolic, and microsomal fractions, respectively. Phosphenolpyruvate carboxykinase was present not only in the mitochondria but also in the cytosol of the chicken liver and the kidney. The intracellular distribution of the liver enzyme differed from that of the kidney enzyme in chicken embryos as well as in growing chickens.

Effect of fasting and fasting and refeeding on hepatic and renal gluconeogenic enzymes in the chicken.

The effect of fasting and fasting and refeeding on hepatic and renal gluconeogenic enzyme activities were studied in six-week-old chickens (Gallus domesticus: New Hampshire male x Columbian female). Hepatic pyruvate carboxylase appeared not to be affected by fasting, but the renal enzyme activity increased in four-day fasted chickens. The hepatic mitochondrial and cytosolic phosphoenolpyruvate carboxykinases were essentially not affected by fasting. The renal mitochondrial phosphoenolpyruvate carboxykinase showed a slight increase in activity only after a four-day fast, but the cytosolic enzyme activity increased markedly already after a two-day fast. Also the activities of the hepatic and renal fructose-1,6-diphosphatase and glucose-6-phosphatase increased markedly on fasting. Refeeding for four days after a four-day fast returned these enzyme activities to near control values.

Development of gluconeogenic, glycolytic, and pentose-shunt enzymes in the chicken kidney.

The activities of the key gluconeogenic, glycolytic, and pentose-shunt enzymes in chicken kidney were determined starting from 8 days before to 58 days after hatching. The activities of pyruvate carboxylase (PC), mitochondrial and cytosolic phosphoenolypruvate carboxykinase (PEPCK), fructose-1,6-diphosphatase (FDPase) and glucose-6-phosphatase (G6Pase) were low in the embryonic tissue but increased towards the time of hatching. After hatching, the activities of PC, mitochondrial PEPCK, and G6Pase continued to increase, but those of FDPase and cytosolic PEPCK decreased. Relatively little change in these activities was observed in chickens over 24 days old. The activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) increased during embryonic growth. After hatching, HK activity continued to increase and then decrease, whereas PFK appeared to decrease and then increase to prehatch levels in 28-day-old birds. LDH activity continued to increase until 8 days after hatching and remained constant thereafter. No definite pattern was discernible in the case of PK. As for the pentose-shunt enzymes, there was no significant change in glucose-6-phosphate dehydrogenase activity (G6PDH), but the activity of 6-phosphogluconate dehydrogenase (6PGDH) increased until the chickens were 14 days old and then remained relatively constant.

Activities of pyruvate and prionyl Coenzyme A carboxylase in chicken tissues during normal growth and biotin deficiency.

Activities of pyruvate and propionyl CoA carboxylase in chicken tissues during normal growth and biotin deficiency were investigated. In normal growing chickens, liver and kidney pyruvate carboxylase activity was high and varied with age. The activity in heart and brain was low and remained relatively constant throughout the experimental period. Propionyl CoA carboxylase activity in kidney and heart appeared to increase with age but remained unchanged in liver and brain. Biotin deficiency progressively decreased both pyruvate and propionyl CoA carboxylase activities in liver, kidney, heart and brain. Most marked effects were observed in liver and kidney.