Derek R. Langslow

Hepatic gluconeogenesis in chickens

SummaryGluconeogenesis by isolated hepatocytes resulted in glucose release but insignificant rates of glycogen synthesis. The effectiveness of precursors was similar for hepatocytes from fed and starved chickens except for impaired gluconeogenesis from pyruvate when compared to lactate in lactate in starved chicken hepatocytes. The impairment was caused by limitations in cytosolic NADH production as a result of the mitochondrial location of phosphoenolpyruvate carboxykinase in chicken liver. The order of effectiveness of precursors on hepatic gluconeogenesis was generally similar to the effects of precursors on increasing the plasma glucose concentration in vivo. The exceptions were caused by interactions with other precursors in vivo.The alteration of the NADH/NAD+ ratio by ethanol and ATP/ADP ratio by adenosine could play significant roles in the control of precursor conversion to glucose. Physiological glucagon concentrations stimulated gluconeogenesis from precursors entering the pathway both above and below the level of triose phosphates, and its effect were mimicked by dibutyryl cyclic AMP.Previous results on the effects of precursor and glucagon injection on the plasma glucose concentration of chickens in vivo can largely be explained by effects at the hepatic level.Isolated chicken and rat hepatocytes share many common features. Qualitatively the ordering of gluconeogenic effectiveness was similar but quantitive differences existed as a result of differing activities and cellular locations of enzymes. Neither preparation readily synthesised glycogen and the sensitivity to glucagon was similar.

Hormonal effects on cyclic nucleotides and carbohydrate and lipid metabolism in isolated chicken hepatocytes

Abstract The basal cAMP content of isolated chicken hepatocytes (0.5–1.5 pmol 10 6 cells) was increased transitorily by both glucagon (up to 60-fold) and adrenaline (up to 20-fold). These hormones stimulated cellular glycogenolysis by up to 200% and gluconeogenesis from lactate by up to 30%, and inhibited lipogenesis from acetate by up to 90%. Half-maximal metabolic effects were induced by hormone concentrations that were less than 10% of the concentrations producing a half-maximal rise in cAMP. Physiological concentrations of insulin and avian pancreatic polypeptide (APP) failed to alter basal or hormone-elevated cAMP concentrations, and were also without effect on glycogenolysis, gluconeogenesis, and lipogenesis. Insulin failed to stimulate glycogen synthesis. None of these hormones affected the cGMP content of the hepatocytes (0.03 pmol/10 6 cells), or altered hepatocyte phosphodiesterase activity. Since high-affinity insulin receptors are present on these chicken hepatocytes we conclude that the insulin resistance of chickens in vivo is due to lack of a hormone effector system in the liver; the lack of metabolic effects resulting from APP is consistent with its lack of receptors on the hepatocytes.

The binding of pancreatic hormones to isolated chicken hepatocytes

Hepatocytes were isolated from chicken liver by a recirculating perfusion method. Cells exhibited physical and metabolic characteristics similar to those of whole liver, and retained their viability, as judged by trypan blue exclusion, oxygen consumption, and nucleotide content, for up to 2 hr when incubated at 37°. Binding of 125I-labeled insulin, glucagon, and avian pancreatic polypeptide (APP) were assessed. The first two had both high- and low-affinity sites, but APP showed only low-affinity binding. Even at the lowest physiological blood insulin concentrations it is likely that many high-affinity binding sites will be occupied; in contrast, only a small percentage of high-affinity glucagon sites will be occupied even at the highest physiological levels of this hormone. It is concluded that avian resistance to insulin lies not in a lack of receptors on hepatocytes, but in the mechanism by which receptor occupancy is manifested as a metabolic response; and that the effects of APP are mediated by tissues other than liver.

The use of viable hepatocytes to study the hormonal control of glycogenolysis in the chicken

SummaryThe rapid isolation of high yields of parenchymal cells from chicken liver is described. Stringent tests of viability show that the isolated hepatocytes are both structurally and metabolically similar to those in intact liver. During incubation viability decreased and the significance of this change on the interpretation of metabolic experiments is discussed. Lactate was a much more effective gluconeogenic precursor than pyruvate even in the presence of additional reducing equivalents. Hepatocytes isolated from fed chickens produced glucose from glycogen degradation. Glycogenolysis was stimulated by glucagon, dibutyryl cyclic AMP and adrenaline. Half maximal glucagon effects were elicited by physiological concentrations of the hormone. Glucagon and dibutyryl cyclic AMP stimulated glucagon, dibutyryl cyclic AMP and adrenaline their action was not additive to that of adrenaline.

The action of hydrocortisone, insulin, and glucagon on chicken liver hexokinase and glucose-6-phosphatase and on the plasma glucose and free fatty acid concentrations.

Abstract Chicken liver hexokinase and glucose-6-phosphatase activities were measured i vitro following the administration of glucagon, insulin, and hydrocortisone in vivo . Insulin lowered hexokinase activity but did not affect glucose-6-phosphatase activity. Neither enzyme was altered by glucagon. Hydrocortisone suppressed hexokinase activity 1, 4, and 24 hr after injection. White the glucose-6-phosphatase activity per unit weight of liver was unaltered by hydrocortisone, the total liver weight was increased and the ratio of enzyme activity to wet weight remained constant. Hydrocortisone caused hyperglycaemic at all three time intervals. Hydrocortisone elevated the plasma FFA concentration up to 4 hr but was increased threefold over the fed control values after 24 hr. The effect of these three hormones on the regulation of glucose flux into and out of liver may be partly explained by actions on hexokinase and glucose-6-phosphatase. It is likely that other liver enzyme activities which alter the flux through glucose-6-phosphatase are also changed. Hydrocortisone greatly increases the flux through gluconeogenesis such that hyperglycaemia and a large increase in glycogen content are achieved.

Glucose phosphorylation and dephosphorylation in chicken liver.

1. Glucokinase was absent from chicken liver and only the low Km hexokinases, inhibited by AMP, ADP but not ATP, were present. 2. The Km of chicken liver glucose-6-phosphatase for glucose-6-phosphate was reduced from 5.65 to 3.75 mM following starvation, and the enzyme was inhibited by glucose. 3. Starvation of chickens for 24 hr slightly lowered the hexokinase activity and doubled glucose-6-phosphatase activity; it did not change subcellular distribution of the enzymes. Oral glucose rapidly restored the activities to fed values. 4. It was concluded that glucose uptake into, and efflux from, chicken hepatocytes, was regulated by the activity and kinetic characteristics of glucose-6-phosphatase and by the glucose-6-phosphate concentration, and that the hexokinases had little regulatory function.

Possible mechanisms for the increased sensitivity to glucagon and catecholamines of chicken adipose tissue during hatching.

Abstract The lipolytic sensitivity of chicken adipocytes to glucagon increases about 10-fold over the hatching period. Experiments were conducted to determine whether this change in sensitivity was related to changes in cyclic AMP metabolism within adipocytes. The capacity of glucagon to increase adipocyte cyclic AMP content was increased in 2-day-old chicks compared to 19-day embryos. This effect was seen at all glucagon concentrations, without any marked shift in the glucagon concentration for half-maximal effect. Cyclic AMP responses to catecholamines and theophylline were also greater in chick adipocytes than in cells from embryos. Lipolysis was maximally activated when cyclic AMP was increased to only a small fraction of maximum. There was some indication that a given increase in cyclic AMP content was associated with higher rates of lipolysis in chick than in embryo adipocytes. Phosphodiesterase activity was at a minimum on the day of hatching, but levels in 2-day-old chicks were similar to those in 19-day embryos. It is concluded that the increase in lipolytic sensitivity to hormones over the hatching period is associated with an increased capacity for cyclic AMP accumulation, probably due to increased adenylate cyclase activity. There may also be an increase in the sensitivity of the lipase activation system to cyclic AMP.

The action of pancreatic hormones on the cyclic AMP content of isolated chicken hepatocytes

Abstract The hormonal control of cyclic AMP content was investigated in hepatocytes isolated by collagenase digestion from chicken liver. Glucagon (2.9 × 10 −7 M ) increased cyclic AMP content within 1 min, and the concentration continued to increase for at least 30 min. More than 90% of the total cyclic AMP measured remained within the cells during this time. Significant stimulation of glycogenolysis was obtained with a glucagon concentration (2.9 × 10 −11 M ) which had no detectable effect on cyclic AMP content. Half-maximal increases in cyclic AMP content and glucose production were produced by glucagon concentrations of approximately 10 −8 and 10 −10 M , respectively. Insulin (0.17–17 × 10 −9 M ) and avian pancreatic polypeptide (0.24–240 × 10 −9 M ) had no effect on cyclic AMP content either in the presence or absence of glucagon. Adrenaline increased cyclic AMP content, the minimum effective concentration being 5 × 10 −8 M . The cyclic AMP response to adrenaline reached a peak within 2.5 min, and the maximum increase was much less than that produced by glucagon.