Interaction of salmon glucagon, glucagon-like peptide, and epinephrine in the stimulation of phosphorylase a activity in fish isolated hepatocytes.

The simultaneous addition of epinephrine and salmon glucagon to catfish (Ictalurus melas) and trout (Salmo gairdneri) hepatocytes did not induce greater increases in glycogen phosphorylase a activity and in glucose release than those caused by epinephrine alone. The effects of epinephrine are greater than those of glucagon. Propranolol added to the hormonal pool blocked the epinephrine effects. In trout cells, epinephrine and glucagon-like peptide (GLP) had similar effects and when they were added simultaneously the stimulation of metabolic indices was higher compared to that obtained with either epinephrine or GLP. However, the effects were not additive. In the presence of epinephrine plus GLP the inhibitory effect of propranolol was not evident, due to the effect induced by GLP, on which propranolol was not effective. This may indicate that epinephrine masks the GLP effect. Results could mean that epinephrine and glucagon-family peptides act in catfish and trout hepatocytes through different receptors on the same pathway leading to glycogen phosphorylase a activation.

Glycogenolytic action of glucagon-family peptides and epinephrine on catfish hepatocytes.

Glycogenolytic effects of salmon and mammalian glucagons, salmon glucagon-like peptide (GLP) and epinephrine were studied on liver cells isolated from catfish (Ictalurus melas). In spring and summer, salmo-glucagon (3×10(-10) to 3×10(-8) M) was more effective than its mammalian counterpart in the stimulation of glucose release and cAMP synthesis in hepatocytes. GLP was less potent as compared to both glucagons. γ-amylase activity was not affected by the treatment with either glucagon-family peptides or epinephrine.The comparison of the glycogenolytic effects of salmon glucagon to those of epinephrine reveals a greater potency of the latter hormone in the stimulation of cAMP synthesis, glycogen-phosphorylase activity and glucose release. Glycogen content in the liver cells was equally depleted after treatment with both of the two hormones.

Adenylate cyclase of catfish hepatocyte membranes: basal properties and sensitivity to catecholamines and glucagon.

Some characteristics of adenylate cyclase of catfish (Ictalurus melas) liver membranes were studied, and the effects of catecholamines and of glucagon were tested. The enzyme has an optimum temperature of 40 degrees C, and a Km for ATP of 0.16 mM at 30 degrees C, and requires Mg2+ for its activity. The enzyme activity is inhibited with a Ca2+ concentration higher than 5 X 10(-5) M, and enhanced with F- higher than 10(-4) M. The response of adenylate cyclase to GTP is biphasic, with a maximum of activity at 10(-5) M GTP. Catecholamines (epinephrine, norepinephrine, isoproterenol, phenylephrine) enhance cyclase activity. Propranolol inhibits the increase in enzyme activity induced by catecholamines, whereas phentolamine is ineffective. This indicates that catecholamines (phenylephrine included) activate adenylate cyclase through a beta-adrenergic mechanism. Glucagon (mammalian) has a smaller effect than epinephrine in increasing the enzyme activity of catfish hepatocyte membranes. This fact is the opposite of that observed for the cyclase activity of rat liver membranes.

Glucagon control of glycogenolysis in catfish tissues.

1. In catfish (Ictalurus melas) after glucagon treatment blood glucose increased until 150 min, then it gradually decreased towards control values at the 5th hr. 2. In glucagon treated fish, liver glycogen levels were significantly lower then in controls 30 min after hormone administration; thereafter, liver glycogen levels returned rapidly to initial values. Glucagon did not induce any significant effect on the glycogen content in white and red muscles. 3. In liver slices, the addition of glucagon to the incubation medium significantly enhanced the glycogen phosphorylase activity and decreased the level of glycogen. Both phosphorylase activity and glycogen content of white and red muscle slices were practically unaffected by glucagon.

Catecholamine effect on cyclic adenosine 3':5'-monophosphate level in isolated catfish hepatocytes.

The effect of catecholamines (epinephrine, norepinephrine, isoproterenol, and phenylephrine) on cyclic adenosine 3':5'-monophosphate (cAMP) level in isolated catfish (Ictalurus melas) liver cells was studied in the presence or absence of alpha (phentolamine) and beta (propranolol)-receptor antagonists. All catecholamines increased the hepatocyte cAMP level: the rank of their potency was epinephrine = isoproterenol greater than norepinephrine greater than phenylephrine. Propranolol completely blocked the catecholamine effect; phentolamine was ineffective. Results confirm previous findings (L. Brighenti, A. C. Puviani, M. E. Gavioli, and C. Ottolenghi, 1987, Gen. Comp. Endocrinol. 66, 306-313) that epinephrine and norepinephrine act via beta-receptor activation. However, the comparison of the effects of isoproterenol and phenylephrine on cAMP with those on phosphorylase alpha and on glycogen breakdown suggests that a more complex mechanism is possibly involved in the catecholamine effect on catfish glycogenolysis.

Mechanisms involved in catecholamine effect on glycogenolysis in catfish isolated hepatocytes.

Isolated catfish hepatocytes were treated with epinephrine, norepinephrine, isoproterenol, and phenylephrine in the presence or in the absence of propranolol or phentolamine as beta and alpha inhibitors, respectively. Glycogen phosphorylase a activity and glycogen content, as well as glucose released from cells, were tested. Phosphorylase activity was stimulated by all the catecholamines and was accompanied by a decrease of glycogen content in cells and by an increase in glucose output into the medium. Whereas phentolamine did not affect the catecholamine action on any parameter considered, propranolol inhibited the effect of epinephrine, norepinephrine, and phenylephrine, but hardly altered that of isoproterenol. The effect of epinephrine and norepinephrine, as modified by propranolol and not by phentolamine, is consistent with a beta action of these catecholamines. The fact that propranolol and not phentolamine inhibited the phenylephrine effect indicates that in catfish hepatocytes phenylephrine behaves as a beta agonist and/or that propranolol may also bind to alpha receptors. Results also indicate that in catfish liver cells isoproterenol, whose effect is scarcely influenced by propranolol, is not a pure beta agonist.

Epinephrine effect on glycogen phosphorylase activity in catfish liver and muscles.

In catfish, the percentage of the active (a) form of glycogen phosphorylase with respect to the total (a + b) form varied in control slices from about 90% to 65% and 20%, respectively for liver, red, and white muscles. Epinephrine added to the incubation medium of liver and white muscle slices caused a significant increase in the specific activity of phosphorylase a in liver and white muscle, but not in red muscle. In liver slices epinephrine showed its effect from the concentration of 3.5 X 10(-8) M. The increase of enzyme activity explains the lowering of the glycogen level in liver and white muscle induced by epinephrine.

Epinephrine effect on carbohydrate metabolism in isolated and perfused catfish liver.

Two doses of epinephrine were infused for 6 hr into isolated catfish liver previously perfused with a glucose-free medium or with a medium containing 10 mM glucose. The hormone induced (a) a continuous decrease in liver glycogen level, both in absence and in presence of glucose in the medium; low dose of epinephrine was without effect on the decay of glycogen; (b) a great release of glucose, both in absence and in presence of glucose in the medium; the low dose of epinephrine induced an effect similar to the maximal dose, only in experiments without glucose in the medium; (c) no effect on lactate uptake by liver; or (d) a prevention of the decline in liver glycogen phosphorylase activity observed during 1 hr incubation of liver slices. It has been concluded that epinephrine caused an increase of glucose in perfusion medium with different mechanisms according to the level of glucose and the dose of epinephrine. High doses of hormone cause the glycogenolysis by activation of glycogen phosphorylase, both in presence and in absence of glucose; low doses of epinephrine probably preferentially promote in liver the gluconeogenetic processes in glucose-free experiments.

Effects of insulin on glycogen metabolism in isolated and perfused catfish liver.

The isolated and perfused catfish liver showed (a) a decrease in liver glycogen, (b) a continuous increase in glucose output, and (c) a decrease of lactate in the medium. Insulin did not influence liver glycogen decay during the first 2 hr; thereafter the hormone induced an increase of glycogen, particularly when glucose was added into perfusate. In insulin treated liver, the glucose output was lower than controls in the first hours of perfusion; thereafter a re-uptake of glucose occurred. After 2-3 hr of perfusion, the lactate present in the medium was increased by insulin towards the starting level. The long lasting effects of insulin on catfish in vivo were confirmed.