Hormone responsiveness of isolated catfish hepatocytes in perifusion system is higher than in flasks incubation.

Hepatocytes were isolated from catfish (lctalurus melas) by conventional collagenase digestion. Sensitivities of liver cells isolated from the same fish to the glycogenolytic action of epinephrine, mammalian glucagon, catfish glucagon, catfish glucagon-like peptide, synthetic fragment 19-29 of anglerfish glucagon I, fragment 19-29 of anglerfish glucagon II, and anglerfish glucagon II were compared in two different systems: perifusion in a Bio-Gel P4 column and flask incubation. Both experimental procedures were continued for a total of 100-120 min, while hormones were applied simultaneously to both preparations for 10 min. Effluent fractions from the columns and incubation media from the flasks were collected for glucose determination. The hormonal effects were clearly enhanced in perifused cells compared to those in cells incubated in flasks, the effect being especially evident at physiological concentrations of hormones. The hormonal effects in both systems were dose-dependent. Epinephrine and mammalian glucagon (10 nM), applied separately to the same column, produced two different peaks, glucagon causing more glucose production than epinephrine. In the presence of 0.4 mM glucose in the perifusion system, hormonal effects were diminished, implying that glucose accumulation during incubation of liver cells in flasks might affect hormonal effects. The results obtained in this study indicate that piscine hepatocytes suspended and perifused in a Bio-Gel column are more sensitive to physiological concentrations of glycogenolytic hormones and may represent a new tool for experimental studies of fish liver metabolism and its hormonal regulation.

Beta-adrenergic receptors in catfish liver membranes: characterization and coupling to adenylate cyclase.

beta-Adrenergic binding sites in catfish liver membranes have been characterized by centrifugal assay, using a beta-adrenergic receptor antagonist, (-)-[3H]dihydroalprenolol ([3H]DHA). Binding of the radioligand was saturable and reversible. At 22 degrees equilibrium conditions were established in 15 min and the half-time for dissociation of bound [3H]DHA was approximately 4 min. Analysis of binding data was compatible with the existence of two classes of binding sites: a low-affinity site had a Kd of 62.3 nM and a Bmax of 452.0 fmol/mg protein, while the high-affinity site had a Kd of 2.04 nM and a Bmax of 46.7 fmol/mg protein. The dissociation constant of (-)-alprenolol for the beta-adrenergic receptors was about 2 nM as determined independently by direct kinetic studies and by inhibition of isoproterenol-stimulated adenylate cyclase activity. Phenylephrine was as potent as other catecholamines in inhibiting [3H]DHA binding, indicating that fish adrenoceptor subtyping is different from that of mammals.

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.

Inhibition of adenylate cyclase of catfish and rat hepatocyte membranes by 9-(tetrahydro-2-furyl)adenine (SQ 22536).

The adenosine analogue 9-(Tetrahydro-2-furyl)adenine, SQ 22536, inhibited adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity of crude membrane preparations from catfish (Ictalurus melas) and rat isolated hepatocytes in a non-competitive manner. The IC50s were reduced in the presence of NaF. SQ 22536 reduced the activity of adenylate cyclase also in the presence of increasing concentrations of GTP, as well as Mg++ and Mn++. In the presence of catecholamines (epinephrine, norepinephrine, isoproterenol, phenylephrine) SQ 22536 reduced their activating effect on adenylate cyclase in both catfish and rat membranes. SQ 22536 also inhibited the effect of glucagon (0.1 microM) on rat membrane cyclase activity.

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.

Epinephrine effects on carbohydrate metabolism in catfish, Ictalurus melas.

Epinephrine injection in catfish, Ictalurus melas, induced (a) a rapid hyperglycemia which lasted for more than 5 hr; and (b) a slight immediate decrease of glycogen in liver and muscles (red and white), followed by an increase at 48th hr in the liver. Epinephrine, added in vitro to tissue slices, increased the output of glucose from liver and enhanced the decrease of glycogen seen in controls. Among the other organs, only white muscle showed a greater decrease of glycogen than the controls. These effects are attributed to an increase of glycogen phosphorylase activity induced by the hormone.

Effect of insulin on glycogen metabolism in isolated catfish hepatocytes.

Insulin effect on carbohydrate metabolism in catfish hepatocytes consisted of a significant decrease of cell glycogen concentration both in the absence and in the presence of glucose in the medium. The hormone did not influence either the output of glucose from the cell or the intracellular glucose level. Experiments with radioactive glucose showed a very low uptake of the sugar by the hepatocytes; correspondingly the incorporation of radioactivity into glycogen was very low and not influenced by insulin. The glycogen content in catfish liver cells was influenced by the hormone in the opposite way to rat liver cells.

"In vivo" effects of insulin on carbohydrate metabolism of catfish (Ictalurus melas).

The effect of insulin was studied on blood glucose, and on the glycogen level of liver, muscles and heart in fed and in starved catfish (Ictalurus melas). Fish received intraperitoneally 60 iu/kg body weight of bovine insulin, or physiological saline and were sacrificed after 2, 4, 8, 24, 72 hr from injection. Insulin caused a decrease of blood glucose level, both in fed and in fasted animals, and the effect is more evident in fed animals. After insulin treatment, liver glycogen shows a decrease which is significant at the 8th and 24th hr in fasted and in fed animals respectively; after 72 hr the glycogen level in livers of fed and fasted animals is still very low. Insulin increases the glycogen level both in white and in dark muscle, both in fed and in fasted fish, although with different characteristics, but at the 72nd hr in all animals, the increases are significant. Hormone treatment does not change heart glycogen levels in fed catfish till the 24th hr, then there is a net decrease; in starved animals the decrease begins at the 2nd hr, but only at the 48th hr is it significant. The role of insulin was discussed in relation to the lowering of glycogen concentration in liver, in connection with the fact that many authors found different and even opposite effects of this hormone in various fish. It is possible that the glycogen depletion observed in liver after insulin injection is not due to a direct action of this hormone, but depends on the stimulated production of other specific glycogenolytic hormones, such as epinephrine and/or glucagon.

[Effect of insulin on carbohydrate metabolism in the catfish (Ictalurus melas). I) In vivo experiments]. / Effetto dell'insulina sul metabolismo glucidico del pesce gatto (Ictalurus melas). - I) Esperimenti "in vivo".

The effects of bovine insulin on blood glucose, liver and muscle glycogen were studied in catfish (Ictalurus melas). 60U/k body weight of bovine insulin injected intraperitoneally, produced a significant lowering of blood glucose and liver glycogen, more evident 48 h after injection. An increase after hormone injection was also observed into white muscle glycogen.

[Effect of insulin on carbohydrate metabolism in the catfish (Ictalurus melas). II) In vitro experiments]. / Effetto dell'insulina sul metabolismo glucidico del pesce gatto (Ictalurus melas). - II) Esperimenti "in vitro".

Isolated hepatocyte preparations were used to study the "in vitro" effects of insulin on catfish (Ictalurus melas) carbohydrate metabolism. Insulin decreases spontaneous glycogen lowering either in absence or in presence of glucose in the medium. In the same time insulin produces a little increase on the cell glucose level, but reduces the cell uptake of glucose from medium. The effect of insulin shows to be less evident on isolated hepatocytes than in the intact animals.