Celestina Ottolenghi

Metabolic effects of salmon glucagon and glucagon-like peptide in coho and chinook salmon ☆

Different doses of glucagon and glucagon-like peptide (GLP) isolated from coho salmon, Oncorhynchus kisutch were tested in vivo and in vitro on juvenile coho and chinook (O. tshawytscha) salmon. Results obtained suggest an involvement of these peptides in the regulation of plasma glucose, plasma fatty acids, liver glycogen, and the hepatic enzymes: glycogen phosphorylase, pyruvate kinase, triacylglycerol lipase, and glucose-6-phosphate dehydrogenase. Metabolic effects were more enhanced in summer than either in spring or in autumn. GLP was less effective than glucagon in stimulating glycogenolysis in vivo. Salmon glucagon, especially in low concentrations, was generally more potent metabolically than mammalian (porcine/bovine) glucagon. The interaction between glucagon-family peptides and insulin seems to be different from the one described in mammals: glucagon and GLP either lowered plasma circulating levels of insulin or showed no effect. Only at the time of parr-smolt transformation did GLP slightly elevate plasma insulin levels in coho salmon.

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.

An update on high-yield hepatocyte isolation methods and on the potential clinical use of isolated liver cells

Isolated hepatocytes are a suitable system for the study of hepatic physiology and metabolism. They are also used for pharmacological and toxicological studies related to hepatic uptake, metabolism, excretion and toxicity of xenobiotics, as well as morphological and metabolic effects induced in the liver as a result of drug or toxic substance exposure. In this paper, the enzymatic methods for hepatocyte isolation in some mammalian species are reviewed, and methods for evaluating cell purification and assessment of cellular morphology and function are also examined. More recently, interest in hepatocyte transplantation has increased, and the clinical experimentation of hepatocyte-based liver support systems has attracted the attention of scientists and hepatologists. From a clinical perspective, using isolated hepatocytes could be useful both for supporting an acutely devastated liver, a chronically diseased liver, and for correcting genetic disorders resulting in metabolically deficient states. Reports of clinical usage of isolated allogenic hepatocytes in hepatocellular transplantation and of xenogenic liver cells in constructing bio-artificial liver support systems are promising, and are renewing interest in the development of methods for isolation and purification of hepatocytes.

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.

β-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.

“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.

Glycogen in liver and other organs of catfish (ictalurus melas): Seasonal changes and fasting effects

Abstract 1. 1. Catfish ( Ictalurus melas ) contain large amounts of glycogen in liver and other organs; these amounts are greater than those present in the corresponding tissues of mammals. 2. 2. All organs show seasonal variations in their glycogen content: in general, the values are lower in spring than in other seasons. 3. 3. Fasting induces variations in the glycogen content of organs according to the season and, for the liver, also according to sex.

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.

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.

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.