Presence Of Glucagon Research Articles

Specificity of cytochemical demonstration of adenylate cyclase in liver using adenylate-(beta, gamma-methylene) diphosphate as substrate.

Adenylate cyclase activity was demonstrated cytochemically in rat liver for the first time under the light microscope using cryostat sections mounted on glass cover slips and fixed with 1% glutaraldehyde for 1 min. Adenylate-(beta, gamma-methylene)diphosphate (AMP-P(CH2)P) was introduced as a new substrate for adenylate cyclase. It was found that adenylate cyclase was distributed heterogenously within the liver lobule. The enzyme activity was stronger in the area surrounding the central vein. A more specific localization at the plasma membrane and less unspecific background was obtained with AMP-P(CH2)P as compared to adenylylimidodiphosphate (AMP-P(NH)P). The specificity of the enzyme reaction using AMP-P(CH2)P was proved by increased formation of reaction product in the presence of 0.05 mg/ml glucagon and 0.125 mg/ml cholera toxin, as well as by inhibition of the reaction with 0.05 mg/ml alloxan. These effects were also observed at the electron microscopic level. On the other hand, no increase in reaction was observed in the presence of glucagon with AMP-P(NH)P as a substrate for adenylate cyclase, and only a weak activation was observed after adding cholera toxin; alloxan-inhibition was not complete. These effects may be due to the presence of enzymes which hydrolyze AMP-P(NH)P nonspecifically, superimposing on the product of adenylate cyclase activity. We therefore suggest the use of AMP-P(CH2)P as substrate for histochemical adenylate cyclase demonstration in the liver.

Effect of Escherichia coli lipopolysaccharide on the glucagon and insulin binding to isolated rat hepatocytes.

Number and affinity constant of low affinity binding sites of insulin and glucagon to isolated hepatocytes decreased when the cells were incubated with Escherichia coli 0111:B4 lipopolysaccharide. This effect agrees with a non-specific binding of lipopolysaccharide to hepatocytes, similar to the well-recognized non-specific binding of albumin. Also, binding of different lectins to their glycoprotein receptors did not affect the [14C]lipopolysaccharide interaction with the cell membrane surface. Endotoxin depresses gluconeogenesis from lactate when the precursor was incubated with the cells for short time intervals. The longer the preincubation interval with lipopolysaccharide, the higher the inhibition of gluconeogenesis in the absence and in the presence of glucagon. The effect of endotoxin was also studied on the glucagon-induced synthesis of cyclic AMP and the glucagon binding. Levels of cyclic AMP and hormone binding decreased with increasing both endotoxin concentrations and preincubation intervals at which cells were in contact with endotoxin.

Islet-activating protein blocks glucagon desensitization in intact hepatocytes.

Treatment of intact hepatocytes with islet-activating protein, from Bordatella pertussis, led to a pronounced increase in the ability of glucagon to raise intracellular cyclic AMP concentrations. Islet-activating protein, however, caused no apparent increase in the intracellular concentration of cyclic AMP under basal conditions. These effects were attributed to an enhanced ability of adenylate cyclase, in membranes from hepatocytes treated with islet-activating protein, to be stimulated by glucagon. When forskolin was used to amplify the basal adenylate cyclase activity, elevated GTP concentrations were shown to inhibit adenylate cyclase activity in membranes from control hepatocytes. This inhibitory effect of GTP was abolished if the hepatocytes had been pre-treated with islet activating protein. In isolated liver plasma membranes, islet-activating protein caused the NAD-dependent ribosylation of a Mr-40000 protein, the putative inhibitory guanine nucleotide regulatory protein, Ni. This effect was inhibited if guanosine 5'-[beta-thio]diphosphate rather than GTP was present in the ribosylation incubations. The ability of glucagon to uncouple or desensitize the activity of adenylate cyclase in intact hepatocytes was also blocked by pre-treating hepatocytes with islet-activating protein. Islet-activating protein thus heightens the response of hepatocytes to the stimulatory hormone glucagon. It achieves this by both inhibiting the expression of desensitization and also removing a residual inhibitory input expressed in the presence of glucagon.

Potentiation of alpha 1-adrenergic responses in rat liver by a cAMP-dependent mechanism.

Treatment of isolated hepatocytes with the alpha 1-adrenergic agonist norepinephrine induced a dose-dependent increase in free cytosolic Ca2+, as judged by fluorescence increases, in cells loaded with the Ca2+ indicator (2-[(2-bis[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8 -bis [carboxymethyl]aminoquinoline (quin-2). Pretreatment with either glucagon or dibutyryl cAMP increased the rate and magnitude of the quin-2 fluorescence response in hepatocytes treated with submaximal doses of norepinephrine and increased the cell sensitivity such that a physiological concentration of norepinephrine (7.5 nM) was able to provoke a quin-2 fluorescence response. Similar enhancement of norepinephrine-induced phosphorylase activation and pyridine nucleotide reduction in isolated hepatocytes and Ca2+ efflux from the perfused liver was also observed in the presence of glucagon. These potentiated responses correlated with a cAMP-dependent increase (mediated by glucagon, dibutyryl cAMP, or forskolin) in the binding of [3H]norepinephrine or [3H]epinephrine to sites present on isolated hepatocytes bearing the characteristics of alpha 1-adrenergic receptors. The data suggest that a cAMP-dependent mechanism is involved in the regulation of alpha 1-agonist binding to liver cells and, thereby, in the control of hepatic carbohydrate metabolism in response to catecholamines.

Inhibition by glucagon of the calcium pump in liver plasma membranes.

The ATP-dependent calcium transport in plasma membrane vesicles prepared from rat liver was inhibited by 0.1 to 10 microM glucagon. Inhibition of the high affinity (Ca2+-Mg2+)-ATPase was observed concomitantly. This effect was neither mimicked by cyclic AMP nor by dibutyryl cyclic AMP. A study of the structure-activity relationships of six glucagon derivatives demonstrated the specificity of glucagon action since only one or two analogs markedly altered the (Ca2+-Mg2+)-ATPase activity. The study also demonstrated the total absence of correlation between adenylate cyclase activation and (Ca2+-Mg2+)-ATPase inhibition induced by these glucagon derivatives. The decrease in the maximal velocities induced by glucagon of both calcium transport and (Ca2+-Mg2+)-ATPase activity were related to a reduction in the rate of dephosphorylation of the Ca-dependent phosphorylated intermediate of the enzyme. This phosphorylated intermediate was characterized as a 32P-labeled 110,000-dalton protein which accumulated to 50 to 150% over the basal level in the presence of glucagon. The present results demonstrate a novel aspect of the role of glucagon as a calcium-mobilizing agent.

Stimulation of malic enzyme formation in hepatocyte culture by metabolites: Evidence favoring a nonglycolytic metabolite as the proximate induction signal

Recent studies have shown that the addition of increasing concentrations of glucose to the medium of primary adult rat hepatocyte cultures results in the progressive induction of malic enzyme. We have undertaken experiments to determine (1) whether metabolism of glucose was an essential prerequisite for such induction, and (2) whether a specific glycolytic intermediate could be shown to constitute the proximate carbohydrate signal triggering such induction. In line with these objectives we investigated the ability of various sugars and glycolytic metabolites to induce malic enzyme in this system and assessed the influence of insulin, glucagon, and thyroid hormone (triiodothyronine, T 3) on this process. Our results show that only those sugars capable of entering the cell and being metabolized induce malic enzyme (glucose, fructose, and galactose). The nonmetabolizable sugars 3-O-methylglucose and 2-deoxyglucose are ineffective. Incubation with 20 mmol/L lactate, pyruvate, dihydroxyacetone, or glycerol resulted in malic enzyme induction, whereas incubation with acetate, citrate, and α-ketoisocaproate was without effect. The induction by all sugars and metabolites required presence of insulin. As previously reported for glucose, addition of T 3, under all metabolic conditions, resulted in a constant 3.6-fold increase in the rate of malic enzyme induction and further supports the proposal that T 3 acts to multiply the effect of a common carbohydrate-generated signal. Glucagon administration led to a dose-dependent inhibition of the carbohydrate effect with a half-maximal effect and maximal effect at 2 and 100 nmol/L, respectively. None of the glycolytic metabolites tested could reverse the glucagon inhibition completely. Our results indicate that metabolism of glucose is essential for induction of malic enzyme and are most consistent with the view that the signal arises from the metabolism of pyruvate at a step before the formation of cytsolic acetyl-CoA. Since these processes are mitochondrial in location, it appears likely that this organelle is the site of origin of the inducing signal. Of some interest in this connection was the finding that dichloroacetate led to a major induction of malic enzyme, even in the absence of insulin and in the presence of glucagon. The ability of dichloroacetate to bypass the glucagon block thus further supports a nonglycolytic origin of the inducing signal.

The Action of Various Hormones and Metabolites upon Glucose Production by Isolated Hepatocytes from Lactating and Non-Lactating Ewes

Glucose production was measured from hepatocytes isolated from the livers of non-mated, 20- and 50-day lactating ewes (4 animals/group) in the presence and absence of various substrates and hormones. The output of glucose was reduced at peak lactation (20-day) compared to both the unmated (P less than 0.05) and 50-day lactating groups (P less than 0.05). There was no significant difference in glucose production between the unmated and 50-day lactating groups. In the presence of glucagon (1 uM), glucose production was stimulated above control levels (P less than 0.05). However, insulin (1 uM) did not reduce glucose production compared to controls, while the effect of insulin and glucagon combined (1:1 uM) was not significantly higher than control values. Propionate, acetate and butyrate (10 mM) significantly increased glucose output above control values (P less than 0.05), in all three groups of animals. Glucose output was relatively unchanged in the presence of alanine and glutamate (10 mM), while fructose supplementation (10 mM) resulted in a significantly reduced glucose output compared to the controls (P less than 0.05).

The effect of glucagon-induced adenosine 3',5'-monophosphate concentrations on bile acid synthesis in isolated rat liver cells

The effect of increased intracellular adenosine 3',5'-monophosphate (cAMP) concentrations on bile acid synthesis in isolated rat hepatocytes was investigated. When the cells were incubated in the presence of glucagon (0.2 μM) and theophylline (1 mM) the observed rise in the level of cAMP was accompanied by an increase in bile acid production. Hepatocyte cAMP concentrations after 1 h of incubation showed a highly significant positive linear correlation with the amounts of bile acid synthesised by the cells during this time. These results suggest that bile acid production is related to the concentration of cAMP in isolated hepatocytes and provide evidence for a role for the cyclic nucleotide in the regulation of bile acid synthesis.

The role of a guanine nucleotide-binding protein in the activation of rat liver plasma-membrane adenylate cyclase by forskolin.

The effects of the photoreactive GTP analogue GTP-gamma-azidoanilide on rat liver plasma-membrane adenylate cyclase are described. U.v. irradiation in the presence of the analogue abolished activation by any effector or combination of effectors that function via the activatory G protein. Partial protection against this inhibition was given by F- and guanosine 5'-[gamma-thio]triphosphate. It is concluded that GTP-gamma-azidoanilide acts by a light-induced covalent reaction with the G protein. In the dark the effects of the analogue were similar to those of GTP. Irradiation in the presence of GTP-gamma-azidoanilide was found to reduce but not to abolish activation of rat liver plasma membrane adenylate cyclase by forskolin. The activation by forskolin and GTP together were greater than the sum of the individual activations. Forskolin doubled adenylate cyclase activity in the presence of glucagon and guanosine 5'-[beta, gamma-imido]triphosphate, which might be expected to activate to the maximum possible extent via the G protein. It is concluded that there are two components to the forskolin activation, a guanine nucleotide-dependent and a guanine nucleotide-independent component.

Actions of glucagon on flux rates in perfused rat liver. 1. Kinetics of the inhibitory effect on glycolysis and the stimulatory effect on glycogenolysis.

Changes in the rates of glucose, lactate and pyruvate production following infusion of glucagon were studied in isolated livers from fed or fasted rats perfused with non-recirculating Krebs-Henseleit bicarbonate buffer. Evidence was presented that under these experimental conditions, the release of lactate plus pyruvate into the perfusate represents 80-90% of the total glycolytic flux; oxidation of pyruvate derived from glucose and/or glycogen accounts for the remaining 10-20%, whereas recycling of pyruvate to glucose is negligible. In the presence of glucagon, rates of lactate plus pyruvate production were diminished to less than 30% in the fed state (glycogen as substrate) and to less than 10% in the fasted state (glucose as substrate). Rates of pyruvate oxidation were unchanged. Although recycling of pyruvate to glucose was enhanced, it could account for not more than 20% of the decrease in lactate plus pyruvate production. The data indicate a strong inhibition of the substrate flux through glycolysis. The glucagon concentration for half-maximal inhibition of glycolysis was 0.2 nM, independent of the substrate (glucose or glycogen) and the nutritional state. The effective concentrations were within the physiological range of glucagon concentrations reported for portal venous blood. Transient state analyses indicated that the inhibition of glycolysis precedes the stimulation of glycogenolysis. When after a delay glycogenolysis was accelerated, it was followed by a transient stimulation of glycolysis. The stimulatory component in the glucagon effect on glycolysis was diminished in glycogen-depleted livers and when glucose was the main substrate. The coordinated control of glycolysis and glycogenolysis by glucagon and the interaction of the two processes in the transient state are discussed.

Conformational and biological properties of glucagon fragments containing residues 1-17 and 19-29.

The 29 amino acid polypeptide hormone glucagon was cleaved into two large fragments by the enzyme clostripain. The conformational properties of these two fragments were monitored by circular dichroism at pH 2 and 12 in both the presence and absence of sodium dodecyl sulfate. Both glucagon (1-17) and glucagon (19-29) have reduced abilities to fold in aqueous solution. However, both fragments can take on structure of higher apparent helical content in acidic solution in the presence of sodium dodecyl sulfate but only the glucagon (19-29) retains this conformation at high pH. Neither of the two fragments react with dimyristoylphosphatidylcholine as the intact peptide does. Only the carboxyl terminal fragment was capable of reacting with an antibody specific for glucagon. The glucagon (1-17) has markedly reduced affinity for binding to the glucagon receptor as well as markedly reduced ability to stimulate adenylate cyclase activity which is not affected by the presence of glucagon (19-29). It is proposed that the intact sequence provides specific groups required for activity as well as the potential for forming a stable amphipathic helix, both of which are necessary for full biological activity at low peptide concentrations.

The presence of glucagon in ascitic fluid in cirrhotic patients.

Glucose, insulin, glucagon, and liver profiles were determined simultaneously in plasma and ascitic fluid after an overnight fast in ten cirrhotic patients to assess the role of ascites in hyperglucagonemia and hyperinsulinemia of hepatic cirrhosis and to investigate if glucagon and insulin diffuse freely across the peritoneum. Plasma in ten normal subjects was studied as well. Glucose levels in plasma were normal in all patients; in ascitic fluid they were higher than in plasma in 7 cases and lower in 3 cases. Plasma glucagon was raised in seven patients with advanced liver dysfunction as documented by the presence of jaundice and reversed albumin/ globulin ratio. Plasma insulin was elevated in patients with hepatic dysfunction and/or esophageal varices. In the absence of jaundice and varices, glucagon and insulin concentrations were normal. Ascitic fluid glucagon was always lower than the plasma with a significant correlation. Ascitic fluid insulin was undetectable in three patients. In the remaining patients, the realtionship between plasma and ascitic fluid insulin levels was not significant. No relationship were observed between liver dysfunction and hormonal concentrations in ascitic fluid. These studies suggest that, (1) both glucagon.and insulin may diffuse freely across the peritoneum into the peritoneal cavity, with a consistent glucagon gradient between plasma and ascitic fluid. The apparent lack of such a relationship for insulin is unexplained and may need further evaluation, and (2) in hepatic cirrhosis the presence of ascites may not influence plasma glucagon or insulin concentrations unless accompanied by jaundice and a reversal of albumin/globulin ratio, both indices of advanced liver dysfunction and the presence of esophageal varices, a sign of moderately severe portosystemic shunting.

GDP does not mediate but rather inhibits hormonal signal to adenylate cyclase.

This study was aimed to elucidate whether GDP can mediate hormonal signal to adenylate cyclase in hepatic glucagon sensitive adenylate cyclase with ATP as substrate. Conversion of added GDP to GTP catalyzed by nucleoside diphosphate kinase was suppressed to less than 0.3% of added GDP by including UDP. Inhibition of this enzyme activity by UDP was accompanied by a preferential loss of the stimulatory effect of glucagon plus GDP on cyclase activity without changes in effects of glucagon plus GTP, glucagon plus guanosine 5'-(beta, gamma-imino)triphosphate, and NaF. Under this condition, i.e. in the presence of UDP, GDP competitively inhibited the actions of GTP (Ki for GDP, 1 microM) and guanosine 5'-(beta, gamma-imino)triphosphate in the presence of glucagon, the inhibition being complete at high GDP concentrations. GDP also inhibited cyclase activity stimulated by NaF with UDP but did only slightly without UDP. It was demonstrated that nucleoside diphosphate kinase is located in membranes in addition to cytosol fraction. However, the activity of membrane-associated enzyme was not affected by the addition of glucagon. Based on these observations, it is concluded that GDP is unable to mediate hormonal signal to adenylate cyclase and that it acts as an inhibitor of cyclase activity stimulated by GTP or its analog along with hormone. The results suggest a possible role of membrane-associated nucleoside diphosphate kinase in determining GTP and GDP levels at or near their binding site so as to replenish GTP and, thereby, decrease the inhibitory action of GDP when hormone is present.

Effect of glucagon and cyclic adenosine 3',5'-monophosphate on protein phosphorylation in rat pancreatic islets.

Isolated rat pancreatic islets, incubated in the presence of extracellular 32Pi to a state of steady 32P incorporation into cellular phosphopeptides, were exposed to glucagon, (Bu)2cAMP, or somatostatin for 10 min. In other experiments, homogenates of rat islets were phosphorylated using [gamma-32P]ATP with or without cAMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and phosphorylation of proteins was measured by liquid scintillation counting of gel slices. Glucagon (2.9 X 10(-7) M) stimulated the phosphorylation of 15 polypeptides (by approximately 20-50%) with major phosphorylation of proteins with mol wts of 138,000, 93,000, 53,000, 49,000, 35,000, 27,000 and 15,000 in intact rat islets and also stimulated insulin release by 202%. Somatostatin (6.6 X 10(-7) M) inhibited all the glucagon-stimulated phosphorylation by approximately 15-30% and also inhibited the glucagon-stimulated insulin release by 46%. (Bu)2cAMP (10(-3) M) stimulated 32P incorporation (by approximately 20-50%) into the same 15 peptides as did glucagon and also stimulated insulin release by 169%. When homogenates of rat islets were used. cAMP (10(-6) M) stimulated the phosphorylation of proteins (by approximately 25-60%) to an extent similar to that seen in the presence of glucagon or (Bu)2cAMP in intact islets. These findings indicate that the glucagon-stimulated phosphorylation of rat islet proteins may be mediated by cAMP-dependent protein kinase and that protein phosphorylation may be important in mediating the glucagon-stimulated insulin release.

Pattern of protein phosphorylation in rat hepatocytes stimulated by glucagon or by the Ca2+-linked hormones.

We have adapted the high-resolution electrophoretic technique of O'Farrell to analyze phosphorylated proteins from rat hepatocytes. Total proteins were extracted from rat hepatocytes which had been incubated in the presence of [32P]phosphate and with two types of stimuli: glucagon on the one hand and the Ca2+-linked hormones on the other hand. About 200 phosphorylated polypeptides have been separated. Glucagon modifies the incorporation of [32P]phosphate in at least 17 polypeptides and dibutyryladenosine 3',5'-monophosphate mimics this hormonal effect, implying a common mechanism of action. Phenylephrine (in the presence of the beta-antagonist propranolol), vasopressin and angiotensin all modify the incorporation of [32P]phosphate in about 13 polypeptides; since the Ca2+ ionophore A23 187 reproduces the effect of these agents it may be concluded that Ca2+ mediates their effect. Not all the substrates affected by the two types of hormones are identical. Both types of stimuli increase the phosphorylation of a same set of seven proteins and decrease the phosphorylation of a same set of three proteins but seven proteins have their phosphorylation uniquely enhanced by glucagon whereas three other specific proteins get more phosphorylated by the Ca2+ -linked hormones. The clear differences between the patterns of protein phosphorylation observed in the presence of glucagon and dibutyryladenosine 3',5'-monophosphate on the one hand and by the Ca2+-linked hormones on the other hand strongly suggest different mechanisms of action for these two types of stimuli.

The time course of glucagon action on the utilization of [U- 14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U- 14C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO 2. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and 14CO 2, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U- 14C]palmitate.

Protective effects of glucagon during the anaphylactic response in guinea-pig isolated heart.

1 Cardiac anaphylaxis and the effects of glucagon pretreatment were studied in guinea-pig isolated hearts actively sensitized to ovalbumin.2 Antigen challenge of the sensitized hearts markedly increased creatine phosphokinase (CPK) activity in the coronary venous effluent. Control values of CPK release from the hearts before challenge were 3.56 +/- 0.15 mu min(-1) mg(-1). In the first 10 min following challenge, CPK release remained stable at increased levels which ranged between 4.88 +/- 0.20 to 5.39 +/- 0.38 mu min(-1) mg(-1). There was no correlation between immunologically released histamine and CPK release.3 Pretreatment of the hearts with glucagon (0.15 mumol l(-1)) exerted a pronounced anti-arrhythmic activity, reducing the conduction arrhythmias and completely preventing automaticity arrhythmias which normally occurred following ovalbumin challenge.4 Anaphylactic histamine release was reduced significantly in the presence of glucagon. The percentage inhibition of histamine release from glucagon pretreated hearts, during the first 10 min after challenge, ranged between 58% and 94% of that from hearts similarly challenged in the absence of glucagon.5 Glucagon significantly elevated sinoatrial nodal automaticity, enhanced atrioventricular conduction, improved coronary flow and reduced contractile force during anaphylaxis. It appears that these effects are caused both by modulating anaphylactic histamine release and by influencing the effects of the released histamine.6 CPK release from the anaphylactic hearts was significantly inhibited in the presence of glucagon. The average percentage inhibition of CPK activity during the first 10 min after challenge ranged between 42% and 98%.7 The findings from this study provide experimental evidence for protective effects of glucagon pretreatment during cardiac anaphylaxis.

Glucagon inhibits triacylglycerol synthesis in isolated hepatocytes by lowering their glycerol 3-phosphate content

Triacylglycerol synthesis by glycogen-depleted hepatocytes from fed rats that have low glycerol 3-phosphate contents was stimulated by the addition of glycerol 3-phosphate precursors. Glucagon decreased triacylglycerol synthesis only when it also lowered glycerol 3-phosphate content. The hyperbolic-like relationship between glycerol 3-phosphate content and rates of triacylglycerol synthesis was identical in the absence or presence of glucagon, indicating that the glucagon effect on triacylglycerol synthesis was not mediated through changes in enzyme activities of the esterification pathway but through changes in cellular glycerol 3-phosphate content.

Metabolite-controlled phosphorylation of phosphofructokinase in rat hepatocytes.

The effect of glucose on the phosphorylation of phosphofructokinase has been investigated in hepatocytes isolated froin fed and 24‐h‐starved rats by measuring the 32P radioactivity associated with the enzyme. In addition, the effects of gluconeogenic precursors and of glucagon or Bt2cAMP were followed under these conditions.The basal incorporation of phosphate into phosphofructokinase in hepatocytes from starved and fed rats was 0.2 ± 0.1 and 0.6±0.2 mol phosphate/mol phosphofructokinase (tetramer) respectively, whereas the maximal incorporation obtained was 1.03 ± 0.2 mol phosphate/mol phosphofructokinase.With 16 mM l‐lactate + 4 mM pyruvate or with 20 mM l‐alanine, the phosphorylation of the enzyme was lower than the basal incorporation found in hepatocytes from fed and starved rats.Glucose (20 mM) addition to isolated hepatocytes from fed rats resulted in an increase in the incorporation phosphate into phosphofructokinase to almost maximal value.In hepatocytes isolated from starved rats, glucose led to a 2.5‐fold increase of phosphorylation as compared to the basal incorporation. l‐Alanine (20 mM) abolished the glucose‐induced phosphorylation in hepatocytes from starved and from fed animals.Glucagon (0.1 μM) did not significantly enhance thc phosphorylation of phospliofructokinase in the presence of l‐lactate + pyruvate in hepatocytes from starved rats, but stimulated the phosphorylation to maximal value when cells had been preincubated in the presence of glucose.In contrast, in liver cells isolated from fed rats. glucagon had no additional effect on phosphofructokinase pliosphorylation in the presence of glucose, but did increase the phosphorylation to maximal value when l‐lactate + pyruvate served as substrates. l‐Alanine inhibited the glucose‐induced phosphorylation of phosphofructokinase also in the presence of glucagon.In contrast, glucagon stimulated the phosphorylation of l‐type pyruvate kinase independently from exogenous precursors and from the nutritional state of the animals.Bt2cAMP (0.1 mM) as glucagon, did not significantly enhance the phosphorylation of phosphofructokinase above the level obtained with glucose alone in hepatocytes from fed rats, but did increase the velocity of the phosphate incorporation in hepatocytes from starved rats, thus leading under both conditions to the maximal incorporation.It is concluded that the degree of phosphorylation of rat liver phosphofructokinase is affected by the metabolic state, and that glucagon (or CAMP) acts indirectly, probably by changing glucose metabolism due to increased glycogenolysis rather than by an activation of a CAMP‐dependent protein kinase.

Hormonal effects on the liver glucose metabolism in rainbow trout ( Salmo gairdneri)

1. 1. Glucagon, adrenaline and dibutyril cyclic AMP increased the release of glucose to the medium during incubation of liver slices from rainbow trout ( Salmo gairdneri) while insulin had no effect. 2. 2. Glycogen content decreased only slightly after cyclic AMP addition and even increased in the presence of glucagon and adrenaline. Consequently, the release of glucose was due mainly to gluconeogenesis. 3. 3. This is corroborated by the reduction of glucose liberation in presence of α-cyanocinnamate, an inhibitor of gluconeogenesis.