Role of AMP on the activation of glycogen synthase and phosphorylase by adenosine, fructose, and glutamine in rat hepatocytes

The mechanism for glycogen synthesis stimulation produced by adenosine, fructose, and glutamine has been investigated. We have analyzed the relationship between adenine nucleotides and glycogen metabolism rate-limiting enzymes upon hepatocyte incubation with these three compounds. In isolated hepatocytes, inhibition of AMP deaminase with erythro-9-(2-hydroxyl-3nonyl)adenine further increases the accumulation of AMP and the activation of glycogen synthase and phosphorylase by fructose. This ketose does not increase cyclic AMP or the activity of cyclic AMP-dependent protein kinase. Adenosine raises AMP and ATP concentration. This nucleotide also activates glycogen synthase and phosphorylase by covalent modification. The correlation coefficient between AMP and glycogen synthase activity is 0.974. Nitrobenzylthioinosine, a transport inhibitor of adenosine, blocks (by 50%) the effect of the nucleoside on AMP formation and glycogen synthase but not on phosphorylase. 2-Chloroadenosine and N6-phenylisopropyladenosine, nonmetabolizable analogues of adenosine, activate phosphorylase (6-fold) without increasing the concentration of adenine nucleotides or the activity of glycogen synthase. Cyclic AMP is not increased by adenosine in hepatocytes from starved rats but is in cells from fed animals. [Ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA) blocks by 60% the activation of phosphorylase by adenosine but not that of glycogen synthase. Glutamine also increases AMP concentration and glycogen synthase and phosphorylase activities, and these effects are blocked by 6-mercaptopurine, a purine synthesis inhibitor. Neither adenosine nor glutamine increases glucose 6-phosphate. It is proposed that the observed efficient glycogen synthesis from fructose, adenosine, and glutamine is due to the generation of AMP that activates glycogen synthase probably through increases in synthase phosphatase activity. It is also concluded that the activation of phosphorylase by the above-mentioned compounds can be triggered by metabolic changes.     

Synthesis of glycogen from fructose in the presence of elevated levels of glycogen phosphorylase a in rat hepatocytes

Incubation of hepatocytes with glucose promoted the increase in the glycogen synthase (-glucose 6-phosphate/+glucose 6-phosphate) activity ratio, the decrease in the levels of phosphorylase a and a marked increase in the intracellular glycogen level. Incubation with fructose alone promoted the simultaneous activation of glycogen synthase and increase in the levels of phosphorylase a. Strikingly, glycogen deposition occurred in spite of the elevated levels of phosphorylase a. When glucose and fructose were added to the media the activation of glycogen synthase was always higher than when the hexoses were added separately. On the other hand the effects on glycogen phosphorylase were a function of the relative concentrations of both sugars. Inactivation of glycogen phosphorylase occurred when the fructose to glucose ratio was low while activation took place when the ratio was high. The simultaneous presence of glucose and fructose resulted, in all cases, in an enhancement in the deposition of glycogen. The effects described were not limited to fructose as D-glyceraldehyde, dihydroxyacetone, L-sorbose, D-tagatose and sorbitol, compounds metabolically related to fructose, provoked the same behaviour.     

Non-hormonal and hormonal control of glycogen metabolism in isolated sheep liver cells

1. Control of glycogen metabolism by various substrates and hormones was studied in ruminant liver using isolated hepatocytes from fed sheep. 2. In these cells glucose appeared uneffective to stimulate glycogen synthesis whereas fructose and propionate activated glycogen synthase owing to (i) a decrease in phosphorylase a activity and (ii) changes in the intracellular concentrations of glucose 6-phosphate and adenine nucleotides. 3. The activation of hepatic glycogenolysis by glucagon and alpha 1-adrenergic agents was associated with increased phosphorylase a and decreased glycogen synthase activities. 4. The simultaneous changes in these two enzyme activities suggest that in sheep liver, activation of phosphorylase a is not a prerequisite step for synthase inactivation. 5. In sheep hepatocytes, in the presence of propionate and after a lag period, insulin activated glycogen synthase without affecting phosphorylase a. 6. This latter result suggests that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase. Insulin also antagonized glucagon effect on glycogen synthesis by counteracting the rise of cAMP.     

Glycogen synthesis from glucose and fructose in hepatocytes from diabetic rats

In rat hepatocytes, the basal glycogen synthase activation state is decreased in the fed and diabetic states, whereas glycogen phosphorylase a activity decreases only in diabetes. Diabetes practically abolishes the time- and dose-dependent activation of glycogen synthase to glucose especially in the fed state. Fructose, however, is still able to activate this enzyme. Glycogen phosphorylase response to both sugars is operative in all cases. Cell incubation with the combination of 20 mM glucose plus 3 mM fructose produces a great activation of glycogen synthase and a potentiated glycogen deposition in both normal and diabetic conditions. Using radiolabeled sugars, we demonstrate that this enhanced glycogen synthesis is achieved from both glucose and fructose even in the diabetic state. Therefore, the presence of fructose plays a permissive role in glycogen synthesis from glucose in diabetic animals. Glucose and fructose increase the intracellular concentration of glucose 6-phosphate and fructose reduces the concentration of ATP. There is a close correlation between the ratio of the intracellular concentrations of glucose 6-phosphate and ATP (G6-P/ATP) and the activation state of glycogen synthase in hepatocytes from both normal and diabetic animals. However, for any given value of the G6-P/ATP ratio, the activation state of glycogen synthase in diabetic animals is always lower than that of normal animals. This suggests that the system that activates glycogen synthase (synthase phosphatase activity) is impaired in the diabetic state. The permissive effect of fructose is probably exerted through its capacity to increase the G6-P/ATP ratio which may partially increase synthase phosphatase activity, rendering glycogen synthase active.     

Effect of fructose 1-phosphate on the activation of liver glycogen synthase.

The activation (dephosphorylation) of glycogen synthase and the inactivation (dephosphorylation) of phosphorylase in rat liver extracts on the administration of fructose were examined. The lag in the conversion of synthase b into a was cancelled, owing to the accumulation of fructose 1-phosphate. A decrease in the rate of dephosphorylation of phosphorylase a was also observed. The latency re-appeared in gel-filtered liver extracts. Similar latency was demonstrated in extracts from glucagon-treated rats. Addition of fructose 1-phosphate to the extract was able to abolish the latency, and the activation of glycogen synthase and the inactivation of phosphorylase occurred simultaneously. Fructose 1-phosphate increased the activity of glycogen synthase b measured in the presence of 0.2-0.4 mM-glucose 6-phosphate. According to kinetic investigations, fructose 1-phosphate increased the affinity of synthase b for its substrate, UDP-glucose. The accumulation of fructose 1-phosphate resulted in glycogen synthesis in the liver by inducing the enzymic activity of glycogen synthase b in the presence of glucose 6-phosphate in vivo and by promoting the activation of glycogen synthase.     

Pathways of glycogen synthesis in Novikoff ascites-hepatoma cells

Affinity of glucose, fructose and mannose for tumour hexokinase and their rates of phosphorylation at saturation concentration have been correlated with rates of glycogen synthesis by intact tumour cells at different concentrations of the three substrates. Competition experiments with one sugar labelled and the other sugar unlabelled indicate inhibition of glycogen synthesis by the sugar with a low K(m) for hexokinase. Glycogen synthesis from glucose 1-phosphate in aged cells and from nucleoside in freshly prepared cells is stimulated by fructose and inhibited by glucose. The decrease in glycogen formation from glucose 1-phosphate by oligomycin is partially overcome by increased fructose concentrations. These results are explained by an activation of alpha-glucan phosphorylase by fructose and an inhibition of this enzyme by glucose. It is suggested that differences in localization of glucose 6-phosphate, available to the intact cell in various ways, determine its transformation into glycogen by either the UDP-glucose-alpha-glucan glucosyltransferase reaction or by the alpha-glucan phosphorylase reaction.     

Activation of hepatocyte glycogen synthase by metabolic inhibitors

Incubation of isolated rat hepatocytes with metabolic inhibitors causes an increase in the -glucose 6-P/+glucose 6-P activity ratio of glycogen synthase after decreasing ATP and increasing AMP levels. Concomitantly, the activity of phosphorylase is increased six-fold by the same treatment. This activation of both enzymes remains after gel filtration of the hepatocyte extracts. Addition of metabolic inhibitors to cells pretreated with an inhibitor of AMP-deaminase results in an accumulation of AMP and, simultaneously, in a further increase in the activation state of glycogen synthase. The correlation coefficient between the intracellular concentration of AMP and glycogen synthase activity is r = 0.93. It is proposed that the covalent activation of glycogen synthase by metabolic inhibitors can be triggered by changes in the level of the intracellular concentrations of adenine nucleotides.     

Antagonism of the morphine-induced locomotor activation of mice by fructose: comparison with other opiates and sugars, and sugar effects on brain morphine

The mouse locomotor activation test of opiate action in a 2+2 dose parallel line assay was used in a repeated testing paradigm to determine the test, opiate and hexose specificities of a previously reported antagonism of morphine-induced antinocociception by hyperglycemia. In opiate specificity studies, fructose (5 g/kg, i.p.) significantly reduced the potency ratio for morphine and methadone, but not for levorphanol, meperidine or phenazocine when intragroup comparisons were made. In intergroup comparisons, fructose significantly reduced the potencies of levorphanol and phenazocine, but not methadone or meperidine. In hexose/polyol specificity studies, tagatose and fructose significantly reduced the potency ratio for morphine, whereas glucose, galactose, mannose and the polyols, sorbitol and xylitol, caused no significant decrease in potency. Fructose, tagatose, glucose and mannose (5 g/kg, i.p.) were tested for effects on brain morphine levels 30 min after morphine (60 min after sugar), and all four sugars significantly increased brain morphine relative to saline-pretreated controls. It is concluded that the antagonism of morphine by acute sugar administration shows specificity for certain sugars and occurs despite sugar-induced increases in the distribution of morphine to the brain. Furthermore, the effects of fructose show an opiate specificity similar to that of glucose on antinociception observed previously in our laboratory, except that methadone was also significantly inhibited in the present study, when a repeated-testing experimental design was used.     

Synergism of glucose and fructose in net glycogen synthesis in perfused rat livers

Synergism of glucose and fructose in net glycogen synthesis was studied in perfused livers from 24-h fasted rats. With either glucose or fructose alone, net glycogen deposition did not occur (p greater than 0.10 for each), whereas the addition of both together resulted in significant glycogen accumulation (net glycogen accumulation was 0.21 +/- 0.03 mumol of glucose/g of liver/min at 2 mM fructose and 30 mM glucose, p less than 0.001). To better understand this synergism, intermediary substrate levels were compared at steady state with various glucose levels in the absence and in the presence of 2 mM fructose. Independent of fructose, hepatic glucose and glucose 6-phosphate increased proportionally when glucose level in the medium was raised (r = 0.86, p less than 0.001). Unlike glucose 6-phosphate, UDP-glucose did not consistently increase with glucose (p greater than 0.10); in fact, there was a small decrease at a very high glucose level (30 mM), a result consistent with the well-established activation of glycogen synthase by glucose. With elevated glucose, the level of glucose 6-phosphate was strongly correlated with glycogen content (r = 0.71, p less than 0.01, slope = 32). Adding fructose increased the "efficiency" of glucose 6-phosphate to glycogen conversion: the effect of a given increment in glucose 6-phosphate upon glycogen accumulation was increased 2.6-fold (r = 0.73, p less than 0.01, slope = 86). A kinetic modeling approach was used to investigate the mechanisms by which fructose synergized glycogen accumulation when glucose was elevated. Based on steady-state hepatic substrate levels, net hepatic glucose output, and net glycogen synthesis rate, the model estimated the rate constants of major enzymes and individual fluxes in the glycogen metabolic pathway. Modeling analysis is consistent with the following scenario: glycogen synthase is activated by glucose, whereas glucose-6-phosphatase was inhibited. In addition, the model supports the hypothesis that fructose synergizes net glycogen accumulation due to suppression of phosphorylase. Overall, our analysis suggests that glucose enhances the metabolic flux to glycogen by inducing a build up of glucose 6-phosphate via combined effects of mass action and glucose-6-phosphatase inhibition and activating glycogen synthase and that fructose enhances glycogen accumulation by retaining glycogen via phosphorylase inhibition.     

Effect of metabolites and phosphorylase on the D to I conversion of glycogen synthase from human polymorphonuclear leukocytes.

The D to I conversion of glycogen synthase from human polymorphonuclear leukocytes was examined both in a gel-filtered homogenate and in a preparation of glycogen particles with adhering enzymes, purified by chromatography on concanavalin A bound to Sepharose. It was found that glucose 6-phosphate as well as mannose 6-phosphate, glucosamine 6-phosphate, and 2-deoxy-glucose 6-phosphate activated the reaction, whereas the corresponding sugars were without effect. Mn2+ and Ca2+ increased the conversion rate by 51% and 27%, respectively, whereas Mg2+ and inorganic phosphate were without effect. Sodium fluoride inhibited the reaction completely. Glycogen inhibited the reaction in physiological concentrations and 0.5 mM glucose 6-phosphate was able to overcome this inhibition. MgATP greatly augmented the inhibition caused by glycogen in the glycogen particle preparation. This combined effect could be overcome by glucose 6-phosphate in concentrations from 0.1 to 1 mM. Phosphorylase alpha purified from human polymorphonuclear leukocytes inhibited the D to I conversion in a glycogen particle preparation. The inhibition was counteracted by glucose 6-phosphate and to a lesser degree by AMP. Phosphorylase beta was also inhibitory, but only at higher concentrations than phosphorylase alpha. No phosphorylase phosphatase activity was found in the glycogen particle preparation, which may indicate that chromatography on concanavalin A-Sepharose separates this enzyme from the synthase phosphatase or partially destroys the activity of a hypothetical common protein phosphatase.     

Lithium's effects on rat liver glucose metabolism in vivo

Oral administration of lithium carbonate to fed-healthy rats strongly decreased liver glycogen content, despite the simultaneous activation of glycogen synthase and the inactivation of glycogen phosphorylase. The effect seemed to be related to a decrease in glucose 6-phosphate concentration and to a decrease in glucokinase activity. Moreover, in these animals lithium markedly decreased liver fructose 2,6-bisphosphate, which could be a consequence of the fall in glucose 6-phosphate and of the inactivation of 6-phosphofructo-2-kinase. Liver pyruvate kinase activity and blood insulin also decreased after lithium administration. Lower doses of lithium carbonate had less intense effects. Lithium administration to starved-healthy and fed-streptozotocin-diabetic rats caused a slight increase in blood insulin, which was simultaneous with increases in liver glycogen, glucose 6-phosphate, and fructose 2, 6-phosphate. Glucokinase, 6-phosphofructo-2-kinase, and pyruvate kinase activities also increased after lithium administration in starved-healthy and fed-diabetic rats. Lithium treatment activated glycogen synthase and inactivated glycogen phosphorylase in a manner similar to that observed in fed-healthy rats. Glycemia was not modified in any group of animals. These results indicate that lithium acts on liver glycogen metabolism in vivo in at least two different ways: one related to changes in insulinemia, and the other related to the direct action of lithium on the activity of some key enzymes of liver glucose metabolism.     

Glycogen hyperaccumulation in Saccharomyces cerevisiae ras2 mutant. A biochemical study.

The mechanism by which yeast ras2 mutant hyperaccumulates glycogen has been investigated. Total glycogen synthase activity was between and 1.3 times higher in the ras2 mutant than in an isogenic strain. In addition, while in the normal strain the glycogen synthase activation state decreased along the exponential phase, in the mutant strain the opposite behaviour was observed: glycogen synthase activation state rose continuously reaching full activation at the beginning of the stationary phase. Glycogen phosphorylase a activity was up to 40 times higher in the mutant than in the normal strain. Glucose 6-phosphate and fructose 2,6-bisphosphate levels were slightly more elevated in the mutants. The increase in total glycogen synthase and, particularly, the full activation of this enzyme may explain glycogen hyperaccumulation in the ras2 mutant even in the presence of elevated levels of glycogen phosphorylase a.     

Studies on glycogen synthesis in pigeon liver homogenates. Incorporation of hexose into glycogen

Liver homogenates of avian species, but not of mammals, form glycogen from glucose, mannose, fructose and galactose. Incorporation of labelled glucose, fructose and mannose, but not of labelled galactose, into glycogen is diluted isotopically by unlabelled glucose. Except for fructose, glycogen formation from other substrates by pigeon liver homogenates compares favourably with that from the same substrates in pigeon liver slices. Optimum conditions for glycogen synthesis from glucose by pigeon liver homogenate are: medium of incubation, 0.175m-sucrose-45mm-potassium chloride-15mm-glycylglycine buffer, pH7.5; concentration of substrate, 15mm; concentration of tissue, less than 120mg./ml.; temperature of incubation, 37-43 degrees ; atmosphere, oxygen. Uncouplers of oxidative phosphorylation, Ca(2+), EDTA, PP(i), 2-deoxyglucose 6-phosphate and microsomal fraction of rat liver are inhibitory to glycogen synthesis from glucose. Starvation of pigeons for 24 and 48hr. leads to a slight stimulation of glycogen synthesis in their liver homogenates as compared with fed controls. Pigeon liver homogenates can be separated into subcellular fractions that on reconstitution can synthesize glycogen. All the enzymes of the glycogen pathway except soluble high-K(m) glucokinase are present in pigeon liver.     

The concentrations of glucose 1,6-bisphosphate and other regulatory metabolites, and the activities of enzymes of the glycogen metabolism in the perfused rabbit psoas muscle

The following parameters were determined in the rabbit psoas muscle after perfusion in the presence of either insulin, propranolol, or isoproterenol: Concentrations of cyclic AMP, glucose 1,6-bisphosphate, fructose 2,6-bisphosphate, glucose-1-phosphate, glucose 6-phosphate, and fructose-1,6-bisphosphate. Maximum and "regulatory" activities of the enzymes glycogen phosphorylase, glycogen synthase, phosphofructokinase, and histone-phosphorylating protein kinase.     

The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35 degrees C without affecting cyclic AMP concentration.     

The glycogenic action of protein targeting to glycogen in hepatocytes involves multiple mechanisms including phosphorylase inactivation and glycogen synthase translocation

Expression of the glycogen-targeting protein PTG promotes glycogen synthase activation and glycogen storage in various cell types. In this study, we tested the contribution of phosphorylase inactivation to the glycogenic action of PTG in hepatocytes by using a selective inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a and sequential activation of glycogen synthase. Similar to CP-91194, graded expression of PTG caused a concentration-dependent inactivation of phosphorylase and activation of glycogen synthase. The latter was partially counter-acted by the expression of muscle phosphorylase and was not additive with the activation by CP-91149, indicating that it is in part secondary to the inactivation of phosphorylase. PTG expression caused greater stimulation of glycogen synthesis and translocation of glycogen synthase than CP-91149, and the translocation of synthase could not be explained by accumulation of glycogen, supporting an additional role for glycogen synthase translocation in the glycogenic action of PTG. The effects of PTG expression on glycogen synthase and glycogen synthesis were additive with the effects of glucokinase expression, confirming the complementary roles of depletion of phosphorylase a (a negative modulator) and elevated glucose 6-phosphate (a positive modulator) in potentiating the activation of glycogen synthase. PTG expression mimicked the inactivation of phosphorylase caused by high glucose and counteracted the activation caused by glucagon. The latter suggests a possible additional role for PTG on phosphorylase kinase inactivation.     

Effect of fructose on glycogen synthesis in the perfused rat liver

The effect of fructose on glycogen synthesis was examined in the perfused liver of starved rats. With increasing fructose concentration in the perfusate, glycogen synthesis and the % a form of glycogen synthase increased to a maximum at 2 mM and then decreased, progressively. The glucose 6-P level increased with the increase in fructose concentration. On the other hand, the ATP content was unchanged at a concentration of 2 mM or less and decreased at 3 mM or more. We also showed that the stimulation of glycogen synthesis by fructose at a concentration of 2 mM or less was due to activation of glycogen synthase by accumulated glucose 6-P and that ATP depletion at a concentration of 3 mM or more caused an increase in phosphorylase a and a decrease in glycogen synthase activity even in the presence of a high concentration of glucose 6-P.     

Glucose 6-phosphate produced by gluconeogenesis and by glucokinase is equally effective in activating hepatic glycogen synthase

Glucose 6-phosphate (Glc-6-P) produced in cultured hepatocytes by direct phosphorylation of glucose or by gluconeogenesis from dihydroxyacetone (DHA) was equally effective in activating glycogen synthase (GS). However, glycogen accumulation was higher in hepatocytes incubated with glucose than in those treated with DHA. This difference was attributed to decreased futile cycling through GS and glycogen phosphorylase (GP) in the glucose-treated hepatocytes, owing to the partial inactivation of GP induced by glucose. Our results indicate that the gluconeogenic pathway and the glucokinase-mediated phosphorylation of glucose deliver their common product to the same Glc-6-P pool, which is accessible to liver GS. As observed in the treatment with glucose, incubation of cultured hepatocytes with DHA caused the translocation of GS from a uniform cytoplasmic distribution to the hepatocyte periphery and a similar pattern of glycogen deposition. We hypothesize that Glc-6-P has a major role in glycogen metabolism not only by determining the activation state of GS but also by controlling its subcellular distribution in the hepatocyte.     

Ketose induced respiratory inhibition in isolated hepatocytes

The addition of 10 mM fructose or 10 mM tagatose to a suspension of hepatocytes caused respiratory inhibition, whereas no change in oxygen uptake was observed following the addition of glucose. However, incubations in the presence of fructose showed a high, aerobic glycolytic activity. Tagatose is phosphorylated to tagatose 1-phosphate but is not further metabolized by cell free liver extract. Moreover, the addition of fructose to glucagon treated cells also caused the Crabtree-like effect. The concentration of adenine nucleotides and inorganic phosphate (Pi) in the mitochondrial and cytosolic compartments during incubation (time 30 min) was determined by the digitonin fractionation procedure. In the presence of 10 mM fructose or tagatose, the total adenine nucleotide pools decreased by 40%; however, glucose produced no change. The addition of ketoses diminished the asymmetric distribution of extramitochondrial (ATP/ADP)e ratio and intramitochondrial (ATP/ADP)i ratio. At the same time the total mitochondrial Pi fell from 17 mM to 6-7 mM. The mitochondrial membrane potential (-161 mV) in the presence of fructose showed no changes during the 30 min experimental period. An increase in the NADH/NAD+ ratio was observed. These results suggest that in hepatocytes the inhibition of respiration is not necessarily linked with the enhanced aerobic glycolysis, by competition for common substrates.     

Intracellular mechanism of action of sympathetic hepatic nerves on glucose and lactate balance in perfused rat liver

In rat liver perfused in situ stimulation of the nerve plexus around the hepatic artery and the portal vein caused an increase in glucose output and a shift from lactate uptake to output. The effects of nerve stimulation on some key enzymes, metabolites and effectors of carbohydrate metabolism were determined and compared to the actions of glucagon, which led to an increase not only of glucose output but also of lactate uptake. 1. Nerve stimulation caused an enhancement of the activity of glycogen phosphorylase a to 300% and a decrease of the activity of glycogen synthase I to 40%, while it left the activity of pyruvate kinase unaltered. Glucagon, similarly to nerve action, led to a strong increase of glycogen phosphorylase and to a decrease of glycogen synthase; yet in contrast to the nerve effect it lowered pyruvate kinase activity clearly. 2. Nerve stimulation increased the levels of glucose 6-phosphate and of fructose 6-phosphate to 200% and 170%, respectively; glucagon enhanced the levels to about 400% and 230%, respectively. The levels of ATP and ADP were not altered, those of AMP were increased slightly by nerve stimulation. 3. Nerve stimulation enhanced the levels of the effectors fructose 2,6-bisphosphate and cyclic AMP only slightly to 140% and 125%, respectively; glucagon lowered the level of fructose 2,6-bisphosphate to 15% and increased the level of cyclic AMP to 300%. 4. In calcium-free perfusions the metabolic responses to nerve stimulation showed normal kinetics, if calcium was re-added 3 min before, but delayed kinetics, if it was re-added 2 min after the onset of the stimulus. The delay may be due to the time required to refill intracellular calcium stores. The hemodynamic alterations dependent on extracellular calcium were normal in both cases. The activation of glycogen phosphorylase, the inhibition of glycogen synthase and the increase of glucose 6-phosphate can well explain the enhancement of glucose output following nerve stimulation. The unaltered activity of pyruvate kinase and the marginal increase of fructose 2,6-bisphosphate cannot be the cause of the nerve-stimulation-dependent shift from lactate uptake to output. The very slight increase of the level of cyclic AMP after nerve stimulation cannot elicit the observed activation of glycogen phosphorylase.(ABSTRACT TRUNCATED AT 400 WORDS)