ABSTRACT
Addition of the commonly used anesthetic pentobarbital to hepatocytes from fed rats resulted in a dose-dependent decrease in the level of fructose 2,6-bisphosphate. At a concentration of pentobarbital (0.4 mM) that lowered fructose 2,6-bisphosphate by 60%, there was no significant change in the level of fructose 6-phosphate, ATP, or L-glycerol 3-phosphate. Higher concentrations of pentobarbital (2 mM) enhanced both glycolysis and glycogenolysis and fructose 2,6-bisphosphate levels were reduced to less than 10% of the control. Concomitant with these changes there was a decrease in ATP, glucose 6-phosphate, and fructose 6-phosphate and a two- and fivefold increase in ADP and AMP, respectively. In hepatocytes from starved rats pentobarbital also lowered ATP levels and inhibited gluconeogenesis but had no effect on either lactate production or the already low level of sugar diphosphate. However, in the fasted case pentobarbital completely prevented the 10-fold elevation of fructose 2,6-bisphosphate brought about by 30 mM glucose. The anesthetic had no effect on cAMP-dependent protein kinase activity or on pyruvate kinase activity in hepatocytes from fed or starved rats but caused reciprocal changes in the activities of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Kinase activity was decreased and bisphosphatase activity was increased. These results suggest that the effects of pentobarbital on gluconeogenesis and glycolysis are due to inhibition of energy metabolism with elevated AMP levels causing activation of 6-phosphofructo-1-kinase and inhibition of fructose 1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Fructosediphosphates/metabolism , Hexosediphosphates/metabolism , Liver/metabolism , Pentobarbital/pharmacology , Adenine Nucleotides/metabolism , Animals , Cell Separation , Gluconeogenesis/drug effects , Glucose/pharmacology , Liver/cytology , Liver/enzymology , Male , Phosphofructokinase-2 , Phosphoric Monoester Hydrolases/metabolism , Phosphotransferases/metabolism , Protein Kinases/metabolism , Pyruvate Kinase/metabolism , Rats , Rats, Inbred Strains , Starvation/metabolismABSTRACT
When glucose was given to starved rats there was an increase in both 6-phosphofructo 2-kinase and pyruvate kinase activity and a decrease in fructose 2,6-bisphosphatase activity 30 min and 60 min later. These changes were accompanied by an increase in glycogen deposition and by modest, but significant increases in fructose 2,6-bisphosphate levels at the same time. Metabolite measurements indicated that flux through 6-phosphofructo 1-kinase and pyruvate kinase were increased. These results suggest that although glycogen deposition may occur via the gluconeogenic pathway, glycolysis is activated at the same time by changes in the phosphorylation state of key regulatory enzymes as well as by the small rise in fructose 2,6-bisphosphate.
Subject(s)
Carbohydrate Metabolism , Fructose-Bisphosphatase/metabolism , Glucose/metabolism , Liver/enzymology , Phosphofructokinase-1/metabolism , Pyruvate Kinase/metabolism , Animals , Dietary Carbohydrates/metabolism , Fasting , Kinetics , Male , Rats , Rats, Inbred StrainsABSTRACT
Chemiluminescence, as a direct measure of oxygen free radical production, induced in isolated cells, hepatocytes, and red cells by the action of alloxan has been measured. The assay system used luminol, 3 microM, for signal amplification. The buffer used was Krebs-Ringer bicarbonate with 16 mM Hepes, pH 7.4. This buffer did not react with alloxan in the absence of cells. Some chemiluminescence was noted from all cells in the absence of alloxan. In the presence of alloxan, reactions occurred within seconds and islet cells were significantly more reactive to alloxan than either red cells or hepatocytes as defined by alloxan dose-response curves with fixed cell numbers or fixed surface areas. These data indicate a cell specificity for an early action of alloxan perhaps mediated at the cell membrane.
Subject(s)
Alloxan/pharmacology , Islets of Langerhans/drug effects , Animals , Dose-Response Relationship, Drug , Erythrocytes/drug effects , Free Radicals , Islets of Langerhans/metabolism , Liver/cytology , Liver/drug effects , Luminescent Measurements , Male , Oxygen/metabolism , RatsABSTRACT
Fructose 2,6-bisphosphate levels were assayed in freeze-clamped livers of anesthesized rats. All doses of pentobarbital that were effective in anesthesizing the rats caused a significant decrease in fructose 2,6-bisphosphate levels. Injection of pentobarbital also resulted in decreased 6-phosphofructo 2-kinase activity and increased fructose 2,6-bisphosphatase activity measured in dialyzed (NH4)2SO4-treated liver extracts but with no change in pyruvate kinase activity. It was concluded that the anesthesia-induced decrease in fructose 2,6-bisphosphate levels results at least in part from increased phosphorylation of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase.
Subject(s)
Fructosediphosphates/metabolism , Hexosediphosphates/metabolism , Liver/metabolism , Pentobarbital/pharmacology , Anesthesia, General , Animals , Fructose-Bisphosphatase/metabolism , Kinetics , Liver/drug effects , Male , Phosphofructokinase-1/metabolism , Pyruvate Kinase/metabolism , Rats , Rats, Inbred StrainsABSTRACT
Fructose-2,6-bisphosphate levels in freeze-clamped livers of starved rats were 0.5 nmol/g liver. Oral administration of 1 g glucose per kg body weight to starved rats increased glycogen levels from 4 mg/g liver to 13.5 mg/g in 2 hr but did not significantly alter fructose-2,6-bisphosphate levels. The low level of this effector is consistent with an active gluconeogenic process and the results support the hypothesis that carbon atoms for glycogen synthesis can be derived from 3-carbon precursors via this pathway, even in the presence of glucose.
Subject(s)
Fructosediphosphates/metabolism , Glucose/metabolism , Hexosediphosphates/metabolism , Liver/metabolism , Animals , Glucose/administration & dosage , Kinetics , Liver Glycogen/metabolism , Male , Rats , Rats, Inbred Strains , StarvationSubject(s)
Carbohydrate Metabolism , Fructosediphosphates/physiology , Hexosediphosphates/physiology , Allosteric Regulation , Animals , Cyclic AMP/metabolism , Epinephrine/physiology , Fructose-Bisphosphatase/metabolism , Fructosediphosphates/metabolism , Glucagon/physiology , Gluconeogenesis , Glycolysis , Insulin/physiology , Liver/enzymology , Liver/metabolism , Phosphofructokinase-1/metabolism , Phosphofructokinase-2 , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Phosphotransferases/metabolism , Rats , Tissue DistributionABSTRACT
(+)- and (-)-catechin showed opposite effects on glycogen metabolism in isolated rat hepatocytes. Addition of 0.5 mM catechin to hepatocytes from fasted rats resulted in the case of the (+)-isomer in a 90% stimulation and the case of the (-)-isomer in a 90% inhibition of net glycogen production. When 0.5 mM of the two isomers were added to hepatocytes from fed rats, (+)-catechin inhibited glycogenolysis by 33%, whereas the (-)-isomer stimulated the same process by 42%. At equal concentrations, the effects of (-)-catechin were stronger than those of the (+)-isomer. (-)-Epicatechin acted in a manner similar to (+)-catechin; however, the effect was less pronounced. (+)-Catechin antagonized the inhibitory action of suboptimal doses of glucagon on glycogen production whereby no change in basal or glucagon-elevated cyclic AMP level was observed. The activities of glycogen synthase a and glycogen phosphorylase a were changed by (+)- and (-)-catechin in a way corresponding to the changes in glycogen production and breakdown. (-)-Catechin, however, stimulated the activities of both glycogen synthase a and glycogen phosphorylase a in hepatocytes from fed rats. A possible interaction of the flavonoids or of their metabolites with glycogen phosphorylase is discussed.
Subject(s)
Benzopyrans/pharmacology , Catechin/pharmacology , Liver Glycogen/metabolism , Liver/drug effects , Animals , In Vitro Techniques , Kinetics , Male , Rats , Rats, Inbred Strains , Stereoisomerism , Structure-Activity RelationshipSubject(s)
Gluconeogenesis , Hormones/physiology , Liver/metabolism , Animals , Cell-Free System , Fructosediphosphates/pharmacology , Glucagon/pharmacology , Gluconeogenesis/drug effects , Glucose/pharmacology , Glucose-6-Phosphate , Glucosephosphates/biosynthesis , Hormones/pharmacology , Rats , Starvation/metabolismABSTRACT
Isolated liver cells from 24 h starved rats were incubated in Krebs-Ringer buffer containing 4% albumin. In the presence of 10, 20 and 30 mM glucose, addition of insulin stimulated net glycogen production by 52, 39 and 20%, respectively. 2 . 10(-9) M insulin was required for half-maximal stimulation. Increases of glycogen production and of glycogen synthase a activity were observed after 15-30 min of incubation with insulin. The stimulatory effect of insulin was additive to that of lithium. In agreement with the literature, insulin antagonized the inhibitory action of suboptimal doses of glucagon on glycogen deposition whereby a decrease of glucagon-elevated cyclic AMP levels was observed. In addition, we found that insulin also decreased the basal cyclic AMP levels in the absence of added glucagon by 22%. It is concluded that physiological concentrations of insulin stimulate net glycogen deposition in hepatocytes from fasted rats; the decrease of basal cyclic AMP levels upon insulin addition may play a role in the mechanism of the hormone action.
Subject(s)
Insulin/pharmacology , Liver Glycogen/biosynthesis , Liver/metabolism , Animals , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Drug Synergism , Glucose/metabolism , Glycogen Synthase/metabolism , Lithium/pharmacology , Liver/cytology , Male , Potassium/pharmacology , RatsABSTRACT
In hepatocytes from starved rats, 10mM-glucose suppressed in incorporation of 2mM labelled alanine into glucose+glycogen by more than 40%, whereas no inhibition was observed with labelled lactate as substrate. Addition of glycerol instead of glucose did not show this inhibition. The inhibitory effect could also be demonstrated in label-free experiments.