Simon J. Pilkis

Effects of glucagon on cyclic AMP and carbohydrate metabolism in livers from diabetic rats

Abstract Cyclic AMP metabolism in normal and diabetic livers was studied using the isolated perfused rat liver and plasma membrane and supernatat fractions from rat liver. Liver from alloxan- or streptozotocin-diabetic rats had increased tissue levels of cyclic AMP and showed increased release of cyclic AMP during perfusion. Addition of glucagon or cyclic AMP to the medium produced little or no increase in the high rates of glucose production and lactate gluconeogenesis in diabetic livers. Low concentrations of glucagon (2·10 −10 M or less) did not increase tissue accumulation or release of cyclic AMP in livers from diabetic rats but were effective in normal livers. Higher concentrations of the hormone produced normal responses in diabetic livers. Basal or fluoride-stimulated adenylate cyclase activity in plasma membranes isolated from diabetic livers was not altered, but the enzyme was subnormally normally responsive to concentrations of glucagon within the range 10 −10 −10 −8 M. Insulin (10 −11 −10 −6 M) added in vitro was without effect on basal or glucagon-stimulated plasma membrane adenylate cyclase. Neither modulation of the Mg 2+ concentration, nor addition of Ca 2+ , GTP, theophylline or ouabain caused the emergence of an insulin effect. Rat liver plasma membranes contained phosphodiesterase activity with two apparent K m values of about 0.5 and 70 μM. The activity of the low K m enzyme was decreased in plasma membranes from diabetic rats and was increased by insulin treatment of the rats in vivo. Both low and high K m activities were also decreased in supernatant fractions from livers of diabetic rats. Insulin at 10 −9 or 10 −7 M concentration had no effect in vitro on plasma membrane phosphodiesterase activities.

The Role of Fructose 2,6-Bisphosphate in the Regulation of Carbohydrate Metabolism

Publisher Summary Fructose 2,6-bisphosphate (fructose 2,6-P 2 ) is a unique sugar diphosphate that is an important regulator of hepatic carbohydrate metabolism. This chapter describes the discovery and identification of the compound and its chemical synthesis; discusses the regulation of its concentration in liver and of the enzymes responsible for its metabolism; summarizes what is known of the mechanism of the effect of fructose 2,6-P 2 in the regulation of 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase activity; and describes the role of this effector in the regulation of hepatic carbohydrate metabolism. Fructose 2,6-P 2 has been found in all mammalian tissues that have been studied. The highest concentrations have been found in liver, brain, and heart muscle. Lower concentrations have been found in skeletal muscle, kidney, and epididymal fat. It has also been detected in rat pancreatic islets, and its concentration increases upon exposure of the islets to glucose. The compound has been isolated also from Saccharomyces cerevisiae grown on glucose, from mung beans, and from spinach leaves.

Hormonal regulation of DNA synthesis in primary cultures of adult rat hepatocytes: Action of glucagon☆

Abstract Primary cultures of adult rat hepatocytes, grown in modified minimal essential medium (Eagles) containing 10% calf serum, could be induced into DNA replication by combinations of epidermal growth factor (EGF), insulin and glucagon. The three hormones acted synergistically, and cells began entering DNA synthesis 48 h after hormone addition. The ability of the hormones to stimulate DNA synthesis was enhanced by plating cells at high cell concentrations or by conditioned medium, and was diminished by daily medium change. The contribution of glucagon to DNA synthesis was replaced by cAMP plus 1-methyl, 3-isobutyl xanthine or by adrenergic agents. Evidence is presented which suggests that all three hormones are required on the first day of culture, and that EGF and insulin are also required after the first day. This appears to be a useful system for studies on the hormonal initiation of growth in quiescent cells.

Fructose 2,6-bisphosphate: A mediator of hormone action at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle

Abstract Fructose 2,6-bisphosphate was discovered in the course of studies on the regulation of hepatic 6-phosphofructo-1-kinase activity by glucagon. The compound is acid-labile but stable to heating in alkali. These properties are due to the presence of a phosphate on the hemiketalic hydroxyl group at C2. Fructose 2,6-bisphosphate can be synthesized chemically by reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide followed by base-catalyzed ring opening of the fructose 1,2-cyclic, 6-bisphosphate intermediate. Fructose 2,6-bisphosphate is a potent allosteric activator of 6-phosphofructo-1-kinase and an inhibitor of fructose 1,6-bisphosphatase. It potentiates the effect of AMP on both enzymes. Since 6-phosphofructo-1-kinase is one of the most important control elements in glycolysis, it is likely that fructose 2,6-bisphosphate is a significant physiological regulator of this process in liver. Likewise, fructose 2,6-bisphosphate is probably a significant physiological regulator of fructose 1,6-bisphosphatase, a key gluconeogenic enzyme. For example, the effect of glucagon to enhance carbon flux through fructose 1,6-bisphosphatase and to inhibit flux through 6-phosphofructo-l-kinase in intact hepatocytes is explained by the ability of the hormone to lower the level of fructose 2,6-bisphosphate. This decrease in fructose 2,6-bisphosphate is brought about, at least in part, by a cAMP-dependent phosphorylation and inactivation of the enzyme responsible for its synthesis, 6-phosphofructo-2-kinase. This novel enzyme catalyzes the transfer of the γ phosphate of ATP to the C2 position of fructose 6-phosphate. Phosphorylation of this enzyme is catalyzed by the cAMP-dependent protein kinase in vitro with concomitant inhibition of enzyme activity. Fructose 1,6-bisphosphatase and 6-phosphofructo-l-kinase are also substrates for the cAMP-dependent protein kinase both in vitro and in vivo. However, a role for phosphorylation in regulating their activity remains uncertain. The discovery of the unique sugar phosphate, fructose 2,6-bisphosphate, has been an important advance in our understanding of the regulation of carbohydrate metabolism in liver.

Hormonal control of hepatic gluconeogenesis.

Publisher Summary This chapter describes the hormonal control of hepatic gluconeogenesis, which is the process whereby lactate, pyruvate, glycerol, and certain amino acids are converted into glucose and glycogen. The liver is the major site of gluconeogenesis although the kidney becomes important during prolonged starvation. The most important function of gluconeogenesis is the maintenance of blood glucose levels during times when food intake is restricted and/or glycogen stores are depleted. Hormonal control of gluconeogenesis can be divided for convenience into three categories. The first involves the regulation of substrate supply. All gluconeogenic substrates reach the liver in subsaturating concentration. Thus, the regulation of substrate release into the blood from the extrahepatic tissues will directly affect hepatic glucose formation. The regulation of the level of cyclic AMP (cAMP) could also explain the inhibitory effect of insulin on gluconeogenesis. There is also evidence that insulin can suppress gluconeogenesis by a mechanism that is independent of changes in cAMP.

Regulation by insulin of gluconeogenesis in isolated rat hepatocytes.

Insulin (10nM) completely suppressed the stimulation of gluconeogenesis from 2 mM lactate by low concentrations of glucagon (less than or equal to 0.1 nM) or cyclic AMP (less than or equal to 10 muM), but it had no effect on the basal rate of gluconeogenesis in hepatocyctes from fed rats. The effectiveness of insulin diminished as the concentration of these agonists increased, but insulin was able to suppress by 40% the stimulation by a maximally effective concentration of epinephrine (1 muM). The response to glucagon, epinephrine, or insulin was not dependent upon protein synthesis as cycloheximide did not alter their effects. Insulin also suppressed the stimulation by isoproterenol of cyclic GMP. These data are the first demonstration of insulin antagonism to the stimulation of gluconeogenesis by catecholamines. Insulin reduced cyclic AMP levels which had been elevated by low concentrations of glucagon or by 1 muM epinephrine. This supports the hypothesis that the action of insulin to inhibit gluconeogenesis is mediated by the lowering of cyclic AMP levels. However, evidence is presented which indicates that insulin is able to suppress the stimulation of gluconeogenesis by glucagon or epinephrine under conditions where either the agonists or insulin had no measurable effect on cyclic AMP levels. Insulin reduced the glucagon stimulation of gluconeogenesis whether or not extracellular Ca2+ were present, even though insulin only lowered cyclic AMP levels in their presence. Insulin also reduced the stimulation by epinephrine plus propranolol where no significant changes in cyclic AMP were observed without or with insulin. In addition, insulin suppressed gluconeogenesis in cells that had been preincubated with epinephrine for 20 min, even though the cyclic AMP levels had returned to near basal values and were unaffected by insulin. Thus insulin may not need to lower cyclic AMP levels in order to suppress gluconeogenesis.

Control of pyruvate kinase activity by glucagon in isolated hepatocytes

Abstract Incubation of hepatocytes with 10 nM glucagon led to an increase in the K0.5 for phosphoenolpyruvate for pyruvate kinase measured in homogenates of the cells. Incubation of partially purified rat liver pyruvate kinase with protein kinase and ATP led to a similar result. In addition, both the phosphorylated enzyme and homogenates prepared from cells incubated with glucagon exhibited an apparently decreased sensitivity to stimulation by fructose diphosphate when activity was measured in the presence of physiological concentrations of ATP and alanine. These similarities suggest that the effect of glucagon to inhibit hepatocyte pyruvate kinase may be mediated at least in part by a phosphorylation-dephosphorylation mechanism.

Stimulation by glucagon of the incorporation of U-14 C-labeled substrates into glucose by isolated hepatocytes from fed rats☆

The effect of glucagon on the incorporation of U-14C-labeled lactate, pyruvate or alanine into glucose has been studied using isolated hepatocytes from livers of fed rats. Rates of incorporation into glucose were about the same as observed in perfused liver preparations provided precautions were taken to avoid depletion of certain metabolities by the preparative procedures. With each substrate, stimulation of the incorporation into glucose by a maximally effective concentration of glucagon (10 nM) was associated with about a 75% reduction in the substrate concentration required for a half-maximal rate and with about a 30% increase in maximum rate. Consequently, the hormone caused a substantial (2--4-fold) stimulation when any one of the above substrates was present at a near physiological concentration, but brought about only a relatively small stimulation (1.4-fold) when very high substrate concentrations were used. Provision of cytoplasmic reducing equivalents (by ethanol addition), or of precursor for acetyl-coenzyme A formation (by acetate addition)-stimulated incorporation of labeled alanine into glucose and their effects were additive with that of glucagon. This suggested that provision of either of these intermediates was not a means by which the hormone increased the incorporation of labeled substrate into glucose. NH4+ stimulated the incorporation of 20 mM [U-14C] lactate into glucose 2-fold, probably by promoting glutamate synthesis and thus enhancing the transamination of oxaloacetate to aspartate. Evidence was obtained to support the view that glucagon also increases glutamate production (presumably from endogenous protein). However, the stimulation of incorporation into glucose from 20 mM [U-14C] lactate by NH4+ plus glucagon was synergistic. This suggested that glucagon also stimulated the incorporation of labeled substrate into glucose by additional means. Stimulation of the incorporation of [U-14C] alanine into glucose by beta-hydroxybutyrate plus glucagon was also synergistic. This suggested that another action of glucagon may be to provide more intramitochondrial reducing potential.

Effect of Diabetes, Insulin, Starvation, and Refeeding on the Level of Rat Hepatic Fructose 2,6-Bisphosphate

The influence of alloxan diabetes and starvation for 72 h on the level of rat hepatic fructose 2,6-bisphosphate was investigated. Both diabetes and starvation decreased the level to 10% of the value found in livers of normal, fed rats (10 nmol/g liver). The activity of the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, 6-phosphofructo 2-kinase, was also decreased in livers of diabetic rats. Insulin administration for 24 h to diabetic rats restored the level of fructose 2,6-bisphosphate to normal. Refeeding a high carbohydrate diet for 24 h to starved rats resulted in fructose 2,6-bisphosphate levels that were 2.5-fold higher than that in livers of fed rats. The level of fructose 2,6-bisphosphate in diabetes and starvation, and after refeeding correlates well with the rate of glycolysis and gluconeogenesis in these states and thereby provides further support for its role in regulating hepatic carbohydrate metabolism.

Regulation of phosphofructokinase activity by glucagon in isolated rat hepatocytes

Abstract Glucagon addition to isolated hepatocytes from fed rats resulted in an inhibition of the activity of phosphofructokinase measured in extracts of the cells. Glucagon caused a shift in the fructose 6-phosphate concentration curve to the right resulting in an increase in the K 0.5 for F6P from 0.09 mM to 0.31 mM. No effect of glucagon was seen when the enzyme was assayed with saturating concentrations of fructose 6-phosphate or in the presence of 1 mM AMP. The effect of glucagon was seen within minutes and the concentration of hormone giving half-maximal inhibition was 0.2 nM. This effect of glucagon on phosphofructokinase activity may contribute to the effect of glucagon on substrate cycling at the fructose 6-phosphate-fructose bisphosphate level.