Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase

Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.     

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.     

The role of inhibition of pyruvate kinase in the stimulation of gluconeogenesis by glucagon: a reevaluation

We have reexamined the concept that glucagon controls gluconeogenesis from lactate-pyruvate in isolated rat hepatocytes almost entirely by inhibition of flux through pyruvate kinase, thereby making gluconeogenesis more efficient. 1. We tested and refined the 14C-tracer technique that has previously yielded the opposite conclusion, that is, that inhibition of pyruvate kinase is a relatively unimportant mechanism. The tracer procedure, as used by us, was found to be insensitive to the size of the pyruvate pool, and experiments using modifications of the technique to obviate a number of other potential errors support the earlier conclusion that control of pyruvate kinase is not the predominant mechanism. 2. Any stimulation of formation of glucose that results from inhibition of pyruvate kinase is the consequence of elevation of the steady-state concentrations of phosphoenolpyruvate and all subsequent intermediates in the gluconeogenic pathway. During ongoing stimulation of glucose synthesis by glucagon in isolated hepatocytes, the concentrations of all measured intermediate compounds between phosphoenolpyruvate and glucose were elevated except triose phosphates and fructose 1,6-bisphosphate. The failure of these compounds to rise above control levels indicates that not all gluconeogenic reactions beyond pyruvate kinase were accelerated thermodynamically as would occur with predominant control at pyruvate kinase. We conclude, therefore, that although glucagon inhibits flux through the pyruvate kinase reaction, this does not account for most of the stimulation of gluconeogenesis. Major control sites are also within the pyruvate-phosphoenolpyruvate segment and the fructose 1,6-bisphosphate cycle.     

Hepatic pyruvate kinase. Regulation by glucagon, cyclic adenosine 3'-5'-monophosphate, and insulin in the perfused rat liver.

A reversible interconversion of two kinetically distinct forms of hepatic pyruvate kinase regulated by glucagon and insulin is demonstrated in the perfused rat liver. The regulation does not involve the total enzyme content of the liver, but rather results in a modulation of the substrate dependence. The forms of pyruvate kinase in liver homogenates are distinguished by measurements of the ratio of the enzyme activity at a subsaturating concentration of P-enolpyruvate (1.3 mM) to the activity at a saturating concentration of this substrate (6.6 mM). A low ratio form of pyruvate kinase (ratio between 0.1 and 0.2) is obtained from livers perfused with 10(-7) M glucagon or 0.1 mM adenosine 3':5'-monophosphate (cyclic AMP). A high ratio form of the enzyme is obtained from livers perfused with no hormone (ratio = 0.35 to 0.45). The regulation of pyruvate kinase by glucagon and cyclic AMP occurs within 2 min following the hormone addition to the liver. Insulin (22 milliunits/ml) counteracts the inhibition of pyruvate kinase caused by 5 X 10(-11) M glucagon, but has only a slight influence on the enzyme properties in the absence of the hyperglycemic hormone. The low ratio form of pyruvate kinase obtained from livers perfused with glucagon or cyclic AMP is unstable in liver extracts and will revert to a high ratio form within 10 min at 37 degrees or within a few hours at 0 degrees. Pyruvate kinase is quantitatively precipitated from liver supernatants with 2.5 M ammonium sulfate. This precipitation stabilizes the enzyme and preserves the kinetically distinguishable forms. The kinetic properties of the two forms of rat hepatic pyruvate kinase are examined using ammonium sulfate precipitates from the perfused rat liver. At pH 7.5 the high ratio form of the enzyme has [S]0.5 = 1.6 +/- 0.2 mM P-enolpyruvate (n = 8). The low ratio form of enzyme from livers perfused with glucagon or cyclic AMP has [S]0.5 = 2.5 +/- 0.4 mM P-enolpyruvate (n = 8). The modification of pyruvate kinase induced by glucagon does not alter the dependence of the enzyme activity on ADP (Km is approximately 0.5 mM ADP for both forms of the enzyme). Both forms are allosterically modulated by fructose 1,6-bisphosphate, L-alanine, and ATP. The changes in the kinetic properties of hepatic pyruvate kinase which follow treating the perfused rat liver with glucagon or cyclic AMP are consistent with the changes observed in the enzyme properties upon phosphorylation in vitro by a clyclic AMP-stimulated protein kinase (Ljungstr?m, O., Hjelmquist, G. and Engstr?m, L. (1974) Biochim. Biophys. Acta 358, 289--298). However, other factors also influence the enzyme activity in a similar manner and it remains to be demonstrated that the regulation of hepatic pyruvate kinase by glucagon and cyclic AMP in vivo involes a phosphorylation.     

Metabolic control of hepatic gluconeogenesis during exercise.

Prolonged exercise increased the concentrations of the hexose phosphates and phosphoenolpyruvate and depressed those of fructose 1,6-bisphosphate, triose phosphates and pyruvate in the liver of the rat. Since exercise increases gluconeogenic flux, these changes in metabolite concentrations suggest that metabolic control is exerted, at least, at the fructose 6-phosphate/fructose 1,6-bisphosphate and phosphoenolpyruvate/pyruvate substrate cycles. Exercise increased the maximal activities of glucose 6-phosphatase, fructose 1,6-bisphosphatase, pyruvate kinase and pyruvate carboxylase in the liver, but there were no changes in those of glucokinase, 6-phosphofructokinase and phosphoenolpyruvate carboxykinase. Exercise changed the concentrations of several allosteric effectors of the glycolytic or gluconeogenic enzymes in liver; the concentrations of acetyl-CoA, ADP and AMP were increased, whereas those of ATP, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were decreased. The effect of exercise on the phosphorylation-dephosphorylation state of pyruvate kinase was investigated by measuring the activities under conditions of saturating and subsaturating concentrations of substrate. The submaximal activity of pyruvate kinase (0.5 mM-phosphoenolpyruvate), expressed as percentage of Vmax., decreased in the exercised animals to less than half that found in the controls. These changes suggest that hepatic pyruvate kinase is less active during exercise, possibly owing to phosphorylation of the enzyme, and this may play a role in increasing the rate of gluconeogenesis.     

Binding of hexose bisphosphates to muscle phosphofructokinase

On the basis of kinetic activation assays, the apparent affinity of muscle phosphofructokinase for fructose 2,6-bisphosphate was about 9-fold greater than that for fructose 1,6-bisphosphate, which in turn was about 10 times higher than that for glucose 1,6-bisphosphate. Equilibrium binding experiments showed that both fructose bisphosphates bind to phosphofructokinase with negative cooperativity; the affinity for fructose 2,6-bisphosphate was about 1 order of magnitude greater than the affinity for fructose 1,6-bisphosphate. Binding of fructose 2,6-bisphosphate to phosphofructokinase was antagonized by fructose 1,6-bisphosphate and glucose 1,6-bisphosphate and vice versa. Both fructose bisphosphates promoted aggregation of the enzyme to higher polymers as indicated by sucrose density gradient centrifugation. Other indicators of phosphofructokinase conformation such as thiol reactivity and maximum activation of in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase gave identical results in the presence of fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, or glucose 1,6-bisphosphate, indicating a common conformation is produced by all three ligands. It is concluded that the sugar bisphosphates bind to a single site on the enzyme.     

Purification and regulation of fructose-1,6-bisphosphatase from Bacillus licheniformis.

The fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) from the spore-forming bacterium Bacillus licheniformis was purified approximately 800-fold (with a 20% yield of activity) by a procedure that included ammonium sulfate precipitation, precipitation by MnCl2, and gamma-alumina gel absorption. Catalysis by this enzyme in vitro was specific for fructose 1,6-bisphosphate (Km of approximately 20 muM) and proceeded optimally at pH 8.0 to 8.5. Fructose-1,6-bisphosphatase was found to be rapidly inactivated by incubation in the presence of AMP or in the absence of Mn2+. The AMP inactivation was prevented by adding P-enolpyruvate to the incubation mixture. The enzyme was slowly inactivated when incubated in the presence of stabilizing concentrations of Mn2+ (5 mM) at protein concentrations of less than 8 mg of protein per ml. An additional system is produced during sporulation which specifically inactivates fructose bisphosphatase in vitro. This system, which is distinctly different from the AMP inactivating system, can be blocked by P-enolpyruvate. This fructose bisphosphatase, like fructose bisphosphatases from other sources, was strongly inhibited by AMP, exhibiting a Ki of approximately 5 muM. This inhibition, however, could be completely overcome by P-enolpyruvate. P-enolpyruvate was also found to be an activator of the enzyme and exhibited a Km of approximately 2 muM. This activation was prevented in a competitive manner by AMP, exhibiting a Ki of approximately 5 muM. No other effector of fructose bisphosphatase was identified in an extensive search. The specific activity of fructose bisphosphatase in crude extracts was found to be independent of the stage of the life cycle of the bacterium or of the nature of the carbon-energy source supporting growth. Immunoprecipitation studies indicate that no new species of fructose biphosphatase is produced during gluconeogenic growth or sporulation. The enzyme extracted from cells under a variety of physiological conditions exhibited a molecular weight of about 5 times 10-5 as determined by sucrose density centrifugation. Therefore, it is proposed that a single constitutively synthesized fructose bisphosphatase is present in B. licheniformis. Measurements of the intracellular level of fructose 1,6-bisphosphate indicate that the variation in the level of substrate throughout growth (1 mM) and sporulation (0.3 mM) does not regulate the in vivo activity of this enzyme, since the Km of the enzyme for fructose 1,6-bisphosphate is approximately 10-fold lower than the lowest in vivo concentration of substrate. P-enolpyruvate is proposed as the major regulator of fructose bisphosphatase activity in vivo.     

Use of 31P nuclear magnetic resonance spectroscopy and 14C fluorography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis

High-resolution 31P nuclear magnetic resonance spectroscopy and 14C fluorography have been used to identify and quantitate intermediates of the Embden-Meyerhof pathway in intact cells and cell extracts of Streptococcus lactis. Glycolysing cells contained high levels of fructose 1,6-bisphosphate (a positive effector of pyruvate kinase) but comparatively low concentrations of other glycolytic metabolites. By contrast, starved organisms contained only high levels of 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate. The concentration of Pi (a negative effector of pyruvate kinase) in starved cells was fourfold greater than that maintained by glycolysing cells. The following result suggest that retention of the phosphoenolpyruvate pool by starved cells is a consequence of Pi-mediated inhibition of pyruvate kinase: the increase in the phosphoenolpyruvate pool (and Pi) preceded depletion of fructose 1,6-bisphosphate, and reduction in intracellular Pi (by a maltose-plus-arginine phosphate trap) caused the restoration of pyruvate kinase activity in starved cells. Time course studies showed that Pi was conserved by formation of fructose 1,6-bisphosphate during glycolysis. Conversely, during starvation high levels of Pi were generated concomitant with depletion of intracellular fructose 1,6-bisphosphate. The concentrations of Pi and fructose 1,6-bisphosphate present in starved and glycolysing cells of S. lactis varied inversely. The activity of pyruvate kinase in the growing cell may be modulated by the relative concentrations of the two antagonistic effectors.     

Phosphorylation- and ligand-induced conformational changes of rat liver fructose-1,6-bisphosphatase

The effects of cyclic AMP-dependent phosphorylation on the structural properties of rat liver fructose-1,6-bisphosphatase were investigated by uv difference spectroscopy and circular dichroism. The incorporation of 4 mol of phosphate per mole of fructose-1,6-bisphosphatase induces a significant increase in the alpha-helix content of the enzyme without affecting its spectrophotometric properties. The addition of fructose 1,6-bisphosphate or fructose 2,6-bisphosphate also affects the conformation of the enzyme. However, both the phosphorylated and the nonphosphorylated forms exhibit similar ligand-induced conformational changes. These results show that cyclic AMP-dependent phosphorylation of fructose-1,6-bisphosphatase induces a specific conformational change. They also suggest that this modification does not alter the interaction of the enzyme protein with fructose 1,6-bisphosphate and fructose 2,6-bisphosphate.     

Hormonal control of fructose 2,6-bisphosphate concentration in isolated rat hepatocytes.

The ability of glucagon and of adrenaline to affect the concentration of fructose 2,6-bisphosphate in isolated hepatocytes was re-investigated because of important discrepancies existing in the literature. We were unable to detect a significant difference in the sensitivity of the hepatocytes with regard to the effect of glucagon to initiate the interconversion of phosphorylase, pyruvate kinase, 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase, and also to cause the disappearance of fructose 2,6-bisphosphate. In contrast, we have observed differences in the time-course of these various changes, since the interconversions of phosphorylase and of pyruvate kinase were at least twice as fast as those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. When measured in a cell-free system in the presence of MgATP, the cyclic AMP-dependent interconversion of pyruvate kinase was 5-10-fold more rapid than those of 6-phosphofructo-2-kinase and of fructose 2,6-bisphosphatase. These data indicate that 6-phosphofructo-2-kinase and fructose 2,6-bisphosphatase are relatively poor substrates for cyclic AMP-dependent protein kinase; they also support the hypothesis that the two catalytic activities belong to a single protein. Adrenaline had only a slight effect on the several parameters under investigation, except for the activation of phosphorylase. In the absence of Ca2+ ions from the incubation medium, however, adrenaline had an effect similar to that of glucagon.     

Competition among oxidizable substrates in brains of young and adult rats. Whole homogenates.

A new purification procedure for rat liver fructose-1,6-bisphosphatase that involves use of Procion Red-Sepharose is described. The purified enzyme was homogeneous, had a subunit Mr of 40 000-41 000 and seemed to be undegraded. The enzyme could be phosphorylated by cyclic AMP-dependent protein kinase with a stoicheiometry of one per subunit. Phosphorylation caused a 2-fold decrease in the Km of the enzyme for fructose 1,6-bisphosphate, but did not affect its allosteric responses to AMP, Mg2+ and fructose 2,6-bisphosphate.     

Hormonal regulation of L-type pyruvate kinase in hepatocytes from phosphorylase kinase-deficient (gsd/gsd) rats.

The hormonal regulation of L-type pyruvate kinase in hepatocytes from phosphorylase b kinase-deficient (gsd/gsd) rats was investigated. Adrenaline (10 microM) and glucagon (10 nM) each led to an inactivation and phosphorylation of pyruvate kinase. Dose-response curves for adrenaline-mediated inactivation of pyruvate kinase, phosphorylation of pyruvate kinase and the stimulation of gluconeogenesis from 1.8 mM-lactate were similar for hepatocytes from control and gsd/gsd rats. Time-course studies indicated that adrenaline-mediated inactivation and phosphorylation of pyruvate kinase proceeded more slowly in phosphorylase kinase-deficient hepatocytes than in control hepatocytes. The age-dependent change in the adrenergic control of pyruvate kinase was similar between control and phosphorylase kinase-deficient hepatocytes. Adrenaline, glucagon and noradrenaline activated the cyclic AMP-dependent protein kinase and inhibited pyruvate kinase in phosphorylase kinase-deficient hepatocytes. Vasopressin (0.2-2 nM), angiotensin (10nM) and A23187 (10 microM) had no effect on the activity ratio of the cyclic AMP-dependent protein kinase or pyruvate kinase in these cells. It is concluded that phosphorylase kinase plays no significant role in the hormonal control of pyruvate kinase and that phosphorylation and inactivation of this enzyme results predominantly from the action of the cyclic AMP-dependent protein kinase.     

Purification and properties of fructose-1,6-bisphosphatase of Bacillus subtilis.

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrase, EC 3.1.3.11) of Bacillus subtilis is a constitutive enzyme that was purified 1000-fold (30% yield) to 80% purity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis where it exhibits a band corresponding to 72,000 daltons. It sediments at 15 S in sucrose density gradients indicating a molecular weight of 380,000, but apparently is very asymmetric. Its activity is irreversibly inactivated in the absence of Mn2+. The enzyme specifically catalyzes dephosphorylation of D-fructose 1,6-bisphosphate with a pH optimum of 8.0. It has 40 to 60% of full activity in the absence of P-enolpyruvate; 20 microM P-enolpyruvate activates it maximally. High concentrations of monovalent cations also activate, NH4+ being most effective. Inhibitors fall into two groups. 1) Nucleoside monophosphates, phosphorylated coenzymes, and polynucleotides inhibit competitively with P-enolpyruvate (AMP (Ki = 2 microM) and dAMP are most effective). 2) The inhibition by nucleoside di- and triphosphates, PPi, and highly phosphorylated nucleotides (guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and adenosine 5'-triphosphate 3'-diphosphate are most effective) is not competed by P-enolpyruvate but is partially overcome by fructose 1,6-bisphosphate (2 microM). Therefore, highly phosphorylated nucleotides (pppGpp and others), produced in over 0.2 mM concentrations upon step down from fast to slow growth rates (Gallant, J., and Lazzarini, R.A. (1976) in Protein Synthesis (McConkey, E.H., ed) Vol. 2, pp. 309-349, Marcel Dekker, Inc., New York), can reduce the conversion rate of fructose 1,6-bisphosphate to fructose 6-phosphate during gluconeogenesis. Comparing glycolytic growth on D-glucose and gluconeogenic growth on L-malate, the intracellular concentrations of fructose 1,6-bisphosphate differ but are both above the Km (13 microM) of the enzyme, those of AMP are similar, whereas those of P-enolpyruvate (0.18 mM versus 1.3 mM) indicate that the enzyme has only 40% of its full activity during glycolysis; nucleotides other than AMP may inhibit additionally. Thus, the futile cycle of fructose 1,6-bisphosphate synthesis and degradation during glycolysis is partially avoided, but the cells are poised for rapid adaptation upon change to gluconeogenic growth conditions.     

Role of fructose 2,6-bisphosphate in the regulation of glycolysis and gluconeogenesis in chicken liver

Glucagon and dibutyryl cyclic AMP inhibited glucose utilization and lowered fructose 2,6-bisphosphate levels of hepatocytes prepared from fed chickens. Partially purified preparations of chicken liver 6-phosphofructo-1-kinase and fructose 1,6-bisphosphatase were activated and inhibited by fructose 2,6-bisphosphate, respectively. The sensitivities of these enzymes and the changes observed in fructose 2,6-bisphosphate levels are consistent with an important role for this allosteric effector in hormonal regulation of carbohydrate metabolism in chicken liver. In contrast, oleate inhibition of glucose utilization by chicken hepatocytes occurred without change in fructose, 2,6-bisphosphate levels. Likewise, pyruvate inhibition of lactate gluconeogenesis in chicken hepatocytes cannot be explained by changes in fructose 2,6-bisphosphate levels. Exogenous glucose caused a marked increase in fructose 2,6-bisphosphate content of hepatocytes from fasted but not fed birds. Both glucagon and lactate prevented this glucose effect. Fasted chicken hepatocytes responded to lower glucose concentrations than fasted rat hepatocytes, perhaps reflecting the species difference in hexokinase isozymes.     

An assessment of the ability of insulin-stimulated cyclic AMP phosphodiesterase to decrease hepatocyte intracellular cyclic AMP concentrations.

Treatment of hepatocytes with either NH4Cl (10mM) or fructose (10mM) blocks insulin's activation of the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of insulin (10 nM) to decrease intracellular cyclic AMP concentrations raised by glucagon (10 nM) was unaffected by pre-treatment with either NH4Cl (10 mM) or fructose (10 mM). It is concluded that the 'dense-vesicle' enzyme does not play a significant role in this action of insulin and that as yet unidentified cyclic AMP phosphodiesterase(s) must be activated by insulin. Treatment of hepatocytes with either NH4Cl or fructose appeared to increase, reversibly, cyclic AMP phosphodiesterase activity. When N6-(phenylisopropyl)adenosine was used to prevent glucagon from blocking insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity, insulin's ability to decrease intracellular cyclic AMP concentrations in glucagon-treated hepatocytes was increased markedly. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity can exert a potent effect in decreasing intracellular cyclic AMP concentrations elevated by glucagon.     

Effects of insulin,glucagon and dexamethasone on pyruvate kinase in cultured hepatocytes

Long-term (24–48 h) and short-term (10–30 min) regulation by hormones of hepatic pyruvate kinase activity was investigated in adult rat hepatocytes cultured under serum-free conditions. In the absence of hormones, pyruvate kinase total activity decreased to 83%, 67% and 39% of the initial level at 24, 48 and 72 h of culture. Insulin (100 nM) maintained total activity significantly above control levels throughout this period. In contrast, glucagon (100 nM) and dexamethasone (100 nM) accelerated the gradual decrease within 24 h (glucagon) or 48 h (dexamethasone) of culture. In these long-term experiments, activity at non-saturating concentrations of phosphoenolpyruvate was decreased by glucagon and dexamethasone but not directly modulated by insulin. However, insulin increased the cellular content of the pyruvate kinase activator fructose-1,6-diphosphate. In short-term experiments on cells cultured under serum- and hormone-free conditions for 48 h, both glucagon and dexamethasone independently caused a rapid, dose-dependent increase of the K_0.5 for phosphoenolpyruvate within 10 min, while V_max was not affected. Insulin inhibited this action of glucagon and dexamethasone and, in their absence, significantly increased substrate affinity for phosphoenolpyruvate within 30 min. Cellular fructose-1,6-diphosphate contents remained unchanged under these conditions. The data identify glucocorticoids and insulin - in addition to glucagon - as short-term regulators of the catalytic properties of pyruvate kinase. All three hormones are effective in the long-term control of total enzyme activity.     

Effects of various metabolic conditions and of the trivalent arsenical melarsen oxide on the intracellular levels of fructose 2,6-bisphosphate and of glycolytic intermediates in Trypanosoma brucei

Upon differential centrifugation of cell-free extracts of Trypanosoma brucei, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase behaved as cytosolic enzymes. The two activities could be separated from each other by chromatography on both blue Sepharose and anion exchangers. 6-phosphofructo-2-kinase had a Km for both its substrates in the millimolar range. Its activity was dependent on the presence of inorganic phosphate and was inhibited by phosphoenolpyruvate but not by citrate or glycerol 3-phosphate. The Km of fructose-2,6-bisphosphatase was 7 microM; this enzyme was inhibited by fructose 1,6-bisphosphate (Ki = 10 microM) and, less potently, by fructose 6-phosphate, phosphoenolpyruvate and glycerol 3-phosphate. Melarsen oxide inhibited 6-phosphofructo-2-kinase (Ki less than 1 microM) and fructose-2,6-bisphosphatase (Ki = 2 microM) much more potently than pyruvate kinase (Ki greater than 100 microM). The intracellular concentrations of fructose 2,6-bisphosphate and hexose 6-phosphate were highest with glucose, intermediate with fructose and lowest with glycerol and dihydroxyacetone as glycolytic substrates. When added with glucose, salicylhydroxamic acid caused a decrease in the concentration of fructose 2,6-bisphosphate, ATP, hexose 6-phosphate and fructose 1,6-bisphosphate. These studies indicate that the concentration of fructose 2,6-bisphosphate is mainly controlled by the concentration of the substrates of 6-phosphofructo-2-kinase. The changes in the concentration of phosphoenolpyruvate were in agreement with the stimulatory effect of fructose 2,6-bisphosphate on pyruvate kinase. At micromolar concentrations, melarsen oxide blocked almost completely the formation of fructose 2,6-bisphosphate induced by glucose, without changing the intracellular concentrations of ATP and of hexose 6-phosphates. At higher concentrations (3-10 microM), this drug caused cell lysis, a proportional decrease in the glycolytic flux, as well as an increase in the phosphoenolypyruvate concentrations which was restricted to the extracellular compartment. Similar changes were induced by digitonin. It is concluded that the lytic effect of melarsen oxide on the bloodstream form of T. brucei is not the result of an inhibition of pyruvate kinase.     

Hormonal control of [14C]glucose synthesis from [U-14C]dihydroxyacetone and glycerol in isolated rat hepatocytes.

The hormonal control of [14C]glucose synthesis from [U-14C-A1dihydroxyacetone was studied in hepatocytes from fed and starved rats. In cells from fed rats, glucagon lowered the concentration of substrate giving half-half-maximal rates of incorporation while it had little or no effect on the maximal rate. Inhibitors of gluconeogenesis from pyruvate had no effect on the ability of the hormone to stimulate the synthesis of [14C]glucose from dihydroxyacetone. The concentrations of glucagon and epinephrine giving half-maximal stimulation from dihydroxacetone were 0.3 to 0.4 mM and 0.3 to 0.5 muM, respectively. The meaximal catecholamine stimulation was much less than the maximal stimulation by glucagon and was mediated largely by the alpha receptor. Insulin had no effect on the basal rate of [14C]clucose synthesis but inhibited the effect of submaximal concentration of glucagon or of any concentration of catecholamine. Glucagon had no effect on the uptake of dihydroxyacetone but suppressed its conversion to lactate and pyruvate. This suppression accounted for most of the increase in glucose synthesis. In cells from gasted rats, where lactate production is greatly reduced and the rate of glucose synthesis is elevated, glucagon did not stimulate gluconeogenesis from dihydroxyacetone. Findings with glycerol as substrate were similar to those with dihyroxyacetone. Ethanol also stimulated glucose production from dihydroxyacetone while reducing proportionately the production of lactate. Ethanol is known to generate reducing equivalents fro clyceraldehyde-3-phosphate dehydrogenase and presumably thereby inhibits carbon flux to lactate at this site. Its effect was additive with that of glucagon. Estimates of the steady state levels of intermediary metabolites and flux rates suggested that glucagon activated conversion of fructose diphosphate to fructose 6-phosphate and suppressed conversion of phosphoenolpyruvate to pyruvate. More direct evidence for an inhibition of pyruvate kinase was the observation that brief exposure of cells to glucagon caused up to 70% inhibition of the enzyme activity in homogenates of these cells. The inhibition was not seen when the enzyme was assayed with 20 muM fructose diphosphate. The effect of glucagon to lower fructose diphosphate levels in intact cells may promote the inhibition of pyruvate kinase. The inhibition of pyruvate kinase may reduce recycling in the pathway of gluconeogenesis from major physiological substrates and probably accounts fromsome but not all the stimulatory effect of glucagon.     

Long-term effect of glucagon administration on rat liver L-type pyruvate kinase.

After 5 h of treatment with glucagon, liver L-type pyruvate kinase (ATP: pyruvate 2-0-phosphotransferase; EC 2.7.1.40) showed a significant decrease of K0.5 and the Hill coefficient (nH) in the absence of fructose 1,6-diphosphate. However, in the presence of fructose 1,6-diphosphate, liver enzymes from treated rats showed a slight decrease of K0.5 but nH remained unchanged. In both circumstances, no changes of Vmax were observed after treatment. These changes in the kinetic properties of liver L-type pyruvate kinase are consistent with the dephosphorylation of the enzyme caused by insulin release in response to treatment with glucagon.     

A study of the allosteric kinetics of Phycomyces pyruvate kinase as judged by the effect of L-alanine and fructose 1,6-bisphosphate

The influence of fructose 1,6-bisphosphate and L-alanine on the kinetics of pyruvate kinase (ATP:pyruvate O2-phosphotransferase, EC 2.7.1.40) from Phycomyces blakesleeanus NRRL 1555 (-) was studied at pH 7.5. By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phosphoenol pyruvate and Mg2+ were abolished and the velocity curves became hyperbolic. In the presence of L-alanine the positive homotropic cooperativity with respect to phosphoenol pyruvate increased with Hill coefficient values close to 4, while the sigmoid kinetics with respect to Mg2+ became hyperbolic. Fructose 1,6-bisphosphate overcomes the inhibition produced by L-alanine, the antagonism between phosphoenol pyruvate and L-alanine also being evident. Inhibition has been found at high Mg2+ concentrations, compatible with the binding of the magnesium ions to an inactive conformational state of the enzyme. The data were analysed on the basis of the two-states concerted-symmetry model of Monod, Wyman and Changeux, and the parameters of the model were calculated. Phosphoenol pyruvate and fructose 1,6-bisphosphate appeared to show exclusive binding to the active conformational state (R), whereas magnesium ions bind preferentially, by a factor of 45, to the R state. L-Alanine binds more readily to the inactive T state of the enzyme.